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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Gustafsson, A. C.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Single-nucleotide polymorphism analysis by pyrosequencing2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 280, no 1, 103-110 p.Article in journal (Refereed)
    Abstract [en]

    There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic: alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

  • 2.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Analysis of the p53 tumor suppressor gene by pyrosequencing2000In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 28, no 1, 140-+ p.Article in journal (Refereed)
    Abstract [en]

    Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one ol both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Further-more, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

  • 3. Andersson, K
    et al.
    Gülich, S
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hämäläinen, M
    Nygren, P A
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Malmqvist, M
    Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space.1999In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 37, no 3Article in journal (Refereed)
    Abstract [en]

    The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.

  • 4.
    Berglund, Helena
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    NMR studies of DNA/RNA-binding proteins1997Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis concerns the structure and internal dynamics inDNA and RNA-binding proteins. While it has long been known thata proper three-dimensional structure is essential for aprotein's ability to interact with other biomolecules, theinfluence of the inherent protein dynamics is less wellunderstood. Using nuclear magnetic resonance (NMR)spectroscopy, both structure and dynamics within a protein canbe assessed. By applying two- and three-dimensional NMRtechniques on uniformly isotopically labelled protein samples,the solution structures of biomolecules or biomolecularcomplexes with molecular weights up to approximately 35 000 Dacan be determined. In addition, protein dynamics on manydifferent timescales can be investigated using severaldifferent experimental techniques, for example relaxationexperiments and amide hydrogen exchange measurements. Inparticular 15N relaxation measurements give a very useful viewof the protein mobility along the peptide backbone.

    DNA/RNA-binding proteins participate in many processes inthe cell and there is still a lot to be learned about how theseproteins exert their function. A characterisation of theinternal dynamics in these proteins and if and how theirmobility influences the biological funcition is an importantstep in this work. This thesis involves the DNA-bindingproteins Sso7d fromSulfolobus solfatancusand the glucocorticoid receptorDNA-binding domain (GRDBD) as wellasthe ribosomal RNA binding protein S 15 fromThennus thermophilus.We have shown that thesequencespecific binding glucocorticoid receptor DNA-binding domain isa well ordered protein also in the uncomplexed state and thatGRDBD is not subjected to any major local folding processesupon DNA complexation. We could also show increased dynamics ina triple mutant with altered DNA binding specificity.Furthermore, arginine side chain dynamics was investigated indifferent protein surroundings; buried in a protein core inGRDBD, solvent-exposed on the Sso7d surface, and finally at theSso7d-DNA interface. We could demonstrate that the side chainmobility on the protein-DNA interface was restricted but to alesser extent than in the core region of a protein. Finally,NMR methods were used to determine the solution structure ofthe ribosomal rRNA binding protein S 15. The structure revealeda predorninandy ot-helical fold and allowed for identificationof a putative RNA-interacting surface.

    Keywords:nuclear magnetic resonance spectroscopy,protein dynamics, nucleic acid binding proteins, NMR relaxationexperiments

  • 5.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Controlling lipase enantioselectivity for organic synthesis2001In: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 18, no 1, 13-22 p.Article, review/survey (Refereed)
    Abstract [en]

    Lipases are used frequently as chiral catalysts in the synthesis of various fine chemicals and intermediates. The increasing need of compounds with high stereochemical purity requires catalysts with an improved and controlled performance. This overview emphasizes some important aspects for the control of lipase enantioselectivity and some examples where the enantioselectivity has been altered or reversed are highlighted. However, in several of these cases the complete explanation for the altered or reversed enantioselectivity remains unclear and needs to be solved. Three different strategies (engineering of the reaction medium, the substrate molecule, and the enzyme) for exploring lipase enantioselectivity at a molecular level are discussed and summarized. These three different approaches represent powerful tools for understanding the molecular basis for lipase enantioselective catalysis and can guide the rational improvement and tailoring of catalyst performance. By combining approaches from chemistry and biology much is learnt about the most important parameters controlling lipase enantioselectivity for organic synthesis.

  • 6. Carlqvist, P.
    et al.
    Eklund, R.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Brinck, Tore
    KTH, Superseded Departments, Chemistry.
    Rational design of a lipase to accommodate catalysis of Baeyer-Villiger oxidation with hydrogen peroxide2003In: Journal of Molecular Modeling, ISSN 1610-2940, E-ISSN 0948-5023, Vol. 9, no 3, 164-171 p.Article in journal (Refereed)
    Abstract [en]

    The mechanism and potential energy surface for the Baeyer-Villiger oxidation of acetone with hydrogen peroxide catalyzed by a Ser105-Ala mutant of Candida antarctica Lipase B has been determined using ab initio and density functional theories. Initial substrate binding has been studied using an automated docking procedure and molecular dynamics simulations. Substrates were found to bind to the active site of the mutant. The activation energy for the first step of the reaction, the nucleophilic attack of hydrogen peroxide on the carbonyl carbon of hydrogen peroxide, was calculated to be 4.4 kcal mol(-1) at the B3LYP/6-31+G* level. The second step, involving the migration of the alkyl group, was found to be the rate-determining step with a computed activation energy of 19.9 kcal mol(-1) relative the reactant complex. Both steps were found to be lowered considerably in the reaction catalyzed by the mutated lipase, compared to the uncatalyzed reaction. The first step was lowered by 36.0 kcal mol(-1) and the second step by 19.5 kcal mol(-1). The second step of the reaction, the rearrangement step, has a high barrier of 27.7 kcal mol(-1) relative to the Criegee intermediate. This could lead to an accumulation of the intermediate. It is not clear whether this result is an artifact of the computational procedure, or an indication that further mutations of the active site are required.

