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  • 1. Akter, Farhima
    et al.
    Mie, Masayasu
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Kobatake, Eiry
    Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 11, p. 5040-5046Article in journal (Refereed)
    Abstract [en]

    The chemical reactions used to make antibody DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of phi X174 gene A* protein and Z(mab2s) (A*-Zmab). The phi X174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab2s) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin gamma 1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-gamma (IFN-gamma) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

  • 2. Allerbring, E. B.
    et al.
    Duru, A. D.
    Uchtenhagen, H.
    Madhurantakam, C.
    Tomek, M. B.
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Mazumdar, P. A.
    Friemann, R.
    Uhlin, M.
    Sandalova, T.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Achour, A.
    Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics2012In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 42, no 11, p. 2990-3000Article in journal (Refereed)
    Abstract [en]

    The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D b in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D b in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D b/gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D b/Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D b/gp33 compared with H-2D b/Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.

  • 3. Allerbring, Eva B.
    et al.
    Duru, Adil D.
    Chadderton, Jesseka
    Markov, Natalia
    Uchtenhagen, Hannes
    Popov, Alexander
    Madhurantakam, Chaithanya
    Sandalova, Tatyana
    Turner, Stephen J.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Achour, Adnane
    Structural and thermodynamic basis underlying in vivo reestablishment of T-cell recognition of a viral escape mutant2015In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 68, no 2, p. 151-151Article in journal (Other academic)
  • 4. Andersson, C.
    et al.
    Hansson, M.
    Power, U.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column2001In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 34, p. 25-32Article in journal (Refereed)
    Abstract [en]

    The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

  • 5. Andersson, M.
    et al.
    Ronnmark, J.
    Arestrom, I.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ahlborg, N.
    Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies2003In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 283, no 02-jan, p. 225-234Article in journal (Refereed)
    Abstract [en]

    Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.

  • 6.
    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.

  • 7.
    Bandmann, Nina
    et al.
    KTH, School of Biotechnology (BIO).
    Löfdahl, Per-Åke
    KTH, School of Biotechnology (BIO).
    Lönneborg, Rosa
    KTH, School of Biotechnology (BIO).
    Älgenäs, Cajsa
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Exploring the use of a combinatorial expression vector library for facilitated soluble recombinant protein productionManuscript (Other academic)
  • 8.
    Bandmann, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichi coli2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5Article in journal (Refereed)
    Abstract [en]

    The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cytoplasm of E. coli. Large expression vector libraries were constructed through either conservative (ExLib1) or free (ExLib2) randomization of a seven-amino-acid window strategically located between a degenerated start codon and a sequence encoding a fluorescently tagged target protein. Flow cytometric sorting and analyses of libraries, subpopulations or individual clones were followed by SDS-PAGE, western blotting, mass spectrometry and DNA sequencing analyses. For ExLib1, intracellular accumulation of soluble protein was shown to be affected by codon specific effects at some positions of the common N-terminal extension. Interestingly, for ExLib2 where the same sequence window was randomized via seven consecutive NN(G/T) tri-nucleotide repeats, high product levels (up to 24-fold higher than a reference clone) were associated with a preferential appearance of novel SID-like sequences. Possible mechanisms behind the observed effects are discussed.

  • 9.
    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.

  • 10. Berggren, K.
    et al.
    Nilsson, A.
    Johansson, G.
    Bandmann, N.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Tjerneld, F.
    Partitioning of peptides and recombinant protein-peptide fusions in thermoseparating aqueous two-phase systems: effect of peptide primary structure2000In: Journal of Chromatography B, ISSN 0378-4347, Vol. 743, no 02-jan, p. 295-306Article in journal (Refereed)
    Abstract [en]

