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  • 1. Ahlqvist, J.
    et al.
    Kumar, A.
    Sundstrom, H.
    Ledung, E.
    Hornsten, E. G.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mattiasson, B.
    Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 2, p. 216-225Article in journal (Refereed)
    Abstract [en]

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  • 2.
    Bandmann, Nina
    et al.
    KTH, Superseded Departments, Biotechnology.
    Collet, Eric
    KTH, Superseded Departments, Biotechnology.
    Leijen, J
    Uhlén, M
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 79, no 2, p. 161-172Article in journal (Refereed)
    Abstract [en]

    The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.

  • 3.
    Bandmann, Nina
    et al.
    KTH, Superseded Departments, Biotechnology.
    van Alstine, James
    KTH, Superseded Departments, Biotechnology.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 93, no 1, p. 1-14Article in journal (Refereed)
    Abstract [en]

    Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated, Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M 13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.

  • 4. Bollok, M
    et al.
    Vasala, A
    Neubauer, A
    Myllyharju, J
    Jahic, M
    Enfors, S O
    Neubauer, P
    Quantitative mRNA analysis as a tool for optimization of recombinant protein production in cell cultures2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 118, p. S83-S84Article in journal (Other academic)
  • 5.
    Bäcklund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Markland, Katrin
    KTH, School of Biotechnology (BIO).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Fedbatch design for periplasmic product retention in Escherichia coli2008In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 135, no 4, p. 358-365Article in journal (Refereed)
    Abstract [en]

    The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h-1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.

  • 6.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, Mariena
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 1, p. 86-98Article in journal (Refereed)
    Abstract [en]

    Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST 1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

  • 7.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Characterization of the stability of Vibrio fluvialis JS17 amine transaminase2018In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 282, p. 10-17Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Vibrio fluvialis (Vf-ATA) is an attractive enzyme with applications within Biocatalysis for the preparation of chiral amines. Various catalytic properties of Vf-ATA have been investigated, but a biophysical characterization of its stability has been lacking. Today, the industrial application of Vf-ATA is limited by its low operational stability. In order to enhance the knowledge regarding the structural stability of ATAs, general characterizations of different ATAs are required. In this work, the stability of Vf-ATA was explored. First, the affinity between enzyme and pyridoxal-5’-phosphate (PLP) (KD value of 7.9 ΌM) was determined. Addition of PLP to enzyme preparations significantly improved the enzyme thermal stability by preventing enzyme unfolding. With the aim to understand if this was due to the PLP phosphate group coordination into the phosphate group binding cup, the effect of phosphate buffer on the enzyme stability was compared to HEPES buffer. Low concentrations of phosphate buffer showed a positive effect on the enzyme initial activity, while higher phosphate buffer concentrations prevented cofactor dissociation. Additionally, the effects of various amine or ketone substrates on the enzyme stability were explored. All tested amines caused a concentration dependent enzyme inactivation, while the corresponding ketones showed no or stabilizing effects. The enzyme inactivation due to the presence of amine can be connected to the formation of PMP, which forms in the presence of amines in the absence of ketone. Since PMP is not covalently bound to the enzyme, it could readily leave the enzyme upon formation. Exploring the different stability effects of cofactor, substrates, additives and buffer system on ATAs seems to be important in order to understand and improve the general performance of ATAs.

  • 8. Doverskog, M.
    et al.
    Jacobsson, U.
    Chapman, B. E.
    Kuchel, P. W.
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective H-1/N-15 NMR in vitro assay2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 79, no 1, p. 87-97Article in journal (Refereed)
    Abstract [en]

    This is the second of two papers [Drews, M., Doverskog, M., Qhman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Haggstrom, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR. J. Biotechnol. 78, 23-37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified H-1/N-15 spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5-N-15] glutamine was selectively incorporated into 2-oxoglutarate forming [2-N-15] glutamate with a specific activity of 4.15 +/- 0.21 nmol [2-N-15] glutamate min (-1) (mg total protein)(-1) in the cell-free extracts. The enzyme activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase.

