Abstract
The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L–1 in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL–1, respectively.
Similar content being viewed by others
References
Akashi H (2003) Translational selection and yeast proteome evolution. Genetics 164:1291–1303
Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) Properties of polyclonal, monoclonal, and recombinant antibodies recognizing the organophosphorus pesticide chlorpyrifos-ethyl. J Agric Food Chem 48:4053–4059
Athmaram TN, Saraswat S, Singh AK, Rao MK, Gopalan N, Suryanarayana VVS, Rao PVL (2012) Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris. Virus Genes 45:440–451
Barbosa J, Cruz C, Martins J, Silva JM, Neves C, Alves C, Ramos F, Noronha da Silveira MI (2005) Food poisoning by clenbuterol in Portugal. Food Addit Contam 22:563–566
Boettner M, Prinz B, Holz C, Stahl U, Lang C (2002) High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J Biotechnol 99:51–62
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Burland TG (2000) DNASTAR’s lasergene sequence analysis software. In: Misener S, Krawetz SA (eds) Methods in molecular biology: bioinformatics methods and protocols, vol 132. Human, Totowa, pp 71–91
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Chan CP-y, Cheung Y-c, Renneberg R, Seydack M (2008) New trends in immunoassays. In: Advances in biochemical engineering/biotechnology: biosensing for the 21st century, vol 109. Springer, Berlin, pp 123–154
Chen S-C, He Y-S, Xu X-Y, Lu T-Z, Wang H (2011) Expression of a single chain Fv antibody against clenbuterol in Pichia pastoris. Chin Sci Technol Food Ind 7:217–219
Chiu Y-W, Chen R, Li QX, Karu AE (2000) Derivation and properties of recombinant Fab antibodies to coplanar polychlorinated biphenyls. J Agric Food Chem 48:2614–2624
Coghlan A, Wolfe KH (2000) Relationship of codon bias to mRNA concentration and protein length in Saccharomyces cerevisiae. Yeast 16:1131–1145
Damasceno L, Ventura R, Cardoso J, Segura J (2002) Diagnostic evidence for the presence of β-agonists using two consecutive derivatization procedures and gas chromatography–mass spectrometric analysis. J Chromatogr B 780:61–71
De Jaeger G, Buys E, Eeckhout D, de Wilde C, Jacobs A, Kapila J, Angenon G, van Montagu M, Gerats T, Depicker A (1999) High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. Eur J Biochem 259:426–434
Fredericks D, Clay R, Warner T, O’Connor A, de Kretser DM, Hearn MTW (2010) Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris. Biotechnol Prog 26:372–383
Gao Z, Li Z, Zhang Y, Huang H, Li M, Zhou L, Tang Y, Yao B, Zhang W (2012) High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization. Biotechnol Lett 34:507–514
Hoekema A, Kastelein RA, Vasser M, de Boer HA (1987) Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol Cell Biol 7:2914–2924
Hu S, Li L, Qiao J, Guo Y, Cheng L, Liu J (2006) Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expr Purif 47:249–257
Hu H, Gao J, He J, Yu B, Zheng P, Huang Z, Mao X, Yu J, Han G, Chen D (2013) Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris. PLoS ONE 8(3):e58393. doi:10.1371/journal.pone.0058393
Idiris A, Tohda H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86:403–417
Kang HA, Choi E-S, Hong W-K, Kim J-Y, Ko S-M, Sohn J-H, Rhee S-K (2000) Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 53:575–582
Kim H-J, Lee S-J, Kim H-J (2010) Optimizing the secondary structure of human papillomavirus type 16 L1 mRNA enhances L1 protein expression in Saccharomyces cerevisiae. J Biotechnol 150:31–36
Koganesawa N, Aizawa T, Shimojo H, Miura K, Ohnishi A, Demura M, Hayakawa Y, Nitta K, Kawano K (2002) Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris. Protein Expr Purif 25:416–425
Korpimäki T, Brockmann E-C, Kuronen O, Saraste M, Lamminmäki U, Tuomola M (2004) Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level. J Agric Food Chem 52:40–47
Lange S, Schmitt J, Schmid RD (2001) High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris. J Immunol Methods 255:103–114
Lauer B, Ottleben I, Jacobsen H-J, Reinard T (2005) Production of a single-chain variable fragment antibody against fumonisin B1. J Agric Food Chem 53:899–904
Lee N, Holtzapple CK, Stanker LH (1998) Cloning, expression, and characterization of recombinant Fab antibodies against dioxin. J Agric Food Chem 46:3381–3388
Li T, Cheng J, Hu B, Liu Y, Qian G, Liu F (2008) Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastoris. World J Microbiol Biotechnol 24:867–874
Li Y, Zhang B, Chen X, Chen Y, Cao Y (2010) Improvement of Aspergillus sulphureus endo-β-1,4-xylanase expression in Pichia pastoris by codon optimization and analysis of the enzymatic characterization. Appl Biochem Biotechnol 160:1321–1331
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCt method. Methods 25:402–408
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270
Maertens B, Spriestersbach A, von Groll U, Roth U, Kubicek J, Gerrits M, Graf M, Liss M, Daubert D, Wagner R, Schäfer F (2010) Gene optimization mechanisms: a multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli. Protein Sci 19:1312–1326
Mayson BE, Kilburn DG, Zamost BL, Raymond CK, Lesnicki GJ (2003) Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica. Biotechnol Bioeng 81:291–298