    Genetic engineering has been used for fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in an aqueous two-phase system. The system was composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. Peptides containing tryptophan, proline, arginine or aspartate residues were fused at the C-terminus of the recombinant protein ZZ-cutinase. The aim was to find effective tags for the lipolytic enzyme cutinase for large-scale extraction. The target protein and peptide tags were partitioned separately and then together in the fusion proteins in order to gain increased understanding of the influence of certain amino acid residues on the partitioning. The salt K2SO4 was used to reduce the charge dependent salt effects on partitioning and to evaluate the contribution to the partition coefficient from the hydrophobic-hydrophilic properties of the amino acid residues. The effect of Trp on peptide partitioning was independent of the difference in primary structure for (Trp)n, (Trp-Pro)n, (Ala-Trp-Trp-Pro)n and was only determined by the number of Trp. The effect of the charged residues, Arg and Asp, was dependent on the surrounding residues, i.e. if they were situated next to Trp or not. The partitioning behaviour observed for the peptides was qualitatively and in some cases also quantitatively the same as for the fusion proteins. The effect of the salts sodium perchlorate and triethylammonium phosphate on the partitioning was also studied. The salt effects observed for the peptides were qualitatively similar to the effects observed for the fusion proteins.

  • 11. Binz, Hans
    et al.
    Ngoc, Thien Nguyen
    Ståhl, Stefan
    Uhlén, Mathias
    Nygren, Per-Åke
    Respiratory syncytial virus protein g expressed on bacterial membrane1994Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    A method for preparing a peptide or protein, wherein (a) a DNA sequence coding for a heterologous polypeptide on a peptide sequence between amino acid residues 130 and 230 of respiratory syncytial virus protein G, sub-groups A and B, or a peptide sequence at least 80 % homologous thereto, and (b) means enabling the expression of the polypeptide on the bacterial membrane surface, are inserted into a bacterium which is not pathogenic for mammals. The resulting conjugate polypeptide and a live bacterium expressing same, pharmaceutical compositions containing them and their use for preparing a vaccine, as well as a DNA sequence coding for said polypeptide, are also disclosed.

  • 12. Binz, Hans
    et al.
    Nguyen, Thien Ngoc
    Andreoni, Christine
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Method for enhancing the immunogenicity of an immunogenic compound or hapten, and use thereof for preparing vaccines1994Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a process for improving the immunogenicity of an immunogen, an antigen or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said antigen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to mammalian serum albumin. The invention also relates to the use, as a medicament, of the product which can be obtained in this way.

  • 13. Bourbeillon, Julie
    et al.
    Orchard, Sandra
    Benhar, Itai
    Borrebaeck, Carl
    de Daruvar, Antoine
    Duebel, Stefan
    Frank, Ronald
    Gibson, Frank
    Gloriam, David
    Haslam, Niall
    Hiltker, Tara
    Humphrey-Smith, Ian
    Hust, Michael
    Juncker, David
    Koegl, Manfred
    Konthur, Zoltan
    Korn, Bernhard
    Krobitsch, Sylvia
    Muyldermans, Serge
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Palcy, Sandrine
    Polic, Bojan
    Rodriguez, Henry
    Sawyer, Alan
    Schlapshy, Martin
    Snyder, Michael
    Stoevesandt, Oda
    Taussig, Michael J.
    Templin, Markus
    Uhlén, Matthias
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    van der Maarel, Silvere
    Wingren, Christer
    Hermjakob, Henning
    Sherman, David
    Minimum information about a protein affinity reagent (MIAPAR)2010In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 28, no 7, p. 650-653Article in journal (Other academic)
  • 14. Danielsen, S.
    et al.
    Eklund, M.
    Deussen, H. J.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Borchert, T. V.
    In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor2001In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 272, no 02-jan, p. 267-274Article in journal (Refereed)
    Abstract [en]

    The 'detergent lipase' Lipolasel((R)), from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase((R)) could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase((R)) could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase((R))-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase((R)) variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.

  • 15. Eklund, M.
    et al.
    Axelsson, L.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Anti-idiotypic protein domains selected from protein A-based affibody libraries2002In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 48, no 3, p. 454-462Article in journal (Refereed)
    Abstract [en]

    Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fe binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K.) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

  • 16.
    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.