  • 7. Cheeseman, J. D.
    et al.
    Tocilj, A.
    Park, Seongsoon
    KTH, Superseded Departments, Biochemistry and Biotechnology. Department of Chemistry, McGill University, Canada .
    Schrag, J. D.
    Kazlauskas, R. J.
    Structure of an aryl esterase from Pseudomonas fluorescens2004In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 60, no 7, 1237-1243 p.Article in journal (Refereed)
    Abstract [en]

    The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 Å by X-ray diffraction and shows a characteristic α/β-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 Cα atoms between PFE and its five closest structural neighbors averaging 0.8 Å. PFE has far less similarity (r.m.s. deviation in 218 Cα atoms of 5.0 Å) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.

  • 8.
    Ehn, Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nourizad, Nader
    KTH, Superseded Departments, Biotechnology.
    Bergstrom, Kristina
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 329, no 1, 11-20 p.Article in journal (Refereed)
    Abstract [en]

    Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

  • 9. Ellegren, H
    et al.
    Savolainen, Peter
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Rosen, B
    The genetical history of an isolated population of the endangered grey wolf Canis lupus: A study of nuclear and mitochondrial polymorphisms1996In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 351, no 1348, 1661-1669 p.Article in journal (Refereed)
    Abstract [en]

    The grey wolf was thought to have been exterminated in the Scandinavian peninsula when the sudden appearance of a few animals in southern Sweden was reported in 1980. These wolves founded a new Swedish population which currently numbers at least 25 individuals, one of the world's smallest populations of the species. The sudden occurrence of the founder animals caused speculation that these had not appeared by 'natural' means but rather were Swedish zoo animals deliberately released by man. To analyse if this was the case and to elucidate the genetic status bi; this small and isolated population, we assessed nuclear and mitochondrial (mt) genetic variability in wild and captive grey wolves, using microsatellite typing and sequence analysis of the mtDNA D-loop. The new population was found to be monomorphic for a mtDNA haplotype which also was present in the Swedish zoo population. A total of four different mtDNA haplotypes were found among all captive and wild wolves (including two animals from an occasional establishment of a few wolves in northern Sweden in the late 1970s), with a maximum sequence divergence of 3.1 %. Despite the mtDNA congruence, animals from the zoo population could most likely be excluded as founders for the wild population since the latter group of animals displayed several unique microsatellite alleles (i.e. alleles not found in the zoo population). Moreover, a phylogenetic analysis of individual wolves, using microsatellite allele sharing as distance measure, placed all wild animals on a branch separated from that of the captive animals. The average degree of nuclear variability as well as allelic diversity was similar in the wild and the captive populations, respectively, but was lower than that reported for North-American populations of grey wolves. Polymorphism has declined in wild wolves born in recent years suggesting that this small population is currently suffering from a loss of genetic variability due to inbreeding. Inbreeding depression is documented in captive wolves and the long-term survival of the wild Swedish population may therefore depend on immigration of animals from Russia. This study illustrates the usefulness of microsatellites for dissecting close genetic relationships and for addressing the genetic status of individuals.

  • 10. Eriksson, J.
    et al.
    Karamohamed, S.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method for real-time detection of inorganic pyrophosphatase activity2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 293, no 1, 67-70 p.Article in journal (Refereed)
    Abstract [en]

    A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.

  • 11. Gambelunghe, G.
    et al.
    Ghaderi, M.
    Brozzetti, A.
    Del Sindaco, P.
    Gharizadeh, B.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hjelmstrom, P.
    Nikitina-Zake, L.
    Sanjeevi, C. B.
    Falorni, A.
    Umbria Type 1 Diabetes, Registry
    Lack of association of CCR2-64I and CCR5-Delta 32 with type 1 diabetes and latent autoimmune diabetes in adults2003In: Human Immunology, ISSN 0198-8859, E-ISSN 1879-1166, Vol. 64, no 6, 629-632 p.Article in journal (Refereed)
    Abstract [en]

    It is well known that type I diabetes mellitus (T1DM) is a complex genetic disease resulting from the autoimmune destruction of pancreatic beta cells. Several genes have been associated with susceptibility and/or protection for T1DM, but the disease risk is mostly influenced by genes located in the class II region of the major histocompatibility complex. The attraction of leukocytes to tissues is essential for inflammation and the beginning of autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytolines. Some studies have shown that CCR2-64I and CCR5-Delta32 might be important for protection of susceptibility to some immunologically-mediated disorders. In the present study, we demonstrate the lack of association between CCR2-64I and CCR5-Delta32 gene polymorphism and TIDM and we desrcibe a new method for a simple and more precise genotyping of the CCR2 gene.

