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Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains

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In Vitro Mutagenesis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 634))

Abstract

Recombinant antibody fragments are significant therapeutic and diagnostic reagents. As such, their efficacy depends heavily on their affinities and biophysical properties. Thus, mutagenesis approaches have been extensively applied to recombinant antibodies to improve their affinity, stability, and solubility. Among the existing recombinant antibody variants, human VH domains stand out as the ones with the general need of solubility engineering at some point during their development; this solubility engineering step transforms VHs into nonaggregating, functional entities, rendering them useful as therapeutic and diagnostic reagents. Here, we present one of several approaches that have been employed to develop nonaggregating human VH domains. We apply an in vitro site-directed mutagenesis approach to an aggregating human VH domain by means of a splice overlap extension technique. The resultant mutant VHs are nonaggregating in contrast to the parent wild type VH and less prone to aggregation following thermal unfolding.

This is National Research Council of Canada Publication 50001.

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Notes

  1. 1.

    All plasmid constructs are available to researchers free of charge.

References

  1. Carter P (1986) Site-directed mutagenesis. Biochem J 237:1–7

    PubMed  CAS  Google Scholar 

  2. Costa GL, Bauer JC, McGowan B, Angert M, Weiner MP (1996) Site-directed mutagenesis using a rapid PCR-based method. Methods Mol Biol 57:239–248

    PubMed  CAS  Google Scholar 

  3. Crameri A, Cwirla S, Stemmer WP (1996) Construction and evolution of antibody-phage libraries by DNA shuffling. Nat Med 2:100–102

    Article  PubMed  CAS  Google Scholar 

  4. Jackel C, Kast P, Hilvert D (2008) Protein design by directed evolution. Annu Rev Biophys 37:153–173

    Article  PubMed  CAS  Google Scholar 

  5. Ling MM, Robinson BH (1997) Approaches to DNA mutagenesis: an overview. Anal Biochem 254:157–178

    Article  PubMed  CAS  Google Scholar 

  6. Lipovsek D, Plückthun A (2004) In-vitro protein evolution by ribosome display and mRNA display. J Immunol Methods 290:51–67

    Article  PubMed  CAS  Google Scholar 

  7. Wark KL, Hudson PJ (2006) Latest technologies for the enhancement of antibody affinity. Adv Drug Deliv Rev 58:657–670

    Article  PubMed  CAS  Google Scholar 

  8. Yuan L, Kurek I, English J, Keenan R (2005) Laboratory-directed protein evolution. Microbiol Mol Biol Rev 69:373–392

    Article  PubMed  CAS  Google Scholar 

  9. Yuen CM, Liu DR (2007) Dissecting protein structure and function using directed evolution. Nat Methods 4:995–997

    Article  PubMed  CAS  Google Scholar 

  10. Davies J, Riechmann L (1995) Antibody VH domains as small recognition units. Biotechnology (N Y) 13:475–479

    Article  CAS  Google Scholar 

  11. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136

    Article  PubMed  CAS  Google Scholar 

  12. Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM (2003) Domain antibodies: proteins for therapy. Trends Biotechnol 21:484–490

    Article  PubMed  CAS  Google Scholar 

  13. Revets H, De Baetselier P, Muyldermans S (2005) Nanobodies as novel agents for cancer therapy. Expert Opin Biol Ther 5:111–124

    Article  PubMed  CAS  Google Scholar 

  14. Saerens D, Ghassabeh GH, Muyldermans S (2008) Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 8:600–608

    Article  PubMed  CAS  Google Scholar 

  15. Arbabi-Ghahroudi M, MacKenzie R, Tanha J (2009) Selection of non-aggregating VH binders from synthetic VH phage display libraries. Methods Mol Biol 525:187–216, xiii

    Article  PubMed  CAS  Google Scholar 

  16. Arbabi-Ghahroudi M, To R, Gaudette N, Hirama T, Ding W, MacKenzie R, Tanha J (2009) Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points. Protein Eng Des Sel 22:59–66