  • 9. Drews, M.
    et al.
    Doverskog, M.
    Ohman, L.
    Chapman, B. E.
    Jacobsson, U.
    Kuchel, P. W.
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 78, no 1, p. 23-37Article in journal (Refereed)
    Abstract [en]

    H-1/N-15 and C-13 NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-N-15]glutamine, [5-N-15]glutamine, [2-N-15]glutamate, (NH4Cl)-N-15, [2-N-15]alanine, and [1-C-13]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. H-1/N-15 NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media (NH4+)-N-15 was consumed and incorporated into alanine. (NH4+)-N-15 was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. C-13 NMR revealed that the [1-C-13] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

  • 10.
    Eklund, Malin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Sandström, Kristofer
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex2004In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 109, no 3, p. 277-286Article in journal (Refereed)
    Abstract [en]

    Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cell ulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1(Cel6A)) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1(Cel6A) fusion protein had been previously applied. This showed that the SPA-CBM1(Cel6A) fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.

  • 11.
    Enfors, Sven-Olof
    et al.
    KTH, Superseded Departments, Biotechnology.
    Jahic, M.
    Rozkov, A.
    Xu, B.
    Hecker, M.
    Jurgen, B.
    Kruger, E.
    Schweder, T.
    Hamer, G.
    O'Beirne, D.
    Noisommit-Rizzi, N.
    Reuss, M.
    Boone, L.
    Hewitt, C.
    McFarlane, C.
    Nienow, A.
    Kovacs, T.
    Tragardh, C.
    Fuchs, Laszlo
    Revstedt, J.
    Friberg, P. C.
    Hjertager, B.
    Blomsten, G.
    Skogman, H.
    Hjort, S.
    Hoeks, F.
    Lin, H. Y.
    Neubauer, P.
    van der Lans, R.
    Luyben, K.
    Vrabel, P.
    Manelius, A.
    Physiological responses to mixing in large scale bioreactors2001In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 85, no 2, p. 175-185Article in journal (Refereed)
    Abstract [en]

    Escherichia coli fed-batch cultivations at 22 m(3) scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/reassimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

  • 12.
    Eriksson, Ulrika
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Hassel, Jenny
    Lüllau, Elke
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, no 1, p. 76-86Article in journal (Refereed)
    Abstract [en]

    Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B 1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45 kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25 kDa, as well as precursor forms above 48 kDa. Metalloproteinase bands below the main band at 48 kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor DL-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10 kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.

  • 13.
    Gharizadeh, Baback
    et al.
    Stanford Univ, Stanford Genome Technol Ctr.
    Akhras, Michael
    KTH, School of Biotechnology (BIO), Biochemistry.
    Nourizad, Nader
    KTH, School of Biotechnology (BIO), Biochemistry.
    Ghaderi, Mehran
    Univ Örebro, Dept Caring Sci, Div Biomed.
    Yasuda, Kenji
    Univ Tokyo, Grad Sch Arts & Sci, Dept Life Sci.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Pourmand, Nader
    Stanford Univ, Stanford Genome Technol Ctr.
    Methodological improvements of pyrosequencing technology2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 124, no 3, p. 504-511Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.

  • 14.
    Gräslund, Susanne
    et al.
    KTH, Superseded Departments, Biotechnology.
    Eklund, Malin
    KTH, Superseded Departments, Biotechnology.
    Falk, Ronny
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Nygen, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, no 1, p. 41-50Article in journal (Refereed)
    Abstract [en]

    An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

  • 15.
    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, p. 93-102Article 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.

  • 16.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Jonsson, Andreas
    Affibody AB, Bromma.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gunneriusson, Elin
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Herne, Nina
    Affibody AB, Bromma.
    Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 128, no 1, p. 162-183Article in journal (Refereed)
    Abstract [en]

    Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.

  • 17.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineered affinity proteins-Generation and applications2009In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 140, no 3-4, p. 254-269Article, review/survey (Refereed)
    Abstract [en]

    The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.

  • 18. Gulich, S.
    et al.
    Linhult, M.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Stability towards alkaline conditions can be engineered into a protein ligand2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, no 2, p. 169-178Article in journal (Refereed)
    Abstract [en]

    One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.

  • 19. Gulich, S.
    et al.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, no 03-feb, p. 233-244Article in journal (Refereed)
    Abstract [en]

    One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower a-helical content, as well as a lower thermal and chemical stability compared to the parent 2-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.