  • 12. Gambelunghe, G.
    et al.
    Ghaderi, M.
    Gharizadeh, Baback
    KTH, Superseded Departments, Biotechnology.
    Brozzetti, A.
    Tortoioli, C.
    Del Sindaco, P.
    Sanjeevi, C. B.
    Hjelmstrom, P.
    Sirsjo, A.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Santeusanio, F.
    Falorni, A.
    Lack of association of human chemokine receptor gene polymorphisms CCR2-64I and CCR5-Delta 32 with autoimmune Addison's disease2004In: European journal of immunogenetics, ISSN 0960-7420, E-ISSN 1365-2370, Vol. 31, no 2, 73-76 p.Article in journal (Refereed)
    Abstract [en]

    The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5-Delta32 and CCR2-64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2-64I and by polymerase chain reaction and detecting gel for CCR5-Delta32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2-64I and CCR5-Delta32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.

  • 13. Garcia, C. A.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene2000In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 253, no 2, 249-257 p.Article in journal (Refereed)
    Abstract [en]

    The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing(TM) is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

  • 14.
    George, Stefan
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Effects of high sugar concentrations in production scale bioreactor: a scale-down study of s. cervisiae1997Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Substrate concentration gradients exist in large-scalebioreactors, due to a combination of insufficient mass transferand mixing, and microbial activities. Zones with high substrateconcentration may arise close to a feed point. It is largelyunknown to what extent these gradients influence themicroorganisms and what the microbial response might be. Inthis work a scale-down method was developed, for studies ofsuch responses. A coupled reactor system was constructed, inwhich effects of repeated short term exposures to concentrationgradients can be studied. Zones of different kinds, welldefined with respect to substrate concentration and residencetime, can be created by applying different feeding and aerationtechniques.

    The microbial test system used was the aerobic ethanolproduction ofSaccharomyces cerevisiae,due to high sugarconcentrations. Effects of repeated exposures to suchconcentrations were demonstrated. Influences of the culturewere observed at many different levels: a decrease in biomassyield and an increased ethanol production, an increase in themaximum respiration rate, and enhancement of the quality of theBaker's yeast, expressed as the gassing power.

    The dynamics of the actual response to high sugarconcentrations were studied in the reactor system: Transientsin specific rates of glucose consumption and ethanol productionwere demonstrated during the first minute of such an exposure,explained by a short term regulation of the glucose uptake. Theresponse was also shown to be influenced by the history of thecells. The response for cells formerly not exposed to highsugar concentrations differed considerably from that of cellsrepeatedly exposed to such conditions.

    Methods for examining a large scale bioreactor environmentwere developed. A study of a215m3 bubble column reactor was performed, with respectto concentration gradients (sugar, oxygen and intracellularadenosine nucleotides) and hydrodynamic flow pattern (gashold-up and liquid velocities). The liquid velocity profileexhibited the expected gulf-stream pattern. Themicroenvironment for the cell was however probablycharacterized by environmental fluctuations, since axialgradients of oxygen, sugar and adenosine nucleotides weredemonstrated.

    Keywords:scale-down, plug flow reactor, S. cerevisiae,Crabtree effect, Baker's yeast, large scale study,concentration gradients, mixing, overflow metabolism.

  • 15. Gharizadeh, B.
    et al.
    Eriksson, J.
    Nourizad, N.
    Nordstrom, Tommy
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improvements in pyrosequencing technology by employing sequenase polymerase2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, no 2, 272-280 p.Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.

  • 16. Gharizadeh, B.
    et al.
    Ghaderi, M.
    Donnelly, D.
    Amini, B.
    Wallin, K. L.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Multiple-primer DNA sequencing method2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 08-jul, 1145-1151 p.Article in journal (Refereed)
    Abstract [en]

    A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or m ore sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as,multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing(TM) technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.

  • 17. Gharizadeh, B.
    et al.
    Kalantari, M.
    Garcia, C. A.
    Johansson, B.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Typing of human papillomavirus by pyrosequencing2001In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 81, no 5, 673-679 p.Article in journal (Refereed)
    Abstract [en]

    The possibility of using a new bioluminometric UNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.

  • 18. Gharizadeh, B.
    et al.
    Nordstrom, T.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer2002In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 301, no 1, 82-90 p.Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.

  • 19. Gharizadeh, B.
    et al.
    Ohlin, A.
    Molling, P.
    Backman, A.
    Amini, B.
    Olcen, P.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Multiple group-specific sequencing primers for reliable and rapid DNA sequencing2003In: Molecular and Cellular Probes, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 17, no 4, 203-210 p.Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing(TM) technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

  • 20.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Ehn, Maria
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Gunnel, Lundin
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hedhammar, My
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner2002In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, no 1-2, 157-166 p.Article in journal (Refereed)
    Abstract [en]

    To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

  • 21.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hedhammar, My
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, 93-102 p.Article in journal (Refereed)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 22.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundin, Gunnel
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Charge engineering of a protein domain to allow efficient ion-exchange recovery2000In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, no 10, 703-709 p.Article in journal (Refereed)
    Abstract [en]

    We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

  • 23.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nilsson, Joakim
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lindberg, Michael
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein1997In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, 125-132 p.Article in journal (Refereed)
    Abstract [en]

    A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.