  • 17.
    Engfeldt, Torun
    et al.
    KTH, School of Biotechnology (BIO).
    Renberg, Björn
    KTH, School of Biotechnology (BIO).
    Brumer, Harry
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO).
    Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

  • 18. Fredriksson, A.
    et al.
    Johnstrom, P.
    Stone-Elander, S.
    Jonasson, P.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ekberg, K.
    Johansson, B. L.
    Wahren, J.
    Labeling of human C-peptide by conjugation with N-succinimidyl-4- F-18 fluorobenzoate2001In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 44, no 7, p. 509-519Article in journal (Refereed)
    Abstract [en]

    We have labeled proinsulin connecting peptide (C-peptide) with fluorine-18 (t(1/2) = 109.7min) in order to perform in vivo biodistribution and pharmacokinetic studies with position emission tomography (PET). This study reports the optimization of the conjugation labeling in the N-terminal with N-succinimidyl-4-[F-18]fluorobenzoate ([F-18]SFB). In preparative runs N-4-[F-18]fluorobenzoyl-C-peptide ([F-18]FB-C-peptide) was produced in 8-12% decay-corrected yields, counted from resolubilized [F-18]F-, in less than 5h. The specific radioactivity of [F-18]FB-C-peptide, determined using ELISA for one of the preparations, was around 70 GBq/mu mol at end of synthesis.

  • 19.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Klooster, Rinse
    Bisschop, Ilona J.M.
    Gruselius, Joel
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    van der Maarel, Silvère M.
    Single domain affinity proteins for the detection of the genome organizer protein SATB1Manuscript (preprint) (Other academic)
    Abstract [en]

    Affibody molecules and VHH antibody fragments are two classes of affinity proteins, both characterized by a small size and a single subunit domain structure. Here, we report the selection and characterization of affinity proteins from both classes against the special AT rich sequence binding protein SATB1, a suggested marker protein for aggressive and metastasizing breast cancer. The selected VHH antibody fragments originate from a nonimmune phage display library, while affibody molecules were selected from a library constructed in vitro and displayed on ribosomes. It was observed that selected molecules recognizing one of three conserved DNA-binding domains of SATB1, also recognized its close homologue SATB2 while several of the selected molecules from both classes binding to other regions selectively recognized SATB1. Two of these SATB1 selective molecules, VHH clone 2D2 and affibody molecule clone ZSATB1:2, performed well in differentimmunotechnology applications including ELISA, WB, IF and pull-out experiments and gave a selective staining of endogenous SATB1 in Jurkat T cells. These molecules may thus become useful tools, either alone or in combination, for the selective detection of SATB1 in breast tumor specimens. Due to their small size in comparison to immunoglobulins, such single domain binding proteins may be suitable for high resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy, where the resolution may get constrained by the size of the affinity reagent.

  • 20.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO).
    Shibasaki, Seiji
    KTH, School of Biotechnology (BIO).
    Vernet, Erik
    KTH, School of Biotechnology (BIO).
    Skogs, Marie
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Selection and characterization of affibody molecules interfering with the interaction between Ras and RafManuscript (Other academic)
  • 21.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Yu, Feifan
    KTH, School of Biotechnology (BIO), Proteomics.
    Shibasaki, Seiji
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Proteomics.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro2010In: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, no 6, p. 766-773Article in journal (Refereed)
    Abstract [en]

    Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

  • 22.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Salahshour, Samaneh
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolutionManuscript (preprint) (Other academic)
    Abstract [en]

    The use of in vitro protein library technologies for the generation of high affinity binding proteins often includes an affinity maturation step, involving the construction of secondary libraries from which second generation variants with improved affinities are selected. We describe a novel approach for the affinity maturation of affibody molecules based on stepwise in vitro molecular evolution, involving cycles of whole gene error-prone PCR amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display for selection. Starting with a human Raf-1 binding affibody molecule of an initial 2 μM dissociation constant (KD), the in vitro evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor based monitoring of the collective target binding ability of expressed pools obtained after each selection cycle. After a first cycle of diversification and selection, no increase in the hRaf-1 target binding of the pool was observed, whereas after two cycles, a significant increase in the binding response was seen. DNA sequencing showed that an alanine to valine substitution in an earlier variegated position had been effectively enriched, and was present in 11% and 83% of all clones after cycle one and two, respectively, either alone or in combination with other substitutions. Further studies on a subset of individual variants isolated after cycle two showed that the observed A27V substitution alone accounted for a 13-fold increase in affinity, predominantly through increased on-rate kinetics. Additional substitutions in framework or non-framework positions typically resulted in a further two-fold increase in affinity. Interestingly, thermal melting point (Tm) analyses showed that an increased affinity could be associated with either slightly higher or lower Tm values, compared to the parental variant. All investigated variants showed excellent refolding properties, and the selectivity of the affinity matured hRaf-1 binders had not been compromised by the substitutions, as analyzed using a multiplexed bead-based binding assay involving 77 recombinant human control protein fragments.