  • 20.
    Gyarmati, Peter
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Song, Yajing
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chemical fragmentation for massively parallel sequencing library preparation2013In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 168, no 1, p. 95-100Article in journal (Refereed)
    Abstract [en]

    Fragmentation is essential in most library preparation protocols for use with massively parallel sequencing systems. Complexes that generate hydroxyl radicals, such as iron-EDTA, can be used to introduce random DNA cleavage. Here we describe a chemical fragmentation method that can be incorporated into library preparation protocols for next-generation sequencing workflows. This protocol has been validated by whole genome, amplicon and exome sequencing. Chemical fragmentation is a cost-effective alternative to current fragmentation methods that has no observable sequence bias and requires no instrumentation.

  • 21.
    Hagrot, Erika
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Oddsdottir, Hildur Aesa
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hosta, Joan Gonzalez
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jacobsen, Elling W.
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model-Application to amino acid depletion in CHO cell culture2017In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 259, p. 235-247Article in journal (Refereed)
  • 22.
    Hagrot, Erika
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Oddsdóttir, Hildur Aesa
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hosta, Joan Gonzalez
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jacobsen, Elling W.
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - Application to amino acid depletion in CHO cell culture2016In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 228, p. 37-49Article in journal (Refereed)
    Abstract [en]

    Mammalian cell lines are characterized by a complex and flexible metabolism. A single model that could describe the variations in metabolic behavior triggered by variations in the culture conditions would be a precious tool in bioprocess development. In this paper, we introduce an approach to generate a poly-pathway model and use it to simulate diverse metabolic states triggered in response to removal, reduction or doubling of amino acids in the culture medium of an antibody-producing CHO cell line. Macro-reactions were obtained from a metabolic network via elementary flux mode enumeration and the fluxes were modeled by kinetic equations with saturation and inhibition effects from external medium components. Importantly, one set of kinetic parameters was estimated using experimental data of the multiple metabolic states. A good fit between the model and the data was obtained for the majority of the metabolites and the experimentally observed flux variations. We find that the poly-pathway modeling approach is promising for the simulation of multiple metabolic states.

  • 23.
    Hagrot, Erika
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Oddsdóttir, Hildur Æsa
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hosta, Joan Gonzalez
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jacobsen, Elling W.
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Chotteau, Véronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Retraction notice to “Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model – Application to amino acid depletion in CHO cell culture” (Journal of Biotechnology (2016) 228 (37–39)(S0168165616301213)(10.1016/j.jbiotec.2016.03.015))2018In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 265Article in journal (Refereed)
    Abstract [en]

    This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). The authors of the paper wish to retract the paper due to the discovery of a calculation error in the processing of the raw data. The discovered error concerns the calculation of the specific uptake/secretion rates for several metabolites in one of the experimental conditions, i.e. glutamine omission (called Q0). In other words, in Figure 2, the variations of the metabolic fluxes for the condition Q0 are not correct. When this error is corrected, the resulting mathematical model changes (in particular for the results associated with Q0 conditions), several figures and tables are modified, and the interpretation of the fluxes in Q0 has to be slightly modified. Therefore the authors wish to retract the article. However, the error does not affect the modelling approach or the methodology presented in the article. Therefore, a revised version with the correct data has since been published: http://www.sciencedirect.com/science/article/pii/S0168165617302663. We apologize to the scientific community for the need to retract the article and the inconvenience caused.

  • 24. Han, L.
    et al.
    Doverskog, M.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by C-13 NMR spectroscopy2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 92, no 3, p. 237-249Article in journal (Refereed)
    Abstract [en]

    Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia colt from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium. Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610)(.)3). When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen. The 'glycine effect' was not due to CO2 produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production. The metabolism of glycine was further investigated in cultures supplied with [2-C-13] labelled glycine, and the redistribution of label in the [1-C-13], [2-C-13], and [1,2-C-13] isotopomeres of excreted acetate was analysed by C-13 NMR. The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1). Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of [2-C-13] acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine. At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate. Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate. This switch in metabolism appears to be regulated by quorum sensing.

