Construction of recombinant Pichia pastoris expressing single-chain antibody fragment against extracellular domain of EpCAM

How to Cite:Fatemeh MohammadgholizadAtieh HashemiConstruction of recombinant Pichia pastoris expressing single-chain antibody fragment against extracellular domain of EpCAM.koomesh.21(4):e153140.

Abstract

Introduction: Epithelial cell adhesion molecule (EpCAM) is highly expressed on epithelial tumors. So, EpCAM is a valuable antigen for targeted therapy. Using monoclonal antibodies (mabs) is an attractive approach for targeted cancer therapy. Importantly, limitations of intact mabs including large size led to the development of antibody fragments such as single chain fragment variable (scfv). Pichia pastoris is considered as a suitable host for large-scale production of recombinant proteins because of post-translation modification system and low cost. Here, the Pichia expressing a scfv against EpCAM extracellular domain (EpEX) was constructed. Materials and Methods: The codon optimized gene encoding anti-EpEX-scfv protein was cloned in the XhoI and XbaI sites of the pPICZαB vector. The recombinant plasmid was confirmed by restriction enzyme analysis and sequencing. The constructed plasmid was integrated into GS115 strain by electroporation. Positive clones were evaluated by PCR using three sets of primers. Results: Restriction enzyme analysis utilizing HindIII and BamHI (2788, 1537 bp bands), as well as XhoI, XbaI (3506, 819 bp bands) confirmed construction of recombinant pPICZαB-anti EpEX- scfv. In this way, PCR based screening results of integrants showed two bands (2200 and 1321 bp), when AOX1 primer set was used as well as one band (968 bp) when the 3´ insert-specific primer paired with 5´ AOX1 primer. There was one band (751 bp) in PCR result of confirmed integrants when insert-specific primer set was utilized. Conclusion: These findings imply that the engineered strain was constructed. The anti-EpEX-scfv protein can be used as a potential candidate in cancer immunotherapy.

Keywords

Pichia pastoris

Anti-EpEX-scfv

Monoclonal Antibodies

EpCAM

Cancer Immunotherapy.

References

1.
Koprowski H, Steplewski Z, Mitchell K, Herlyn M, Herlyn D, Fuhrer P. Colorectal carcinoma antigens detected by hybridoma antibodies. Somat Cell Genet 1979; 5: 957-971.
2.
Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, Prins F, et al. Epithelial cell adhesion molecule (Ep-CAM) modulates cellcell interactions mediated by classic cadherins. J Cell Biol 1997; 139: 1337-1348.
3.
Munz M, Fellinger K, Hofmann T, Schmitt B, Gires O. Glycosylation is crucial for stability of tumour and cancer stem cell antigen EpCAM. Front Biosci 2008; 13: 5195-5201.
4.
Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee S-M, et al. Single-chain antigen-binding proteins. Science 1988; 242: 423-426.
5.
Yokota T, Milenic DE, Whitlow M, Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 1992; 52: 3402-3408.
6.
Di Paolo C, Willuda J, Kubetzko S, Lauffer I, Tschudi D, Waibel R, et al. A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity. Clin Cancer Res 2003; 9: 2837-2848.
7.
Frenzel A, Hust M, Schirrmann T. Expression of recombinant antibodies. Front Immunol 2013; 4: 217.
8.
MacauleyPatrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005; 22: 249-270.
9.
Butler M, Meneses-Acosta A. Recent advances in technology supporting biopharmaceutical production from mammalian cells. Appl Microbiol Biot 2012; 96: 885-894.
10.
Martnez JL, Liu L, Petranovic D, Nielsen J. Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation. Curr Opin Biotech 2012; 23: 965-971.
11.
Yousefian S, Dehnavi E, Borjian Burojeni M. Secretive expression of bacterial -xylosidase gene including hexahistidin-tag in Pichia pastoris. Koomesh 2013; 14: 389-395. (Persian).
12.
Loke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 2011; 50: 325-328.
13.
Malpiedi LP, Daz CA, Nerli BB, Pessoa Jr A. Single-chain antibody fragments: Purification methodologies. Process Biochem 2013; 48: 1242-1251.
14.
Knappik A, Plckthun A. Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng Des Sel 1995; 8: 81-89.
15.
Krauss J, Arndt MA, Martin AC, Liu H, Rybak SM. Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized antiCD22 singlechain Fv fragment. Protein Eng 2003; 16: 753-759.
16.
Tanfous NGB, Kallel H, Jarboui MA, Fathallah DM. Expression in Pichia pastoris of a recombinant scFv form of MAb 107, an anti human CD11b integrin antibody. Enzyme Microb Tech 2006; 38: 636-642.
17.
Emberson LM, Trivett AJ, Blower PJ, Nicholls PJ. Expression of an anti-CD33 single-chain antibody by Pichia pastoris. J Immunol Methods 2005; 305: 135-151.
18.
Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangemeister-Wittke U, et al. High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res 1999; 59: 5758-5767.
19.
Foroumadi S, Rajabibazl M, Hosseini SH, Rajabi S, Shahidi S, Daraei A, et al. Expression and characterization of recombinant human epidermal growth factor receptor antigene (HER-2) as an indicator of breast cancer in yeast fermented systems. Koomesh 2016; 18: 110-116. (Persian).
20.
Zarei N, Vaziri B, Shokrgozar MA, Mahdian R, Fazel R, Khalaj VJ, et al. High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris. Appl Microbiol Biot 2014; 98: 10023-10039.
21.
Chan MK, Lim SK, Miswan N, Chew AL, Noordin R, Khoo BY, et al. Expression of stable and active human DNA topoisomerase I in pichia pastoris. Protein Expres Purif 2018; 141: 52-62.
22.
Tsai CW, Duggan PF, Shimp Jr RL, Miller LH, Narum DL. Overproduction of Pichia pastoris or Plasmodium falciparum protein disulfide isomerase affects expression, folding and O-linked glycosylation of a malaria vaccine candidate expressed in P. pastoris. J Biotechnol 2006; 121: 458-470.
23.
Qian P, Li X, Tong G, Chen HJVg. High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) in Pichia pastoris. Virus Genes 2003; 27: 189-196.
24.
Liu Y, Wu C, Wang J, Mo W, Yu M. Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris. Appl Microbiol Biotechnol 2013; 97: 10349-10358.
25.
He M, Wu D, Wu J, Chen J. Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production. J Ind Microbiol Biotechnol 2014; 41: 105-114.
26.
Li Y, Li D, Xu X, Cui M, Zhen H, Wang Q. Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris. Genet Mol Res 2014; 13: 4990-5000.
27.
Hu S, Li L, Qiao J, Guo Y, Cheng L, Liu J. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expres Purif 2006; 47: 249-257.
28.
Liu X, Wu D, Wu J, Chen J. Optimization of the production of Aspergillus niger -glucosidase expressed in Pichia pastoris. World J Microb Biotechnol 2013; 29: 533-540.
29.
Yadava A, Ockenhouse CF. Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun 2003; 71: 4961-4969.
30.
Tsygankov M, Padkina MJ. Influence of PDI Gene Overexpression on the Production of Heterologous Proteins in Yeast Pichia pastoris. Russ J Genet 2018; 8: 197-205.
31.
Mala J, Puthong S, Maekawa H, Kaneko Y, Palaga T, Komolpis K, et al. Expression and characterization of functional single-chain variable fragment against norfloxacin in Pichia pastoris GS115. Int Food Res J 2018; 25: 1726-1732.##.

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