Skip to main content
SpringerLink
Log in

Production of Single-Domain Antibodies in Pichia pastoris

  • Protocol
  • First Online:
Single-Domain Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2446))

Abstract

Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448

    Article  CAS  PubMed  Google Scholar 

  2. Greenberg AS, Avila D, Hughes M et al (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173

    Article  CAS  PubMed  Google Scholar 

  3. Davies J, Riechmann L (1994) ‘Camelising’ human antibody fragments: NMR studies on VH domains. FEBS Lett 339:285–290

    Article  CAS  PubMed  Google Scholar 

  4. Harmsen MM, Ruuls RC, Nijman IJ et al (2000) Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features. Mol Immunol 37:579–590

    Article  CAS  PubMed  Google Scholar 

  5. Tanha J, Xu P, Chen Z et al (2001) Optimal design features of camelized human single-domain antibody libraries. J Biol Chem 276:24774–24780

    Article  CAS  PubMed  Google Scholar 

  6. Ewert S, Cambillau C, Conrath K et al (2002) Biophysical properties of camelid VHH domains compared to those of human VH3 domains. Biochemistry 41:3628–3636

    Article  CAS  PubMed  Google Scholar 

  7. Zavrtanik U, Lukan J, Loris R et al (2018) Structural basis of epitope recognition by heavy-chain camelid antibodies. J Mol Biol 430:4369–4386

    Article  CAS  PubMed  Google Scholar 

  8. Bond CJ, Marsters JC, Sidhu SS (2003) Contributions of CDR3 to VHH domain stability and the design of monobody scaffolds for naive antibody libraries. J Mol Biol 332:643–655

    Article  CAS  PubMed  Google Scholar 

  9. Mitchell LS, Colwell LJ (2018) Comparative analysis of nanobody sequence and structure data. Proteins 86:697–706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

    Article  CAS  PubMed  Google Scholar 

  11. Liu Y, Huang H (2018) Expression of single-domain antibody in different systems. Appl Microbiol Biotechnol 102:539–551

    Article  CAS  PubMed  Google Scholar 

  12. Cortez-Retamozo V, Lauwereys M, Hassanzadeh GG et al (2002) Efficient tumor targeting by single-domain antibody fragments of camels. Int J Cancer 98:456–462

    Article  CAS  PubMed  Google Scholar 

  13. Scully M, Cataland SR, Peyvandi F et al (2019) Caplacizumab treatment for acquired thrombotic thrombocytopenic purpura. N Engl J Med 380:335–346

    Article  CAS  PubMed  Google Scholar 

  14. Morrison C (2019) Nanobody approval gives domain antibodies a boost. Nat Rev Drug Discov 18:485–487

    Article  CAS  PubMed  Google Scholar 

  15. Duggan S (2018) Caplacizumab: first global approval. Drugs 78:1639–1642

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Martinez-Delgado G (2020) Inhaled nanobodies against COVID-19. Nat Rev Immunol 20:593–593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Vaneycken I, D’huyvetter M, Hernot S et al (2011) Immuno-imaging using nanobodies. Curr Opin Biotechnol 22:877–881

    Article  PubMed  Google Scholar 

  18. Debie P, Lafont C, Defrise M et al (2020) Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours. J Control Release 317:34–42

    Article  CAS  PubMed  Google Scholar 

  19. Muyldermans S (2020) Applications of nanobodies. Annu Rev Anim Biosci 9:401–421

    Article  PubMed  Google Scholar 

  20. Jailkhani N, Ingram JR, Rashidian M et al (2019) Noninvasive imaging of tumor progression, metastasis, and fibrosis using a nanobody targeting the extracellular matrix. Proc Natl Acad Sci U S A 116:14181–14190

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Keyaerts M, Xavier C, Heemskerk J et al (2016) Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma. J Nucl Med 57:27–33

    Article  CAS  PubMed  Google Scholar 

  22. Rasmussen SGF, Choi H-J, Fung JJ et al (2011) Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469:175–180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Manglik A, Kobilka BK, Steyaert J (2017) Nanobodies to study G protein–coupled receptor structure and function. Annu Rev Pharmacol Toxicol 57:19–37

    Article  CAS  PubMed  Google Scholar 

  24. Jiang X, Smirnova I, Kasho V et al (2016) Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation. Proc Natl Acad Sci U S A 113:12420–12425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Uchański T, Pardon E, Steyaert J (2020) Nanobodies to study protein conformational states. Curr Opin Struct Biol 60:117–123

    Article  PubMed  Google Scholar 

  26. Baral TN, Arbabi-Ghahroudi M (2012) Expression of single-domain antibodies in bacterial systems. Methods Mol Biol 911:257–275

    CAS  PubMed  Google Scholar 

  27. Xue X, Fan X, Qu Q et al (2016) Bioscreening and expression of a camel anti-CTGF VHH nanobody and its renaturation by a novel dialysis–dilution method. AMB Express 6:72

    Article  PubMed  PubMed Central  Google Scholar 

  28. Xu L, Song X, Jia L (2017) A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli. Biotechnol Appl Biochem 64:895–901

    Article  CAS  PubMed  Google Scholar 

  29. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rahbarizadeh F, Rasaee MJ, Forouzandeh M et al (2006) Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris. Mol Immunol 43:426–435

    Article  CAS  PubMed  Google Scholar 

  31. Ezzine A, M'Hirsi el Adab S, Bouhaouala-Zahar B et al (2012) Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system. Biotechnol Appl Biochem 59:15–21