    Article  PubMed  CAS  Google Scholar 

  17. Chen W, Zhu Z, Feng Y, Xiao X, Dimitrov DS (2008) Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain. J Mol Biol 382:779–789

    Article  PubMed  CAS  Google Scholar 

  18. Jespers L, Schon O, Famm K, Winter G (2004) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165

    Article  PubMed  CAS  Google Scholar 

  19. Tanha J, Nguyen TD, Ng A, Ryan S, Ni F, MacKenzie R (2006) Improving solubility and refolding efficiency of human VHs by a novel mutational approach. Protein Eng Des Sel 19:503–509

    Article  PubMed  CAS  Google Scholar 

  20. Christ D, Famm K, Winter G (2007) Repertoires of aggregation-resistant human antibody domains. Protein Eng Des Sel 20:413–416

    Article  PubMed  CAS  Google Scholar 

  21. Famm K, Hansen L, Christ D, Winter G (2008) Thermodynamically stable aggregation-resistant antibody domains through directed evolution. J Mol Biol 376:926–931

    Article  PubMed  CAS  Google Scholar 

  22. Tanha J, Xu P, Chen ZG, Ni F, Kaplan H, Narang SA, MacKenzie CR (2001) Optimal design features of camelized human single-domain antibody libraries. J Biol Chem 276:24774–24780

    Article  PubMed  CAS  Google Scholar 

  23. To R, Hirama T, Arbabi-Ghahroudi M, MacKenzie R, Wang P, Xu P, Ni F, Tanha J (2005) Isolation of monomeric human VHs by a phage selection. J Biol Chem 280:41395–41403

    Article  PubMed  CAS  Google Scholar 

  24. Hemsley A, Arnheim N, Toney MD, Cortopassi G, Galas DJ (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res 17:6545–6551

    Article  PubMed  CAS  Google Scholar 

  25. Higuchi R, Krummel B, Saiki RK (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16:7351–7367

    Article  PubMed  CAS  Google Scholar 

  26. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  PubMed  CAS  Google Scholar 

  27. Nelson RM, Long GL (1989) A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem 180:147–151

    Article  PubMed  CAS  Google Scholar 

  28. Vallette F, Mege E, Reiss A, Adesnik M (1989) Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res 17:723–733

    Article  PubMed  CAS  Google Scholar 

  29. Coulon S, Pellequer JL, Blachere T, Chartier M, Mappus E, Chen SW, Cuilleron CY, Baty D (2002) Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modeling: modulation of binding properties and prominent role of the VL domain in estradiol recognition. J Mol Recognit 15:6–18

    Article  PubMed  CAS  Google Scholar 

  30. Dilsiz N, Crabbe MJ (1994) A high-yield modification of mutation by overlap extension using three primers. Anal Biochem 222:510–511

    Article  PubMed  CAS  Google Scholar 

  31. Dolk E, van der Vaart M, Lutje HD, Vriend G, de Haard H, Spinelli S, Cambillau C, Frenken L, Verrips T (2005) Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo. Appl Environ Microbiol 71:442–450

    Article  PubMed  CAS  Google Scholar 

  32. Hulett MD, Witort E, Brinkworth RI, McKenzie IF, Hogarth PM (1994) Identification of the IgG binding site of the human low affinity receptor for IgG Fc gamma RII. Enhancement and ablation of binding by site-directed mutagenesis. J Biol Chem 269:15287–15293

    PubMed  CAS  Google Scholar 

  33. Kusters-van Someren M, Kishi K, Lundell T, Gold MH (1995) The manganese binding site of manganese peroxidase: characterization of an Asp179Asn site-directed mutant protein. Biochemistry 34:10620–10627

    Article  PubMed  CAS  Google Scholar 

  34. Peng RH, Xiong AS, Yao QH (2006) A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis. Appl Microbiol Biotechnol 73:234–240