  • 23.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Salahshour, Samaneh
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity2011In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, New biotechnology, ISSN 1876-4347, Vol. 29, no 5, p. 534-542Article in journal (Refereed)
    Abstract [en]

    The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.

  • 24.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Yu, Feifan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies2011In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 48, no 3, p. 263-276Article in journal (Refereed)
    Abstract [en]

    Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) recognition and dissociation constants (K (D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

  • 25.
    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.

  • 26.
    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, p. 157-166Article 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.

  • 27.
    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.

  • 28.
    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, p. 703-709Article 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.

  • 29.
    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, p. 125-132Article 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.

  • 30. 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.

  • 31. Henning, P
    et al.
    Andersson, K M E
    Frykholm, K
    Ali, A
    Magnusson, M K
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Granio, O
    Hong, S S
    Boulanger, P
    Lindholm, L
    Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains2005In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 12, no 3, p. 211-224Article in journal (Refereed)
    Abstract [en]

    Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5' s constructed, Ad5/R7- Z(wt)- Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A ( abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/ neu on SK-OV-3 and SK-BR-3, CA242 ( epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA ( prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus - receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

  • 32. Henning, P.
    et al.
    Magnusson, M. K.
    Gunneriusson, E.
    Hong, S. S.
    Boulanger, P.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Lindholm, L.
    Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from Staphylococcal protein A2002In: Human Gene Therapy, ISSN 1043-0342, E-ISSN 1557-7422, Vol. 13, no 12, p. 1427-1439Article in journal (Refereed)
    Abstract [en]

    The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody(TM) ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

  • 33. Jansson, Ronnie
    et al.
    Thatikonda, Naresh
    Lindberg, Diana
    Rising, Anna
    Johansson, Jan
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Hedhammar, My
    Recombinant Spider Silk Genetically Functionalized with Affinity Domains2014In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 5, p. 1696-1706Article in journal (Refereed)
    Abstract [en]

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  • 34. 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.

  • 35.
    Jonsson, Andreas
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Dogan, Jakob
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Harne, Nina
    Abrahmsén, Lars
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering of a femtomolar affinity binding protein to human serum albumin2008In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 8, p. 515-527Article in journal (Refereed)
    Abstract [en]

    We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.

  • 36. Karlstrom, A.
    et al.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Dual labeling of a binding protein allows for specific fluorescence detection of native protein2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 295, no 1, p. 22-30Article in journal (Refereed)
    Abstract [en]

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal. protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn(23)Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-LAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl) amino) hexanoate (NBD-X, SE), respectively. Biosensor analysis of Purified doubly labeled protein showed that high-affinity binding to the Fe region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of FC3(1)) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc(3(1)), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  • 37.
    Larsson, Gen
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Endotoxin analysis2005Patent (Other (popular science, discussion, etc.))
  • 38. 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, p. 87-102Article 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.

  • 39. Lehtio, J.
    et al.
    Teeri, Tuula T.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold2000In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 41, no 3, p. 316-322Article in journal (Refereed)
    Abstract [en]

    A disulfide bridge-constrained cellulose binding domain (CBD,) derived from the cellobiohydrolase Ce17A from Trichoderma reesei has been investigated for use in scaffold engineering to obtain novel binding proteins. The gene encoding the wild-type 36 aa CBDWT domain was first inserted into a phagemid vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein with retained cellulose binding activity. A combinatorial library comprising 46 million variants of the CBD domain was constructed through randomization of 11 positions located at the domain surface and distributed over three separate beta -sheets of the domain. Using the enzyme porcine alpha-amylase (PPA) as target in biopannings, two CBD variants showing selective binding to the enzyme were characterized. Reduction and iodoacetamide blocking of cysteine residues in selected CBD variants resulted in a loss of binding activity, indicating a conformation dependent binding. Interestingly, further studies showed that the selected CBD variants were capable of competing with the binding of the amylase inhibitor acarbose to the enzyme. In addition, the enzyme activity could be partially inhibited by addition of soluble protein, suggesting that the selected CBD variants bind to the active site of the enzyme.

  • 40.
    Lendel, Christofer
    et al.
    KTH, Superseded Departments, Biotechnology.
    Dincbas-Renqvist, Vildan
    KTH, Superseded Departments, Biotechnology.
    Flores, Alexander
    KTH, Superseded Departments, Biotechnology.
    Wahlberg, Elisabet
    KTH, Superseded Departments, Biotechnology.
    Dogan, Jakob
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Härd, Torleif
    Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A2004In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 8, p. 2078-2088Article in journal (Refereed)
    Abstract [en]

    Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

  • 41.
    Lendel, Christofer
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wahlberg, Elisabet
    KTH, Superseded Departments, Biotechnology.
    Berglund, Helena
    KTH, Superseded Departments, Biotechnology.
    Eklund, Malin
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Härd, Torleif
    KTH, Superseded Departments, Biotechnology.
    1H, 13C and 15N resonance assignments of an affibody-target complex2002In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 24, no 3, p. 271-272Article in journal (Refereed)
  • 42. Lindholm, L
    et al.
    Andersson, K
    Boulanger, P
    Frykholm, K
    Henning, P
    Hoeben, R
    Hong, S S
    Johannisson, J
    Magnusson, M
    Myhre, S
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Pettersson, E
    Stahl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Vellinga, J
    Wikman, Maria
    KTH, Superseded Departments, Biotechnology.
    Genetic re-targeting of adenovirus using a hyperstable scFv domain and an affibody (R) molecule against Her2/neu2004In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 9, p. S250-S250Article in journal (Refereed)
    Abstract [en]

    One important goal in gene therapy is to develop adenovirus (Ad) vectors that are genetically de-targeted as well as re-targeted. Genetic re-targeting of Ad using complex cell-binding ligands has previously not been possible. We have previously demonstrated that ligands for genetic re-targeting of adenoviruses must be able to fold correctly in the cytoplasm of virus producing cells, a milieu that is not conducive to the formation of disulphide bonds.

    Here, we describe functional Ad5 viruses with fibers and pIX capsid proteins genetically modified to contain two types of complex ligands. One is affibody® molecules, corresponding to small (6 kDa) binding proteins developed by combinatorial protein engineering using a single three-helix bundle staphylococcal protein A domain. The other type is hyperstable antibody scFv domains.

    The affibody molecule used here (ZH2N) is directed against Her2/neu. Recombinant viruses were constructed with ZH2N in three different positions: (i) at the C-terminus of shaft repeat 7 of de-knobbed fibers; (ii) at the C-terminus of pIX; and (iii) in the HI-loop of the fiber knob. Each of the viruses exhibited a characteristic phenotype regarding fiber content, growth and ability to infect Her2/neu expressing cells.

    In order to test the potentials of scFv liganded Ad vectors, a hyperstable antibody scFv against b-galactosidase was genetically incorporated into knobless fibers, in tandem with a mutated protein A domain reactive with IgG1 Fc that targeted the virus to Fc-expressing 293 cells. These fibers could be rescued into viable virions that retained the original antigen binding specificity of the scFv, demonstrating the basic potential of hyperstable scFvs for genetic re-targeting of Ad. Quite unexpectedly, the fiber content of Ad with knobless, scFv containing fibers was close to normal in contrast to other Ad with knobless fibers that generally has a much reduced fiber content. The hyperstable scFv was further fused to the C-terminus of the capsid protein pIX. The recombinant molecules could be rescued into viable viruses with wt fibers. The scFv retained its binding-specificity on the recombinant virions.

    The results demonstrate that, contrary to current beliefs, it is possible to construct Ad that genetically incorporates functional scFvs and other complex ligands into the virus fiber and pIX. The feasibility is demonstrated by the creation of different viruses that are re-targeted to Her2/neu. These viruses are currently in pre-clinical development.