    Article  CAS  PubMed  Google Scholar 

  32. Baghban R, Gargari SLM, Rajabibazl M et al (2016) Camelid-derived heavy-chain nanobody against clostridium botulinum neurotoxin E in Pichia pastoris. Biotechnol Appl Biochem 63:200–205

    Article  CAS  PubMed  Google Scholar 

  33. Chen Q, Zhou Y, Yu J et al (2019) An efficient constitutive expression system for anti-CEACAM5 nanobody production in the yeast Pichia pastoris. Protein Expr Purif 155:43–47

    Article  CAS  PubMed  Google Scholar 

  34. Miyamoto K, Aoki W, Ohtani Y et al (2019) Peptide barcoding for establishment of new types of genotype–phenotype linkages. PLoS One 14:e0215993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Xian Z, Ma L, Zhu M et al (2019) Blocking the PD-1-PD-L1 axis by a novel PD-1 specific nanobody expressed in yeast as a potential therapeutic for immunotherapy. Biochem Biophys Res Commun 519:267–273

    Article  CAS  PubMed  Google Scholar 

  36. Daly R, Hearn MTW (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  CAS  PubMed  Google Scholar 

  37. Macauley-Patrick S, Fazenda ML, McNeil B et al (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270

    Article  CAS  PubMed  Google Scholar 

  38. Clare JJ, Rayment FB, Ballantine SP et al (1991) High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Nat Biotechnol 9:455–460

    Article  CAS  Google Scholar 

  39. Lueking A, Holz C, Gotthold C et al (2000) A system for dual protein expression in Pichia pastoris and Escherichia coli. Protein Expr Purif 20:372–378

    Article  CAS  PubMed  Google Scholar 

  40. Burgard J, Grünwald-Gruber C, Altmann F et al (2020) The secretome of Pichia pastoris in fed-batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time. Microb Biotechnol 13:479–494

    Article  CAS  PubMed  Google Scholar 

  41. Hardy E, Martı́Nez E, Diago D et al (2000) Large-scale production of recombinant hepatitis B surface antigen from Pichia pastoris. J Biotechnol 77:157–167

    Article  CAS  PubMed  Google Scholar 

  42. Taguchi S, Ooi T, Mizuno K et al (2015) Advances and needs for endotoxin-free production strains. Appl Microbiol Biotechnol 99:9349–9360

    Article  CAS  PubMed  Google Scholar 

  43. Thermo Fisher Scientific (2014) Pichia expression kit: User guide for expression of recombinant proteins in Pichia pastoris. https://tools.thermofisher.com/content/sfs/manuals/pich_man.pdf. Accessed 1 May 2021

  44. Prabha L, Govindappa N, Adhikary L et al (2009) Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris. Protein Expr Purif 64:155–161

    Article  CAS  PubMed  Google Scholar 

  45. Choi B-K, Bobrowicz P, Davidson RC et al (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci U S A 100:5022–5027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Rothbauer U, Zolghadr K, Tillib S et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889

    Article  CAS  PubMed  Google Scholar 

  47. Kumar R (2019) Simplified protocol for faster transformation of (a large number of) Pichia pastoris strains. Yeast 36:399–410

    Article  CAS  PubMed  Google Scholar 

  48. Wu S, Letchworth GJ (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36:152–154

    Article  CAS  PubMed  Google Scholar 

  49. Theron CW, Berrios J, Steels S et al (2019) Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris. Yeast 36:285–296

    Article  CAS  PubMed  Google Scholar 

  50. Brady JR, Whittaker CA, Tan MC et al (2020) Comparative genome-scale analysis of Pichia pastoris variants informs selection of an optimal base strain. Biotechnol Bioeng 117:543–555

    Article  CAS  PubMed  Google Scholar 

  51. Scorer CA, Clare JJ, McCombie WR et al (1994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high–level foreign gene expression. Nat Biotechnol 12:181–184

    Article  CAS  Google Scholar 

  52. Romanos MA, Clare JJ, Beesley KM et al (1991) Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties. Vaccine 9:901–906

    Article  CAS  PubMed  Google Scholar 

  53. Barrero JJ, Casler JC, Valero F et al (2018) An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microb Cell Fact 17:161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Laroche Y, Storme V, Meutter JD et al (1994) High–level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Nat Biotechnol 12:1119–1124

    Article  CAS  Google Scholar 

  55. De Schutter K, Lin Y-C, Tiels P et al (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566

    Article  PubMed  Google Scholar 

  56. Damasceno LM, Pla I, Chang HJ et al (2004) An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif 37:18–26

    Article  CAS  PubMed  Google Scholar 

  57. Carpenter JF, Crowe JH (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25:244–255

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by JST CREST (grant number JPMJCR16G2), JST FOREST (grant number JPMJFR204K), and COI-NEXT (grant number JPMJPF2008).

Author information

Authors and Affiliations

  1. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

    Yusei Matsuzaki, Kaho Kajiwara, Wataru Aoki & Mitsuyoshi Ueda

  2. Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan

    Wataru Aoki & Mitsuyoshi Ueda

Authors
  1. Yusei Matsuzaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kaho Kajiwara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wataru Aoki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mitsuyoshi Ueda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mitsuyoshi Ueda .

Editor information

Editors and Affiliations

  1. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Greg Hussack

  2. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Kevin A. Henry

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Matsuzaki, Y., Kajiwara, K., Aoki, W., Ueda, M. (2022). Production of Single-Domain Antibodies in Pichia pastoris. In: Hussack, G., Henry, K.A. (eds) Single-Domain Antibodies. Methods in Molecular Biology, vol 2446. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2075-5_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2075-5_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2074-8

  • Online ISBN: 978-1-0716-2075-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation