Construction of recombinant yeast expressing EpEX as a suitable candidate in cancer diagnosis and therapy

authors:

avatar Mozhdeh Zamani , avatar Atieh Hashemi ORCID , * , avatar Najmeh Zarei , avatar Hoda Jahandar


how to cite: Zamani M, Hashemi A, Zarei N, Jahandar H. Construction of recombinant yeast expressing EpEX as a suitable candidate in cancer diagnosis and therapy. koomesh. 2019;21(4):e153139. 

Abstract

Introduction: Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is overexpressed on the majority of tumor cells of epithelial origin and thereby can be used as a target of immunodetection and immunotherapy of cancer. So, it is important to produce this protein in its native form. Interestingly, during the last two decades, the yeast Pichia pastoris (P. pastoris) has become a popular host for the production of recombinant proteins because it combines the advantages of both mammalian and prokaryotic expression systems. In this study, the Pichia expressing EpCAM extracellular domain (EpEX) was constructed. Materials and Methods: The codon optimized gene encoding EpEX protein was cloned in the XhoI and XbaI restriction sites of the pPICZαB vector. The constructed plasmid was integrated into GS115 strain by electroporation. Positive clones were evaluated by PCR using AOX1 primers. Results: Sequencing as well as restriction enzyme analysis utilizing XhoI and XbaI (3506, 843 bp bands), as well as BamHI (3651, 698 bp bands) confirmed construction of recombinant EpEX pPICZαB. PCR based screening results of integrants showed two bands (2200 and 1345 bp), when AOX1 primer set was used. Conclusion: These findings imply that the engineered strain was constructed. The constructed strain can be used in EpEX recombinant protein production for diagnostic and therapeutic purposes.

References

  • 1.

    Baeuerle P, Gires O. EpCAM (CD326) finding its role in cancer. Br J Cancer 2007; 96: 417.

  • 2.

    Tutlewska K, Lubinski J, Kurzawski G. Germline deletions in the EPCAM gene as a cause of Lynch syndromeliterature review. Hered Cancer Clin Pract 2013; 11: 9.

  • 3.

    Schmetzer O, Moldenhauer G, Nicolaou A, Schlag P, Riesenberg R, Pezzutto A. Detection of circulating tumor-associated antigen depends on the domains recognized by the monoclonal antibodies used: N-terminal trimmed EpCAM-levels are much higher than untrimmed forms. Immunol Lett 2012; 143: 184-192.

  • 4.

    Chen H, Werner S, Tao S, Zrnig I, Brenner H. Blood autoantibodies against tumor-associated antigens as biomarkers in early detection of colorectal cancer. Cancer Lett 2014; 346: 178-187.

  • 5.

    Simon M, Stefan N, Plckthun A, Zangemeister-Wittke U. Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy. Expert Opin Drug Deliv 2013; 10: 451-468.

  • 6.

    Mellstedt H, Fagerberg J, Frdin JE, HjelmSkog AL, Liljefors M, Markovic K, et al. Ga733/EpCAM as a target for passive and active specific immunotherapy in patients with colorectal carcinoma. Ann N Y Acad Sci 2000; 910: 254-262.

  • 7.

    Lv M, Qiu F, Li T, Sun Y, Zhang C, Zhu P, et al. Construction, expression, and characterization of a recombinant immunotoxin targeting EpCAM. Mediat Inflamm 2015; 2015.

  • 8.

    Abe H, Kuroki M, Imakiire T, Yamauchi Y, Yamada H, Arakawa F, et al. Preparation of recombinant MK-1/Ep-CAM and establishment of an ELISA system for determining soluble MK-1/Ep-CAM levels in sera of cancer patients. J Immunol Methods 2002; 270: 227-233.

  • 9.

    Verch T, Hooper DC, Kiyatkin A, Steplewski Z, Koprowski H. Immunization with a plant-produced colorectal cancer antigen. Cancer Immunol Immunother 2004; 53: 92-99.

  • 10.

    Damasceno LM, Huang C-J, Batt CA. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 2012; 93: 31-39.

  • 11.

    MacauleyPatrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005; 22: 249-270.

  • 12.

    Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 2014; 98: 5301-5317.

  • 13.

    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).

  • 14.

    Loke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 2011; 50: 325-328.

  • 15.

    Cereghino GPL, Cregg JM. Applications of yeast in biotechnology: protein production and genetic analysis. Curr Opin Biotechnol 1999; 10: 422-427.

  • 16.

    Egli T, Van Dijken J, Veenhuis M, Harder W, Fiechter A. Methanol metabolism in yeasts: regulation of the synthesis of catabolic enzymes. Arch Microbiol 1980; 124: 115-121.

  • 17.

    Cregg JM, Cereghino JL, Shi J, Higgins DR. Recombinant protein expression in Pichia pastoris. Mol Biotechnol 2000; 16: 23-52.

  • 18.

    Zhao T, Li Z, Guo Z, Wang A, Liu Z, Zhao Q, et al. Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors. Protein Expr Purif 2018; 150: 12-16.

  • 19.

    Li H, Ali Z, Liu X, Jiang L, Tang Y, Dai J. Expression of recombinant tachyplesin I in Pichia pastoris. Protein Expr Purif 2019; 157: 50-56.

  • 20.

    Park SH, Kim AY, Ma SH, Kim HM, Kang HS, Maeng JS, et al. Purification of human carcinoma antigen GA733-2 expressed in Escherichia coli and production of its polyclonal antibody in rabbit. Anim Cells Syst 2015; 19: 188-193.

  • 21.

    Ullenhag GJ, Frdin J-E, Mosolits S, Kiaii S, Hassan M, Bonnet MC, et al. Immunization of colorectal carcinoma patients with a recombinant canarypox virus expressing the tumor antigen Ep-CAM/KSA (ALVAC-KSA) and granulocyte macrophage colony-stimulating factor induced a tumor-specific cellular immune response. Clin Cancer Res 2003; 9: 2447-2456.

  • 22.

    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).

  • 23.

    Mohammadgholizad F, Hashemi A. Construction of recombinant Pichia pastoris expressing single-chain antibody fragment against extracellular domain of EpCAM. Koomesh 2019. (Persian).

  • 24.

    Barnett JA, Payne RW, Yarrow D. Yeasts: Characteristics and identification: 1990, 2nd ed. Cambridge Univ 1983.

  • 25.

    He M, Wu D, Wu J, Chen. 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 Expr Purif 2006; 47: 249-257.

  • 28.

    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.

  • 29.

    Tsygankov M, Padkina M. Influence of PDI Gene Overexpression on the Production of Heterologous Proteins in Yeast Pichia pastoris. Russ J Genet 2018; 8: 197-205.

  • 30.

    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.

  • 31.

    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 Expr Purif 2018; 141: 52-62.

  • 32.

    Zarei N, Vaziri B, Shokrgozar MA, Mahdian R, Fazel R, Khalaj V, 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##.