Melwanki MB, Huang S-D, Fuh M-R (2007) Three-phase solvent bar microextraction and determination of trace amounts of clenbuterol in human urine by liquid chromatography and electrospray tandem mass spectrometry. Talanta 72:373–377
Menéndez C, Martínez D, Trujillo LE, Mazola Y, González E, Pérez ER, Hernández L (2013) Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris. Appl Microbiol Biotechnol 97:1201–1212
Minning S, Serrano A, Ferrer P, Solá C, Schmid RD, Valero F (2001) Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 86:59–70
Moghaddam A, Løbersli I, Gebhardt K, Braunagel M, Marvik OJ (2001) Selection and characterisation of recombinant single-chain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries. J Immunol Methods 254:169–181
Mörtl M, Németh G, Juracsek J, Darvas B, Kamp L, Rubio F, Székács A (2013) Determination of glyphosate residues in Hungarian water samples by immunoassay. Microchem J 107:143–151
Nishi K, Ishiuchi M, Morimune K, Ohkawa H (2005) Molecular and immunochemical characteristics of monoclonal and recombinant antibodies selective for the triazine herbicide simetryn and application to environmental analysis. J Agric Food Chem 53:5096–5104
Öberg F, Sjöhamn J, Conner MT, Bill RM, Hedfalk K (2011) Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: the importance of codon optimization and clone selection. Mol Membr Biol 28:398–411
País-Chanfrau JM, García Y, Licor L, Besada V, Castellanos-Serra L, Cabello CI, Hernández L, Mansur M, Plana L, Hidalgo A, Támbara Y, del Abrahantes-Pérez CM, del Toro Y, Valdés J, Martínez E (2004) Improving the expression of mini-proinsulin in Pichia pastoris. Biotechnol Lett 26:1269–1272
Pan K, Wang H, Zhang H-b, Liu H-w, Lei H-t, Huang L, Sun Y-m (2006) Production and characterization of single chain Fv directed against β2-agonist clenbuterol. J Agric Food Chem 54:6654–6659
Rao NH, Balou BP, Rajendra L, Sriraman R, Pang Y-YS, Schiller JT, Srinivasan VA (2011) Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles. Vaccine 29:7326–7334
Romanos M (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6:527–533
Sarramegna V, Talmont F, Demange P, Milon A (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification. Cell Mol Life Sci 60:1529–1546
Sinclair G, Choy FYM (2002) Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif 26:96–105
Singh S, Gras A, Fiez-Vandal C, Ruprecht J, Rana R, Martinez M, Strange PG, Wagner R, Byrne B (2008) Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures. Microb Cell Factories 7:28
Sommaruga S, Lombardi A, Salvadè A, Mazzucchelli S, Corsi F, Galeffi P, Tortora P, Prosperi D (2011) Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells. Appl Microbiol Biotechnol 91:613–621
Sporano V, Grasso L, Esposito M, Oliviero G, Brambilla G, Loizzo A (1998) Clenbuterol residues in non-liver containing meat as a cause of collective food poisoning. Vet Hum Toxicol 40:141–143
Teng D, Fan Y, Yang Y-L, Tian Z-G, Luo J, Wang J-h (2007) Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Appl Microbiol Biotechnol 74:1074–1083
Wang H, Wang Q, Zhang F, Huang Y, Ji Y, Hou Y (2008) Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis. Protein Expr Purif 60:97–102
Wang H, Yang J, Liu X, Liang Y, Lei H, Shen Y, Xu X, Sun Y, Xu Z, He Y (2009) Cloning, expression, and identification of anti-carbofuran single chain Fv gene. Biotechnol Prog 25:1018–1024
Wang H, Liu X, He Y, Dong J, Sun Y, Liang Y, Yang J, Lei H, Shen Y, Xu X (2010) Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli. Protein Expr Purif 72:26–31
Ward VK, Schneider PG, Kreissig SB, Hammock BD, Choudary PV (1993) Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine. Protein Eng Des Sel 6:981–988
Wu AB, Chen HD, Tang ZZ, Ye BW, Liu WJ, Jia HY, Zhang DB (2008) Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris. Chem Biol Interact 175:403–405
Yang M, Johnson SC, Murthy PPN (2012) Enhancement of alkaline phytase production in Pichia pastoris: influence of gene dosage, sequence optimization and expression temperature. Protein Expr Purif 84:247–254
Yu H, Yan X, Shen W, Hong Q, Zhang J, Shen Y, Li S (2009) Expression of methyl parathion hydrolase in Pichia pastoris. Curr Microbiol 59:573–578
Zhang T, Gong F, Peng Y, Chi Z (2009) Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase. Process Biochem 44:1335–1339
Zhao K-N, Gu W, Fang NX, Saunders NA, Frazer IH (2005) Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo. Mol Cell Biol 25:8643–8655
Zhu T, Guo M, Sun C, Qian J, Zhuang Y, Chu J, Zhang S (2009) A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol Lett 31:679–684
Acknowledgments
J.-X. Dong and X. Xie contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant 30871755, 31271866), Science and Technology Plan Projects in Guangdong Province (2012A020100002, 2010A032000001–4), and the Doctoral Innovation Program of Hopson Zhujiang Education Fund (H2011001).
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 98 kb)
Rights and permissions
About this article
Cite this article
Dong, JX., Xie, X., Hu, DW. et al. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris . Appl Microbiol Biotechnol 98, 3679–3689 (2014). https://doi.org/10.1007/s00253-013-5324-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-5324-9