  • 24. Gustavsson, M.
    et al.
    Lehtio, J.
    Denman, S.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Martinelle, Mats
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris2001In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 14, no 9, 711-715 p.Article in journal (Refereed)
    Abstract [en]

    Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides. The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function. Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed. Three variants were found to be stable throughout 7-day cultivations. The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers. The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wildtype lipase.

  • 25.
    Gustavsson, Malin T.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Persson, P. V.
    Iversen, T.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Martinelle, Mats
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Polyester coating of cellulose fiber surfaces catalyzed by a cellulose-binding module-Candida antarctica lipase B fusion protein2004In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 5, no 1, 106-112 p.Article in journal (Refereed)
    Abstract [en]

    A new approach to introduce polymers to cellulosic materials was developed by using the ability of a cellulose-binding module-Candida antarctica lipase B conjugate to catalyze ring-opening polymerization of epsilon-caprolactone in close proximity to cellulose fiber surfaces. The epsilon-caprolactone was introduced to the cellulose surfaces either by simple addition of liquid monomer or through gas phase. The effects of water activity and temperature on the lipase-catalyzed polymerization process were investigated. Analysis showed that the water content in the system primarily regulated the obtained polymer molecular weight, whereas the temperature influenced the reaction rate. The hydrophobicity of the obtained surfaces did not arise from covalent attachment of the poly(epsilon-caprolactone) to the surface hydroxyl groups but rather from surface-deposited polymers which could be readily extracted. The degree of lipase-catalyzed hydrolysis through introduction of water to the polymer-coated cellulose fiber surfaces was also investigated and shown to be significant.

  • 26.
    Hansson, Marianne
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Design, selection and production of recombinant proteins for prevention of infectious diseases1998Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The development of subunit vaccines is presently one of themain strategies for the generation of new vaccines againstinfectious diseases. The use of recombinant DNA techniques hasfacilitated the develoment of new strategies for constructionand production of subunit vaccines. This thesis describesvarious such techniques, including (i) new general methods forgene assembly applied to antigen-encoding genes, (ii) asuggested strategy for bioprocess improvement applied to theproduction of a malaria vaccine candidate, (iii) thedevelopment of novel live bacterial vaccine delivery systems,based on non-pathogenic staphylococci, and (iv) thein vitroselection of a new type of antigen-bindingprotein.

    Two different strategies forde novogene assembly have been developed. A method forassembly and polymerization of DNA fragments was developed,based on the class-IIS restriction enzymeBspMI. The central and the C-terminal repeat regions ofthePlasmodium falciparummalaria blood-stage antigenPf155/RESA were both polymerized separately (designated M5 andM3, respectively), using this method. The assembled genes weresuccessfully expressed inE. colias fusions to the divalent IgG-binding proteinZZ, from staphylococcal protein A, and the human serum albumin(HSA) binding protein BB from streptococcal protein G,respectively. Full-length fusion proteins wereaffinity-purified on IgG- and HSA-Sepharose, respectively.Three copies of a respiratorial syncytial virus (RSV) G proteinencoding fragment (designated G3) was also assembled using thismethod. For assembly of theP. falciparumblood-stage antigen Ag332 repeat region, asolid-phase gene construction method was developed. The desirednumber of repeats was built up in a controlled and stepwisemanner. Genes encoding two, three or four copies of an Ag332epitope were expressed inE. colias fusions to ZZ and BB. Both these methods forgene assembly, and in particular the solid-phase strategy, havefound broad applications in thede novoconstruction of genes.

    Expanded bed adsorption was, for the first time for arecombinant product, used to recover the secreted ZZ-M5 fusionprotein directly from crudeE. coliculture medium by anion-exchange chromatography.In a single step, more than 90% of the produced recombinantmalaria vaccine candidate was recovered. The study demonstratedthat the initial genetic design can allow integration of unitoperations and thus improve the downstream productionprocess.

    In the context of live bacterial vaccine delivery, twodifferent non-pathogenic food grade staphylococci,Staphylococcus xylosusandS. carnosus, have been investigated, and expressionsystems allowing efficient surface display of foreign antigenicdeterminants have been described. These systems were used todisplay the malarial antigen M3 onS. xylosusandS. carnosus,and the RSV antigen G3 onS. xylosus. New techniques to verify exposure of therecombinant proteins on the bacterial surface were alsodeveloped.S. xylosuscells displaying the RSV G3 protein were usedto immunize mice orally, resulting in the induction of G3specific serum antibodies that were sustained for more than 20weeks.

