Abstract
The interest in the use of monoclonal antibodies as therapeutic molecules has raised in the recent years. Due to their high affinity and specificity towards other biological molecules, antibodies are being widely used to treat a broad range of human diseases such as cancer, rheumatism, and cardiovascular diseases. Currently, the production of IgG-like antibodies is mainly obtained from stable or transient mammalian expression systems that allow proper folding and posttranslational modifications. Despite the technological advances of the last decade, the use of these systems still has a rather high production cost and long processing times. For these reasons, researchers are increasingly interested in alternative antibody production methods as well as alternative antibody formats. Bacterial systems, such as Escherichia coli, are extensively being used for recombinant protein production because their easy manipulation and cheap costs. However, the presence of lipopolysaccharides (LPS) traces in the already fractionated recombinant protein makes these systems not good candidates for the preparation of therapeutic molecules. Yeast systems, such as Pichia pastoris, present the convenient easy manipulation of microbial systems but show some key advantages of eukaryotic expression systems, like improved folding machinery and absence of LPS. They are especially suitable for the production of antibody fragments, which do not need human-like glycosylation, avoiding the high costs of mammalian systems. Here, the protocol for the expression and purification of a single-chain antibody fragment (scFv) in P. pastoris is provided, in deep detail for lab manipulation and briefly for a 5L-bioreactor production.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Griesenauer RH, Kinch MS (2017) An overview of FDA-approved vaccines & their innovators. Expert Rev Vaccines 16(12):1253–1266. https://www.tandfonline.com/doi/full/10.1080/14760584.2017.1383159
Kinch MS, Griesenauer RH (2018) in review: FDA approvals of new molecular entities. Drug Discovery Today 24:1710–1714
Demaria O, Cornen S, Daëron M, Morel Y, Medzhitov R, Vivier E (2019) Harnessing innate immunity in cancer therapy. Nature 574:45–56
Rafiq S, Yeku OO, Jackson HJ, Purdon TJ, van Leeuwen DG, Drakes DJ et al (2018) Targeted delivery of a PD-1-blocking scFV by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36(9):847–858. http://www.ncbi.nlm.nih.gov/pubmed/30102295
Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297(5580):353–356. http://www.ncbi.nlm.nih.gov/pubmed/12130773
Hardy J (2006) Alzheimer’s disease: the amyloid cascade hypothesis: an update and reappraisal. J Alzheimers Dis 9(3 Suppl):151–153. http://www.ncbi.nlm.nih.gov/pubmed/16914853
Walsh DM, Selkoe DJ (2007) A beta oligomers—a decade of discovery. J Neurochem 101(5):1172–1184. http://www.ncbi.nlm.nih.gov/pubmed/17286590
Tomic JL, Pensalfini A, Head E, Glabe CG (2009) Soluble fibrillar oligomer levels are elevated in Alzheimer’s disease brain and correlate with cognitive dysfunction. Neurobiol Dis 35(3):352–358. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2725199&tool=pmcentrez&rendertype=abstract
Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H et al (2000) Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 6(8):916–919
Montoliu-Gaya L, Villegas S (2016) Aβ-Immunotherapeutic strategies: a wide range of approaches for Alzheimer’s disease treatment. Expert Rev Mol Med 18:e13. http://www.journals.cambridge.org/abstract_S1462399416000119
Check E (2002) Nerve inflammation halts trial for Alzheimer’s drug. Nature 415(6871):462. http://www.nature.com/doifinder/10.1038/415462a
Orgogozo J-M, Gilman S, Dartigues J-F, Laurent B, Puel M, Kirby LC et al (2003) Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology 61(1):46–54. http://www.ncbi.nlm.nih.gov/pubmed/12847155
Bacskai BJ, Kajdasz ST, McLellan ME, Games D, Seubert P, Schenk D et al (2002) Non-Fc-mediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy. J Neurosci 22(18):7873–7878. http://www.ncbi.nlm.nih.gov/pubmed/12223540
Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23(9):1126–1136
Esquerda-Canals G, Martí-Clúa J, Villegas S (2019) Pharmacokinetic parameters and mechanism of action of an efficient anti-Aβ single chain antibody fragment. PLoS One 14(5):e0217793. https://doi.org/10.1371/journal.pone.0217793
Esquerda-Canals G, Roda AR, Martí-Clúa J, Montoliu-Gaya L, Rivera-Hernández G, Villegas S (2019) Treatment with scFv-h3D6 Prevented Neuronal Loss and Improved Spatial Memory in Young 3xTg-AD Mice by Reducing the Intracellular Amyloid-β Burden. J Alzheimer’s Dis 70(4):1069–1091
Giménez-Llort L, Rivera-Hernández G, Marin-Argany M, Sánchez-Quesada JL, Villegas S (2013) Early intervention in the 3xTg-AD mice with an amyloid β-antibody fragment ameliorates first hallmarks of alzheimer disease. mAbs 5(5):665–677
Montoliu-Gaya L, Güell-Bosch J, Esquerda-Canals G, Roda AR, Serra-Mir G, Lope-Piedrafita S et al (2018) Differential effects of apoE and apoJ mimetic peptides on the action of an anti-Aβ scFv in 3xTg-AD mice. Biochem Pharmacol 155:380–392. http://www.ncbi.nlm.nih.gov/pubmed/30026023
Ferrer-Miralles N, Villaverde A (2013) Bacterial cell factories for recombinant protein production; expanding the catalogue. Microb Cell Fact 12(1):113. http://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-113
Montoliu-Gaya L, Martínez JC, Villegas S (2017) Understanding the contribution of disulphide bridges to the folding and misfolding of an anti-Aβ scFv. Protein Sci 26(6):1138–1149. http://www.ncbi.nlm.nih.gov/pubmed/28340507
Marín-Argany M, Rivera-Hernández G, Martí J, Villegas S (2011) An anti-Aβ (amyloid β) single-chain variable fragment prevents amyloid fibril formation and cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway. Biochem J 437(1):25–34. http://biochemj.org/lookup/doi/10.1042/BJ20101712
Montoliu-Gaya L, Esquerda-Canals G, Bronsoms S, Villegas S (2017) Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect. PLoS One 12(8):e0181480. http://dx.plos.org/10.1371/journal.pone.0181480
Birch JR, Racher AJ (2006) Antibody production. Adv Drug Deliv Rev 58(5–6):671–685. http://www.ncbi.nlm.nih.gov/pubmed/16822577
Brake AJ, Merryweather JP, Coit DG, Heberlein UA, Masiarz FR, Mullenbach GT et al (1984) Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 81(15):4642–4646. http://www.ncbi.nlm.nih.gov/pubmed/6087338
Julius D, Brake A, Blair L, Kunisawa R, Thorner J (1984) Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell 37(3):1075–1089. http://www.ncbi.nlm.nih.gov/pubmed/6430565
Daly R, Hearn MTW (2005) Expression of heterologous proteins inPichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18(2):119–138. http://www.ncbi.nlm.nih.gov/pubmed/15565717
Higgins DR, Higgins RD (1995) Overview of protein expression in Pichia pastoris. In: Current protocols in protein science. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp 5.7.1–5.7.18. https://doi.org/10.1002/0471140864.ps0507s02
Grinna LS, Tschopp JF (1989) Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast 5(2):107–115. http://doi.wiley.com/10.1002/yea.320050206
Gemmill TR, Trimble RB (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426(2):227–237. http://www.ncbi.nlm.nih.gov/pubmed/9878752
Abelson JN, Simon MI, Guthrie C, Fink GR (2004) Guide to yeast genetics and molecular biology, part A. Elsevier Science, Amsterdam, p 961
Higgins DR, Cregg JM (1998) Methods in molecular biology pichia protocols, vol 103. Humana Press, Totowa, New Jersey
Invitrogen. Life Technologies. Pichia Fermentation Process Guidelines Overview Introduction. http://tools.thermofisher.com › sfs › pichiaferm_prot
Cámara E, Monforte S, Albiol J, Ferrer P (2019) Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter. Biotechnol Bioeng 116(7):1710–1720. http://www.ncbi.nlm.nih.gov/pubmed/30712270
Ibáñez-Pérez R, Guerrero-Ochoa P, Al-Wasaby S, Navarro R, Tapia-Galisteo A, De Miguel D et al (2019) Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin. Oncoimmunology 8(11):1641392. http://www.ncbi.nlm.nih.gov/pubmed/31646080
Blanco-Toribio A, Lacadena J, Nuñez-Prado N, Álvarez-Cienfuegos A, Villate M, Compte M et al (2014) Efficient production of single-chain fragment variable-based N-terminal trimerbodies in Pichia pastoris. Microb Cell Fact 13(1):116. http://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-014-0116-1
Liang H, Li X, Chen B, Wang B, Zhao Y, Zhuang Y et al (2015) A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy. J Control Release 209:101–109. http://www.ncbi.nlm.nih.gov/pubmed/25916496
Schellmann N, Panjideh H, Fasold P, Bachran D, Bachran C, Deckert PM et al (2012) Targeted tumor therapy with a fusion protein of an antiangiogenic human recombinant scFv and yeast cytosine deaminase. J Immunother 35(7):570–578. http://www.ncbi.nlm.nih.gov/pubmed/22892453
Anuleejun S, Palaga T, Katakura Y, Kuroki M, Kuroki M, Napathorn SC (2014) Optimal production of a fusion protein consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in fed-batch culture of Pichia pastoris. Anticancer Res 34(8):3925–3935. http://www.ncbi.nlm.nih.gov/pubmed/25075014
Xiong C, Mao Y, Wu T, Kang N, Zhao M, Di R et al (2018) Optimized expression and characterization of a novel fully human bispecific single-chain diabody targeting vascular endothelial growth factor165 and programmed death-1 in Pichia pastoris and evaluation of antitumor activity in vivo. Int J Mol Sci 19:10