    Article  PubMed  CAS  Google Scholar 

  35. Salins LL, Ware RA, Ensor CM, Daunert S (2001) A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein. Anal Biochem 294:19–26

    Article  PubMed  CAS  Google Scholar 

  36. Steenbergen SM, Vimr ER (2003) Functional relationships of the sialyltransferases involved in expression of the polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C. J Biol Chem 278:15349–15359

    Article  PubMed  CAS  Google Scholar 

  37. Walker SL, Wonderling RS, Owens RA (1997) Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity. J Virol 71:2722–2730

    PubMed  CAS  Google Scholar 

  38. Waschutza G, Dengler U, Villmann C, Bottinger H, Otto B (1998) Interferon-gamma variants with deletions in the AB surface loop. Flexibility is a critical point for receptor binding. Eur J Biochem 256:303–309

    Article  PubMed  CAS  Google Scholar 

  39. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68

    Article  PubMed  CAS  Google Scholar 

  40. Dan MD, Earley EM, Griffin MC, Maiti PK, Prashar AK, Yuan XY, Friesen AD, Kaplan HA (1995) Human monoclonal antibody BT32/A6 and a cell cycle-independent glioma-associated surface antigen. J Neurosurg 82:475–480

    Article  PubMed  CAS  Google Scholar 

  41. Sambrook J, Fritsch EF, Maniatis T (eds) (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  42. Tanha J, Dubuc G, Hirama T, Narang SA, MacKenzie CR (2002) Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH properties. J Immunol Methods 263:97–109

    Article  PubMed  CAS  Google Scholar 

  43. Tung WL, Chow KC (1995) A modified medium for efficient electrotransformation of E. coli. Trends Genet 11:128–129

    Article  PubMed  CAS  Google Scholar 

  44. Anand NN, Dubuc G, Phipps J, MacKenzie CR, Sadowska J, Young NM, Bundle DR, Narang SA (1991) Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen. Gene 100:39–44

    Article  PubMed  CAS  Google Scholar 

  45. MacKenzie CR, Sharma V, Brummell D, Bilous D, Dubuc G, Sadowska J, Young NM, Bundle DR, Narang SA (1994) Effect of Cλ-Cκ domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs. Biotechnology (N Y) 12:390–395

    Article  CAS  Google Scholar 

  46. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  47. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423

    Article  PubMed  CAS  Google Scholar 

  48. Jespers L, Schon O, James LC, Veprintsev D, Winter G (2004) Crystal structure of HEL4, a soluble, refoldable human VH single domain with a germ-line scaffold. J Mol Biol 337:893–903

    Article  PubMed  CAS  Google Scholar 

  49. van der Linden RHJ, Frenken LGJ, de Geus B, Harmsen MM, Ruuls RC, Stok W, de Ron L, Wilson S, Davis P, Verrips CT (1999) Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta 1431:37–46

    Article  PubMed  Google Scholar 

  50. Davies J, Riechmann L (1996) Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. Protein Eng 9:531–537

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are indebted to Tomoko Hirama, for helping us prepare the manuscript, and to Tom Devecseri, for preparing publication quality figures.

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Authors and Affiliations

  1. Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada

    Mehdi Arbabi-Ghahroudi & Roger MacKenzie

  2. Department of Environmental Biology, Ontario Agricultural College, University of Guelph, Guelph, ON, Canada

    Roger MacKenzie & Jamshid Tanha

  3. Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada

    Jamshid Tanha

Authors
  1. Mehdi Arbabi-Ghahroudi
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  2. Roger MacKenzie
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  3. Jamshid Tanha
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Corresponding author

Correspondence to Jamshid Tanha .

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Editors and Affiliations

  1. Stratagene, North Torrey Pines Rd. 11011, La Jolla, 92037, USA

    Jeff Braman

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© 2010 Humana Press

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Arbabi-Ghahroudi, M., MacKenzie, R., Tanha, J. (2010). Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_22

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  • DOI: https://doi.org/10.1007/978-1-60761-652-8_22

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-651-1

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