    Using phage-display technology, a binding protein (affibody)capable of specific recognition of an RSV vaccine candidate wasselectedin vitro. Biopanning of a phage-displayed combinatoriallibrary, based on the IgG-binding Z domain, against a 101 aminoacid region (G2nata) of the G protein of RSV subgroup A,resulted in the isolation of an affibody, ZRSV1, recognizingthe G proteins of both subgroup A and B RSV. The binding siteof the ZRSV1-affibody was mapped and found to be in a regionproposed to be involved in virus-to-cell attachment and inwhich protective epitopes have been described. The potentialuse of affibodies as diagnostic tools or devices forpassive-vaccination applications, is discussed.

    Key words:affibody, bacterial surface display, expandedbed adsorption, fusion protein, gene assembly,in vitroselection, live bacterial vector, phagedisplay,Plasmodium falciparum, respiratory syncytial virus,staphylococcal protein A,Staphylococcus carnosus,Staphylococcus xylosus, streptococcal protein G, subunitvaccine

    © Marianne Hansson, 1998

  • 27.
    Hedin, Eva . M. K.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Hoyrup, P.
    Patkar, S. A.
    Vind, J.
    Svendsen, A.
    Fransson, L.inda
    KTH, Superseded Departments, Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 48, 14185-14196 p.Article in journal (Refereed)
    Abstract [en]

    The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry, 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using, a water-soluble spin relaxation agent. chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (< 0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.

  • 28.
    Hedin, Eva M. K.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Mouritsen, Ole G.
    Hoyrup, P.
    Low microwave-amplitude ESR spectroscopy: Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins2004In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 60, no 2, 117-138 p.Article in journal (Refereed)
    Abstract [en]

    Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T-2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.

  • 29.
    Hedin, Eva. M. K.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Patkar, S. A.
    KTH, Superseded Departments, Biotechnology.
    Vind, J.
    KTH, Superseded Departments, Biotechnology.
    Svendsen, A.
    KTH, Superseded Departments, Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Selective reduction and chemical modification of oxidized lipase cysteine mutants2002In: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 80, no 6, 529-539 p.Article in journal (Refereed)
    Abstract [en]

    Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) Were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.

  • 30.
    Hober, Sophia
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Forsberg, G
    Palm, G
    Hartmanis, M
    Nilsson, B
    Disulfide exchange folding of insulin-like growth factor I.1992In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 31, no 6Article in journal (Refereed)
    Abstract [en]

    The disulfide exchange folding properties of insulin-like growth factor I (IGF-I) have been analyzed in a redox buffer containing reduced (10 mM) and oxidized (1 mM) glutathione. Under these conditions, the 3 disulfide bridges of the 70 amino acid peptide were not quantitatively formed. Instead, five major forms of IGF-I were detected, and these components were concluded to be in equilibrium as their relative amounts were similar starting from either reduced, native, or a mismatched variant of IGF-I containing two non-native disulfides. The different components in the mixtures were trapped by thiol alkylation using vinylpyridine and subsequently isolated by reverse-phase HPLC. The purified variants were further characterized using plasma desorption mass spectrometry and peptide mapping. Two of the five different forms were identified as native and mismatched IGF-I. One form was a variant with only one disulfide bond, and the other two major components had two disulfides formed. In a separate experiment, early refolding intermediates were trapped by pyridylethylation after only 90 s of refolding in the glutathione buffer, starting from reduced IGF-I. The intermediates were identical to the components observed at equilibrium, but at different relative concentrations. On the basis of the disulfide bond patterns of the different components in the equilibrium mixtures, we conclude that the disulfide between cysteines-47 and -52 in IGF-I is an unfavorable high-energy bond that may exist in the native molecule in a strained configuration.

  • 31.
    Hober, Sophia
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hansson, A
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nilsson, B
    Folding of insulin-like growth factor I is thermodynamically controlled by insulin-like growth factor binding protein.1994In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 33, no 22Article in journal (Refereed)
    Abstract [en]

    Insulin-like growth factor I (IGF-I) is thermodynamically unable to quantitatively form its native disulfides under reversible redox conditions in vitro [Hober et al. (1992) Biochemistry 31, 1749-1756]. These results prompted the question of how IGF-I may overcome this energetic problem in its folding in vivo. Here, we report that an IGF-I precursor, IGF-I-Ea, shows disulfide-exchange folding properties similar to those of mature IGF-I and, thus, is concluded not to overcome the identified folding problem of mature IGF-I. However, correct disulfide bonds are formed very efficiently when insulin-like growth factor binding protein 1 is added in equimolar amounts to IGF-I to the refolding mixture. On the basis of these results, we propose that one important function of at least one of the six homologous insulin-like growth factor binding proteins is to assist in the formation and maintenance of the native disulfides of IGF-I. To our knowledge, this is the first example where the folding of a mammalian protein or peptide in circulation has been demonstrated to be thermodynamically controlled by its binding protein. Speculatively, this could provide a mechanism to regulate the half-life of IGF-I in vivo by altering the interaction with insulin-like growth factor binding proteins.