  • 25.
    Hedhammar, My
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Stenvall, Maria
    KTH, School of Biotechnology (BIO), Proteomics.
    Lönneborg, Rosa
    KTH, School of Biotechnology (BIO), Proteomics.
    Nord, Olof
    KTH, School of Biotechnology (BIO), Proteomics.
    Sjölin, Olle
    KTH, School of Biotechnology (BIO), Proteomics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Uhlén, Matthias
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, no 2, p. 133-146Article in journal (Refereed)
    Abstract [en]

    A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

  • 26.
    Henriksson, Gunnar
    et al.
    KTH, Superseded Departments, Pulp and Paper Technology.
    Johansson, G.
    Pettersson, G.
    A critical review of cellobiose dehydrogenases2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 78, no 2, p. 93-113Article, review/survey (Refereed)
    Abstract [en]

    Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron accepters including quinones, phenoxyradicals, Fe3+, Cu2+ and tiiodide ion. Monosaccharides, maltose and molecular oxygen are:poop substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron accepters, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.

  • 27. Henriksson, H
    et al.
    Stahlberg, J
    Koivula, A
    Pettersson, G
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Valtcheva, L
    Isaksson, R
    The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition1997In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 57, no 1-3, p. 115-125Article in journal (Refereed)
  • 28.
    Jahic, Mehmedalija
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Gustavsson, Malin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Jansen, Ann-Katrin
    Martinelle, Mats
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology. KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes2003In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 102, no 1, p. 45-53Article in journal (Refereed)
    Abstract [en]

    A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degreesC during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

  • 29. Jonasson, P.
    et al.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Jornvall, H.
    Johansson, B. L.
    Wahren, J.
    Uhlen, M.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, no 03-feb, p. 215-226Article in journal (Refereed)
    Abstract [en]

    An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.

  • 30.
    Kaczmarzyk, Danuta
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Anfelt, Josefine
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Särnegrim, Amanda
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp PCC68032014In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 182, no 1, p. 54-60Article in journal (Refereed)
    Abstract [en]

    Among phenotypes of interest for an industrial cyanobacteria host are improved tolerance to temperature, salt, and solvent stress. Cellular responses to many stresses are controlled by the network of sensory receptors and downstream regulatory proteins. We applied transcription factor engineering to Synechocystis and tested mutant strains for tolerance to temperature and the biofuel 1-butanol. Histidine kinases (Hik), response regulators (Rre), and an RNA polymerase sigma factor (SigB) were overexpressed or deleted. Overexpression of SigB increased both temperature and butanol tolerance and lowered the intracellular concentration of reactive oxygen species. This report demonstrates that alteration of regulatory proteins in a cyanobacterium can be a useful tool to improve stress tolerance.

  • 31. Kepka, C.
    et al.
    Collet, E.
    Persson, J.
    Stahl, A.
    Lagerstedt, T.
    Tjerneld, F.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Pilot-scale extraction of an intracellular recombinant cutinase from E-coli cell homogenate using a thermoseparating aqueous two-phase system2003In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 103, no 2, p. 165-181Article in journal (Refereed)
    Abstract [en]

    A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 1 to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO50PO50 (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)4 tag was used for enhanced target protein partitioning to the EO50PO50 phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO50PO50 phase from the first step was supplemented with a non-ionic detergent (C12-18EO5) and heated to the cloud point (CP) temperature (45 degreesC). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.

  • 32. Kujawa, M
    et al.
    Leitner, C
    Halada, P
    Volc, J
    Hallberg, B M
    Divne, Christina
    Peterbauer, C K
    Haltrich, D
    Pyranose oxidase from Trametes multicolour: application in biocatalysis2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 118, p. 89-89Article in journal (Refereed)
  • 33. Larsson, M.
    et al.
    Graslund, S.
    Li, Y. B.
    Brundell, E.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hoog, C.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    High-throughput protein expression of cDNA products as a tool in functional genomics2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, no 2, p. 143-157Article in journal (Refereed)
    Abstract [en]

    A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.

  • 34.
    Laurell, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Comparative analysis of a 3' end tag PCR and a linear RNA amplification approach for microarray analysis2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 127, no 4, p. 638-646Article in journal (Refereed)
    Abstract [en]

     Background: Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA.