  • 43. Linhult, M.
    et al.
    Gulich, S.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand2003In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 16, no 12, p. 1147-1152Article in journal (Refereed)
    Abstract [en]

    Alkaline conditions are generally preferred for sanitization of chromatography media by cleaning-in-place (CIP) protocols in industrial biopharmaceutical processes. The use of such rigorous conditions places stringent demands on the stability of ligands intended for use in affinity chromatography. Here, we describe efforts to meet these requirements for a divalent proteinaceous human serum albumin (HSA) binding ligand, denoted ABD* dimer. The ABD* dimer ligand was constructed by genetic head-to-tail linkage of two copies of the ABD* moiety, which is a monovalent and alkali-stabilized variant of one of the serum albumin-binding motifs of streptococcal protein G. Dimerization was performed to investigate whether a higher HSA-binding capacity could be obtained by ligand multimerization. We also investigated the influence on alkaline stability and HSA-binding capacity of three variants (VDANS, VDADS and GGGSG) of the inter-domain linker. Biosensor binding studies showed that divalent ligands coupled using non-directed chemistry demonstrate an increased molar HSA-binding capacity compared with monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. Significantly higher molar binding capacities were also detected when using the directed coupling chemistry. These results were confirmed in affinity chromatography binding capacity experiments, using resins containing thiol-coupled ligands. Interestingly, column sanitization studies involving exposure to 0.1 M NaOH solution ( pH 13) showed that of all the tested constructs, including the monovalent ligand, the divalent ligand construct containing the VDADS linker sequence was the most stable, retaining 95% of its binding capacity after 7 h of alkaline treatment.

  • 44. Ljungqvist, Charlotta
    et al.
    Nord, Karin
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Receptor structures1999Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to modified polypeptide derivatives of the B domain or Z domain of staphylococcal protein A (SPA). The derivatives contain amino acid substitutions that result in the ability of the B domain or Z domain to interact with at least one domain of a human Factor VIII protein, without substantially disrupting the structure and stability of the B domain or Z domain.

  • 45.
    Lundqvist, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Thalén, Niklas
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Volk, Anna-Luisa
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    von Otter, Eric
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Chromophore prematuration for improved speed and sensitivity of split-GFP in vitro applicationsManuscript (preprint) (Other academic)
  • 46.
    Löfdahl, Per-Åke
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Nord, Olof
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Janzon, Lars
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay2009In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 26, no 5, p. 251-259Article in journal (Refereed)
    Abstract [en]

    Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.

  • 47.
    Löfdahl, Per-Åke
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, p. 111-120Article in journal (Refereed)
    Abstract [en]

    The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.

  • 48. Myhre, S.
    et al.
    Henning, P.
    Granio, O.
    Tylo, A. S.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Olofsson, S.
    Boulanger, P.
    Lindholm, L.
    Hong, S. S.
    Decreased immune reactivity towards a knobless, affibody-targeted adenovirus type 5 vector2007In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 14, no 4, p. 376-381Article in journal (Refereed)
    Abstract [en]

    In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (>= 1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.

  • 49.
    Neiman, Maja
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, H.
    Lehtiö, Janne
    Karolinska Institutet, Solna, Sweden .
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Selectivity analysis of single binder assays used in plasma protein profiling2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 23-24, p. 3406-3410Article in journal (Refereed)
    Abstract [en]

    The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.

  • 50.
    Neiman, Maja
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysisManuscript (preprint) (Other academic)
    Abstract [en]

    The increasing availability of antibodies towards human proteins drives the generation of new and exploratory data, which can be generated by profiling e.g. plasma samples using multiplexed arrays. However, antibody assays can lead to erroneous results due to cross-­‐reactivity to off-­‐targets. Here, we describe an approach in which an antibody-­‐based suspension bead array is combined with subsequent validation of on-­‐ target binding using a coupled affinity purification assay. Based on differential heat treatment of samples, antibody performance was investigated and the results for antibodies directed towards several complement factors provide insights on the detection of proteins in sera from patients with complement deficiency. In conclusion, the combined parallel flow and blot based immunocapture analysis serves an important, first line tool for resolving differently detected serum or plasma protein profiles proposed by antibody arrays. 

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