  • 32.
    Hober, Sophia
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundström Ljung, J
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nilsson, B
    Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions.1999In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 443, no 3Article in journal (Refereed)
    Abstract [en]

    Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.

  • 33.
    Holmquist, Mats
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improved lipase enantioselectivity by combinatorial and rational redesign.2000In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 219, no 1, U163-U163 p.Article in journal (Refereed)
  • 34.
    Hult, Karl
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Engineered enzymes for improved organic synthesis2003In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 14, no 4, 395-400 p.Article, review/survey (Refereed)
    Abstract [en]

    Recent developments to modify enzymes for use in organic synthesis have targeted several areas. These include altering the reaction mechanism of the enzyme to catalyse new reactions, switching substrate specificity, expanding substrate specificity, and improving substrate specificity, such as enantioselectivity in kinetic resolutions. Such modifications can be achieved either by rational redesign, which requires knowledge of the enzyme structure, or by random mutagenesis methods followed by screening. Both strategies of enzyme engineering can be successful and are very useful for improving the utility of enzymes for applied catalysis. Several examples illustrating these concepts in a variety of enzyme classes have appeared recently.

  • 35.
    Jahic, Mehmedalija
    et al.
    KTH, Superseded Departments, Biotechnology.
    Rotticci-Mulder, Johanna C.
    Martinelle, Mats
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein2002In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 24, no 6, 385-393 p.Article in journal (Refereed)
    Abstract [en]

    A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O-2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.

  • 36.
    Jansson, Magnus
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Structure-function analysis of insuline-like growth factor I interactions1997Doctoral thesis, comprehensive summary (Other scientific)
  • 37.
    Jansson, Magnus
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Andersson, G
    Uhlén, M
    Nilsson, B
    Kördel, J
    The IGFBP-1 binding epitope of IGF-1 probed by heteronuclear NMR and mutational analysisManuscript (preprint) (Other academic)
  • 38.
    Jansson, Magnus
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Dixelius, J
    Uhlén, M
    Nilsson, B O
    Binding affinities of insulin-like growth factor-I (IGF-I) fusion proteins to IGF binding protein 1 and IGF-I receptor are not correlated with mitogenic activity.1997In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 416, no 3, 259-264 p.Article in journal (Refereed)
    Abstract [en]

    In this report, comparisons between molecular affinities and cellular proliferation activities have been made for insulin-like growth factor-I (IGF-I) and two IGF-I fusion proteins in order to evaluate fusion proteins as tools for receptor binding studies. Binding affinities and growth promoting effects of the N-terminal fusion Z-IGF-I and the C-terminal fusion IGF-I-Z, and native recombinant human IGF-I, were analyzed. Binding kinetic properties of the three IGF-I variants were analyzed using BIAcore kinetic interaction analysis testing for binding to both human IGF binding protein 1 (IGFBP-1) and a soluble form of the human IGF type I receptor extracellular domains (sIGF-IR). The growth promoting effects on SaOS-2 human osteosarcoma cells of the different fusion proteins were analyzed. A comparison of receptor binding affinities and growth promoting effects shows that the fusion protein receptor affinity does not correlate with proliferative potential. The IGF-I-Z fusion, with the lowest receptor affinity, shows similar proliferative potential to native IGF-I. However, the Z-IGF-I fusion protein, with twice the receptor affinity of IGF-I-Z, displays only about 70% of the IGF-I-Z growth promoting activity. Both IGF-I fusion proteins possess similar affinity to IGFBP-1. These results indicate that determinants other than the receptor affinity could be involved in the regulation of IGF-I proliferative action. This study demonstrates that ligand fusion proteins may be useful to study mechanisms of ligand induced receptor activation.

  • 39.
    Jansson, Magnus
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hallén, D
    Koho, H
    Andersson, G
    Berghard, L
    Heidrich, J
    Nyberg, E
    Uhlén, M
    Kördel, J
    Nilsson, B
    Characterization of ligand binding of a soluble human insulin-like growth factor I receptor variant suggests a ligand-induced conformational change.1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 13, 8189-8197 p.Article in journal (Refereed)
    Abstract [en]

    Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted alpha-beta'-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 x 10(8) M-1 (25 degrees C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 degrees C) and in two different buffer systems at 25 degrees C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(alpha-beta'-Z)2) receptor complex in solution are deduced to 0.96 x 10(8) M-1 (25 degrees C). The determined heat capacity change for the process is large and negative, -0.51 kcal (K mol)-1. Further, the entropy change (DeltaS) at 25 degrees C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding.

  • 40.
    Jansson, Magnus
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, M
    Nilsson, B
    Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor.1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 14, 4108-4117 p.Article in journal (Refereed)
    Abstract [en]

    Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.

  • 41. Johansson, K. E.
    et al.
    Duhamel, G. E.
    Bergsjö, L.
    Engvall, E. O.
    Persson, M.
    Pettersson, Bertil
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Fellström, C.
    Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis2004In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 53, no 4, 345-350 p.Article in journal (Refereed)
    Abstract [en]

    It has been suggested that canine intestinal spirochaetes consist of Brachyspira pilosicoli and a group of strains that has been provisionally designated 'Brachyspira canis'. The purpose of the present study was to compare 22 spirochaete isolates that were obtained from intestinal specimens of dogs in Sweden (n = 12), Norway (n = 4), the United States (n = 3), Australia (n = 2) and Germany (n = 1) with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences. In an evolutionary tree derived from 16S rDNA sequences, the canine isolates grouped into three clusters. One cluster included the type strain of porcine B. pilosicoli, whereas a second larger cluster, which was monophyletic, contained a canine strain that was identified previously as 'B. canis'. The third cluster consisted of three canine isolates of Scandinavian origin, which grouped together with the type strain of the species Brachyspira alvinipulli (pathogenic to chicken). These three genotypes, which were identified on the basis of 16S rDNA sequences, corresponded to four phenotypic groups based on biochemical testing. Two biochemical tests, hippurate hydrolysis and alpha-galactosidase production, were sufficient for rapid identification of each canine cluster.

  • 42.
    Jonasson, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Genetic design for facilitated production of human peptide hormones in<I>Escherichia coli</I>1999Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Genetic strategies have become important in the design ofefficient processes for production of recombinant proteins.When constructing a production scheme, inherent properties,purity and quality requirements, as well as the final use ofthe protein, have to be considered. In this thesis, geneticdesign has been applied to differentEscherichia coliproduction processes for human peptidehormones, facilitating the down-stream processing andincreasing the expression yields.

    UponE. coliproduction of insulin-like growth factor I(IGF-I), misfolded and aggregated forms of IGF-I have reducedthe yield of correctly folded IGF-I. Extensive in vitrorefolding schemes have thus been considered necessary forobtaining acceptable yields. We investigated whethercoexpression of the IGF binding protein type 1 (IGFBP-1) wouldincrease the yield of correctly folded IGF-I. In this study,the two fusion proteins BB-IGFBP-1 and Z-IGF-I were used,containing the serum albumin binding affinity tag BB and theIgG-binding affinity tag Z, respectively. It was demonstratedthat correctly folded IGF-I could be recovered by twosubsequent affinity chromatography steps, verifying thatZ-IGF-I/BB-IGFBP-1 heterodimers were formed in vivo.Furthermore, the addition of a glutathione redox buffer duringcultivation significantly improved the relative yields ofcorrectly folded IGF-I, suggesting that affinity-assisted invivo folding could be considered as an attractive strategy forrecombinant proteins secreted to theE. coliperiplasm.

    Aiming for an efficient process for production of both humaninsulin and proinsulin C-peptide, the possibility to integratethe removal of an affinity handle with the processing ofproinsulin to insulin and C-peptide, was investigated.Expression vectors encoding three different ZZ-proinsulinfusion proteins were constructed. Between the two IgG-binding Zdomains and proinsulin, trypsin-sensitive cleavage sites,consisting of either arginine, lysine-arginine or lysine wereengineered. A study of cleavage kinetics, in which the threefusion proteins were treated with trypsin and carboxypeptidaseB, demonstrated that the construct with a single arginineresidue was most efficiently processed. This fusion protein,which was found to be expressed to high levels in a fed-batchcultivation, accumulated intracellularly as inclusion bodies.After solubilization, refolding was performed by oxidativesulfitolysis. IgG affinity purification was used for singlestep recovery of pure proinsulin fusion protein. Afterenzymatic cleavage of the fusion protein, human insulin andC-peptide were recovered with good yields by preparativereversed-phase chromatography.

    To investigate if increased production levels of theC-peptide could be obtained by gene fragment multimerization,DNA constructs encoding one, three or seven copies of theC-peptide gene were genetically fused to BB. Each C-peptidegene was flanked with codons for arginine, enabling enzymaticrelease of native C-peptide by trypsin/carboxypeptidase Btreatment. The three fusion proteins were produced to similarlevels as soluble and proteolytically stable intracellular geneproducts. Analysis of released C-peptide after enzymatictreatment of the fusion proteins, showed a six-fold increasedyield of C-peptide obtained from the heptameric fusion protein,as compared to the one-copy fusion protein. Based on theheptameric fusion protein BB-C7, an integrated process forproduction of proinsulin C-peptide was developed, whichincluded a heat treatmentprocedure for efficient release ofthe soluble fusion protein into the culture medium. The heattreatment also served as a purification step, precipitating themajority of the host cell proteins. In the production processpresented, chromatographic steps suitable for large-scalepurification, were used. The overall yield of native C-peptidewith a purity exceeding 99%, was 400 mg/l culture,corresponding to an overall recovery of 56%.

    Taken together, the genetic strategies investigated havedemonstrated to be useful in schemes for facilitated productionof recombinant human peptide hormones inE. coli.

    Key words:affinity-assisted in vivo folding, affinityfusion, carboxypeptidase B, C-peptide, enzymatic cleavage,Escherichia coli, gene multimerization, heat treatment,human peptide hormone, IGF-I, IGFBP-1, insulin, proinsulin,staphylococcal protein A, streptococcal protein G, trypsin.