Sommaruga S, Lombardi A, Salvadè A, Mazzucchelli S, Corsi F, Galeffi P et al (2011) Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells. Appl Microbiol Biotechnol 91(3):613–621. http://www.ncbi.nlm.nih.gov/pubmed/21538107
Cao X, Yu H, Chen C, Wei J, Wang P (2015) Expression and characterization of recombinant humanized anti-HER2 single-chain antibody in Pichia pastoris for targeted cancer therapy. Biotechnol Lett 37(7):1347–1354. http://springerlink.bibliotecabuap.elogim.com/10.1007/s10529-015-1804-6
Carreras-Sangrà N, Tomé-Amat J, García-Ortega L, Batt CA, Oñaderra M, Martínez-del-Pozo A et al (2012) Production and characterization of a colon cancer-specific immunotoxin based on the fungal ribotoxin α-sarcin. Protein Eng Des Sel 25(8):425–435. http://www.ncbi.nlm.nih.gov/pubmed/22718791
Tomé-Amat J, Olombrada M, Ruiz-de-la-Herrán J, Pérez-Gómez E, Andradas C, Sánchez C et al (2015) Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin α-sarcin in nude mice bearing human colorectal cancer xenografts. SpringerPlus 4(1):168. http://www.springerplus.com/content/4/1/168
Tomé-Amat J, Menéndez-Méndez A, García-Ortega L, Batt CA, Oñaderra M, Martínez-Del-Pozo Á et al (2012) Production and characterization of scFvA33T1, an immunoRNase targeting colon cancer cells. FEBS J 279(17):3022–3032
Wan L, Zhu S, Zhu J, Yang H, Li S, Li Y et al (2013) Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris. Appl Microbiol Biotechnol 97(9):3855–3863. http://www.ncbi.nlm.nih.gov/pubmed/23250227
Zarei N, Vaziri B, Shokrgozar MA, Mahdian R, Fazel R, Khalaj V (2014) High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris. Appl Microbiol Biotechnol 98(24):10023–10039. http://www.ncbi.nlm.nih.gov/pubmed/25239038
Della Cristina P, Castagna M, Lombardi A, Barison E, Tagliabue G, Ceriotti A et al (2015) Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems. Microb Cell Fact 14(1):19. http://www.microbialcellfactories.com/content/14/1/19
Cai Y, Yao S, Zhong J, Zhang J, Jiang H, Deng Y et al (2017) Inhibition activity of a disulfide-stabilized diabody against basic fibroblast growth factor in lung cancer. Oncotarget 8(12):20187–20197. http://www.ncbi.nlm.nih.gov/pubmed/28423625
Parker SA, Diaz ILC, Anderson KA, Batt CA (2013) Design, production, and characterization of a single-chain variable fragment (ScFv) derived from the prostate specific membrane antigen (PSMA) monoclonal antibody J591. Protein Expr Purif 89(2):136–145. http://www.ncbi.nlm.nih.gov/pubmed/23500147
Kazuma SM, Cavalcante MF, Telles AER, Maranhão AQ, Abdalla DSP (2013) Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis. mAbs 5(5):763–775. http://www.ncbi.nlm.nih.gov/pubmed/23924793
Diaz Arias CA, Molino JVD, de Araújo Viana Marques D, Queiroz Maranhão A, Abdalla Saes Parra D, Pessoa Junior A et al (2019) Influence of carbon source on cell size and production of anti LDL (-) single-chain variable fragment by a recombinant Pichia pastoris strain. Mol Biol Rep 46(3):3257–3264
Vallet-Courbin A, Larivière M, Hocquellet A, Hemadou A, Parimala S-N, Laroche-Traineau J et al (2017) A recombinant human anti-platelet scFv antibody produced in pichia pastoris for atheroma targeting. PLoS One 12(1):e0170305. http://dx.plos.org/10.1371/journal.pone.0170305
Liang MH, Zhou SS, Jiang JG (2017) Construction, expression and characterization of a fusion protein HBscFv-IFNγ in Komagatella (Pichia) pastoris X33. Enzyme Microb Technol 102:74–81. http://www.ncbi.nlm.nih.gov/pubmed/28465064
Huber G, Bánki Z, Kunert R, Stoiber H (2014) Novel bifunctional single-chain variable antibody fragments to enhance virolysis by complement: generation and proof-of-concept. Biomed Res Int 2014:971345. http://www.ncbi.nlm.nih.gov/pubmed/24524088
Wang DD, Su MM, Sun Y, Huang SL, Wang J, Yan WQ (2012) Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris. Protein Expr Purif 86(1):75–81. http://www.ncbi.nlm.nih.gov/pubmed/22982755
Yuan LM, X Ping H, Xie M, Jing JS, Jun LL, Xu LD et al (2014) Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis. Biotechnol Lett 36(12):2425–2431. http://www.ncbi.nlm.nih.gov/pubmed/25129049
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Montoliu-Gaya, L., Villegas, S. (2022). Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris. In: Houen, G. (eds) Therapeutic Antibodies. Methods in Molecular Biology, vol 2313. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1450-1_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1450-1_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1449-5
Online ISBN: 978-1-0716-1450-1
eBook Packages: Springer Protocols