    Results: Preservation of expression ratios after amplification was examined comparing 1092 ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach.

    Conclusion: Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.

  • 35.
    Löfdahl, Per-Åke
    KTH, School of Engineering Sciences (SCI), Physics.
    Improved solubility of TEV protease by directed evolution2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 121, no 3, p. 291-298Article in journal (Refereed)
    Abstract [en]

    The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV–GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

  • 36. Nord, K.
    et al.
    Gunneriusson, E.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1(M) and Taq DNA polymerase2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, no 1, p. 45-54Article in journal (Refereed)
    Abstract [en]

    Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Tag DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0.5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.

  • 37. Nord, O.
    et al.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Microbead display of proteins by cell-free expression of anchored DNA2003In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 106, no 1, p. 1-13Article in journal (Refereed)
    Abstract [en]

    Gene expression technologies where nucleic acid sequences remain physically linked to their corresponding gene products are important tools for selection and identification of rare variants in large protein libraries. Here, we describe a gene expression system, which combines the potential of bead-based suspension array technology (SAT) with gene expression and clonal identification. Using streptavidin-coated polystyrene micrometer-sized beads as solid supports for anchored PCR products, we have investigated conditions for cell-free expression and bioaffinity technology to provide clonal co-anchoring of corresponding gene products. Experiments showed that coupled transcription and translation of PCR product expression cassettes resulted in display of affinity-anchored proteins whose binding characteristics could be analyzed via direct and selective interaction with a fluorescently labeled target protein. Interestingly, experiments performed with differently biotinylated PCR products showed that the efficiency of display was dependent on the directionality of the expression cassette relative to the bead surface. In spiked systems, using small immunoglobulin binding proteins as models, we demonstrate efficient flow cytometric sorting of beads corresponding to the target interacting clones, verified by post-sorting analysis and clonal identification at DNA level. The use of this technology, including alternative for-mats, for different proteomics applications is discussed.

  • 38.
    Rajangam, Alex S.
    et al.
    KTH, School of Biotechnology (BIO).
    Vanden Wymelenberg, Amber
    Sabat, Grzegorz
    Martinez, Diego
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Gaskell, Jill
    Kersten, Philip J.
    Cullen, Dan
    The Phanerochaete chrysosporium secretome: database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 118, p. 17-34Article in journal (Refereed)
    Abstract [en]

    The white rot basidiomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose and lignin. Towards the identification of participating enzymes, 268 likely secreted proteins were predicted using SignalP and TargetP algorithms. To assess the reliability of secretome predictions and to evaluate the usefulness of the current database, we performed shotgun LC-MS/MS on cultures grown on standard cellulose-containing medium. A total of 182 unique peptide sequences were matched to 50 specific genes, of which 24 were among the secretome subset. Underscoring the rich genetic diversity of R chrysosporium, identifications included 32 glycosyl hydrolases. Functionally interconnected enzyme groups were recognized. For example, the multiple endoglucanases and processive exocellobiohydrolases observed quite probably attack cellulose in a synergistic manner. In addition, a hemicellulolytic system included endoxylanases, alpha-galactosidase, acetyl xylan esterase, and alpha-L-arabinofuranosidase. Glucose and cellobiose metabolism likely involves cellobiose dehydrogenase, glucose oxidase, and various inverting glycoside hydrolases, all perhaps enhanced by an epimerase. To evaluate the completeness of the current database, mass spectroscopy analysis was performed on a larger and more inclusive dataset containing all possible ORFs. This allowed identification of a previously undetected hypothetical protein and a putative acid phosphatase. The expression of several genes was supported by RT-PCR amplification of their cDNAs.

  • 39.
    Samuelson, Patrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gunneriusson, E.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Display of proteins on bacteria2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 2, p. 129-154Article, review/survey (Refereed)
    Abstract [en]

    Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and disc-asses current uses and possible future trends of these emerging technologies.

  • 40.
    Sjöberg, Gustav
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Guevara-Martínez, Mónica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Univ Mayor San Simon, Ctr Biotechnol, Fac Sci & Technol, Cochabamba, Bolivia..
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome2019In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 305, p. 43-50Article in journal (Refereed)
    Abstract [en]

    In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3-hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.

  • 41.
    Spens, Erika
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Proliferation of NSO cells in protein-free medium: The role of cell-derived proteins, known growth factors and cellular receptors2009In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 141, no 3-4, p. 123-129Article in journal (Refereed)
    Abstract [en]

    NSO cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to Support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may Open for the novel approaches to improve animal cell processes. The following proteins were identified in NSO conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6,TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11. IL-25, MIF, TGF-beta and TNF-beta as well as the known growth factors EGF, IGF-1/11, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NSO cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NSO cell membranes since TGF-beta(1) caused cell death, and IGF-1/11 and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp 130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NSO cells as demonstrated by siRNA gene silencing.

  • 42.
    Sterky, Fredrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Holmberg, Anders
    Alexandersson, G
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Uhlen, Mathias
    KTH, Superseded Departments, Biotechnology.
    Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR1998In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 60, no 1-2, p. 119-129Article in journal (Refereed)
    Abstract [en]

    Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads. An arbitrary primer initiates extension from the unknown region and back towards the known locus. The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification. The PCR products are then directly sequenced by solid-phase or cycle sequencing. The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.

  • 43.
    Sterky, Fredrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Sequence analysis of genes and genomes2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, no 1, p. 1-31Article, review/survey (Refereed)
    Abstract [en]

    A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.

  • 44.
    Strandberg, L
    et al.
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Enfors, S O
    KTH, Superseded Departments, Biotechnology.
    Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase1990In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 13, no 1, p. 83-96Article in journal (Refereed)
    Abstract [en]

    Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.

  • 45. Surribas, Anna
    et al.
    Stahn, Rainer
    Montesinos, Jose Luis
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Valero, Francisco
    Jahic, Mehmedalija
    Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 130, no 3, p. 291-299Article in journal (Refereed)
    Abstract [en]

    Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.

  • 46.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Branneby, C.
    Abedi, V.
    Wells, A.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    CASCAT: Redesign of omega-Transaminases for Synthesis of Chiral Amines2010In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 150, p. S123-S124Article in journal (Other academic)
    Abstract [en]

    Transaminases (EC 2.6.1.18) are attractive biocatalysts for synthesis of chiral amines and alpha-amino acids. These enzymes catalyze transfer of an amine group from a donor substrate to an acceptor compound using the cofactor pyridoxal-5′-phoshate (PLP). omega-Transaminases are a versatile subgroup of the transaminases that does not require a carboxylic acid group in alpha-position (in contradiction toalpha-transaminases) and hence accept a wider spectrum of ketones or amines. The omega-transaminases are employed industrially for production of both R- and S-enantiopure amines.

    One bottleneck is the unfavourable equilibrium in such reactions run in the synthesis mode. We have developed a one-pot multi-enzyme system in a cascade fashion for equilibrium displacement by removing formed acetone.

    Another issue is the fact that most omega-transaminases show S-selectivity, however a few R-selective strains do exist. We have used an S-selective omega-transaminase variant from Arthrobacter citreus and created an R-selective variant by rational redesign using a homology enzyme model. This homology modelling/rational design approach was further explored on an omega-transaminase from Chromobacterium violaceum.

  • 47.
    Tegel, Hanna
    et al.
    KTH, School of Biotechnology (BIO).
    Hedhammar, My
    KTH, School of Biotechnology (BIO).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO).
    Hober, Sophia
    KTH, School of Biotechnology (BIO).
    Flow cytometry-based analysis of promoter effects on solubility of recombinantly expressed proteins2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 131, no 2, p. S9-S9Article in journal (Other academic)
  • 48.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    A human protein atlas for normal and disease tissue2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 118, p. S2-S2Article in journal (Other academic)
  • 49.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lehtiö, Janne
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Engineering of staphylococcal surfaces for biotechnological applications2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, p. 67-78Article, review/survey (Refereed)
    Abstract [en]

    Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.

  • 50.
    Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Inducible versus constitutive expression of a lipase in Pichia pastoris: A comparative study using different fermentation techniques2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 131, no 2, p. S149-S149Article in journal (Other academic)
12 1 - 50 of 53
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