  • 43. Legendre, Daniel
    et al.
    Laraki, Nezha
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Björnvad, Mads E
    Bouchet, Michèle
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Borchert, Torben V
    Fastrez, Jacques
    Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 296, no 1, 87-102 p.Article in journal (Refereed)
    Abstract [en]

    Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial Libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close Link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentils on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very Limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-L-Ala-L-Ala-L-Pro- Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-L-Lys-L-Ala- L-Pro-Phe(P)-diphenylester ter allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

  • 44.
    Liljeqvist, Sissela
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Recombinant subunit vaccines: protein immunogens1998Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Recombinant techniques provide valuable tools for thedevelopment of modern subunit vaccines. In this thesisdifferent systems for production of recombinant subunitvaccines are presented. The strategies investigated includeprotein immunogens, live bacterial vectors and nucleicacids.

    An expression system for recombinant protein production wasdeveloped forSalmonella typhimurium, and when investigating theproduction ofPlasmodium falciparum-derived antigens, yield andquality were comparable to those obtained forE. coli.S. typhimuriumwas further investigated as a livebacterial vaccine vector, comparing the effect on the immuneresponses of expressing a malarial antigen as surface-displayedor periplasmically located. The bacterial vector carrying thesurface-exposed antigen was able to induce antibody responses,upon immunisation of mice, of the same magnitude as thebacterium with periplasmic location of the antigen, althoughwith approximately tenfold lower level of expression, thusdemonstrating beneficial effects of using surface display ofthe foreign antigen.

    Aiming for a bacterial live vector for subunit vaccinedelivery with increased persistence at mucosal sites, differentadhesion molecules were investigated. The characteristics ofthe cholera toxin B subunit (CTB) with N-, C-, or dual fusionswere studied. Three different fusion proteins were constructed,and expressed inE. coli. All fusion proteins were able to pentamerise,and GM1-binding studies showed significant binding ablilty forthe single-fusion CTB proteins. The CTB or bacterially derivedfibronectin-binding domains (FNBDs), were included inexpression systems for non-pathogenic staphylococci, previouslyextensively investigated in the context of vaccine delivery. Onthese staphylococcal cells, CTB and the FNBDs were expressed ina surface displayed fashion. In whole-cell assays, the CTBmoiety and the FNBDs were confirmed on the surface of the livebacteria, displayed in a functional form, with retainedcapacity to bind GM1 and fibronectin, respectively. Suchbacteria will be of future interest for vaccine delivery.

    In a DNA vaccine vector, the influence of a secretion signalpreceding the gene encoding the antigen was investigated. Twoplasmid DNA vaccine vectors encoding a malarial antigen wereconstructed, one containing and the other one lacking signalsequence, and upon immunisation of mice, the levels ofantibodies elicited to the antigen were not affected by thedifference in cellular targeting. Interestingly, looking at IgGsubclasses, as correlates of different Th subsets, apredominant IgG1 response was evoked by the DNA vector encodeda secreted antigen, suggesting a dominating Th2 type ofresponse, whereas animals injected with the DNA vector encodeda intracellular antigen demonstrated a more equal distributionof the two IgG subclasses studied, proposing a mixed Thprofile.

    Taken together, the recombinant strategies investigated havedemonstrated to provide a variety of possibilities in thedesign anddelivery modes in vaccination, and the futureexpansion of the use of recombinant techniques can beenvisioned for the construction of purpose-designed recombinantsubunit vaccines.

    Key words: albumin binding protein, CTB, DNA vaccine,fibronectin binding domain, GM1 binding, live bacterial vaccinevector,Plasmodium falciparum,Salmonella typhimurium, signal sequence, staphylococcalprotein A,Staphylococcus carnosus,Staphylococcus xylosus

  • 45.
    Martinelle, Kristina
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    On the large metabolism and ammonium production in cultured animal cells1997Doctoral thesis, comprehensive summary (Other scientific)
  • 46. Nordstrom, T.
    et al.
    Alderborn, A.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing (TM) technology2002In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 52, no 2, 71-82 p.Article in journal (Refereed)
    Abstract [en]

    A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing(TM) technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.

  • 47. Nordstrom, T.
    et al.
    Gharizadeh, B.
    Pourmand, N.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Ronaghi, M.
    Method enabling fast partial sequencing of cDNA clones2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 292, no 2, 266-271 p.Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

  • 48. Nordstrom, T.
    et al.
    Nourizad, K.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method enabling pyrosequencing on double-stranded DNA2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 282, no 2, 186-193 p.Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlen, and P. Nyren, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA, In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

  • 49. Nordstrom, T.
    et al.
    Ronaghi, M.
    Forsberg, L.
    de Faire, U.
    Morgenstern, R.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing2000In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 31, 107-112 p.Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPXI) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

  • 50. Nourizad, N.
    et al.
    Ehn, M.
    Gharizadeh, B.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)2003In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 27, no 2, 229-237 p.Article in journal (Refereed)
    Abstract [en]

    ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1 mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48 kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

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