Construction of Yeast Pichia pastoris Expressing the Recombinant p53 as a Secreted Protein into Culture
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Abstract
Abstract: Human p53 protein has been known as a tumor suppressor and described as "the guardian of the genome", referring to its role in conserving stability of genome by preventing mutation. The recent studies on p53 protein expression have demonstrated the important role and effectiveness of exogenous p53 protein in tumor suppression. In the world, the expression of p53 for therapeutically interest was extensively study but in Vietnam it has not been noticed. In the other hand, Pichia pastoris showed a good expression system for many exogenous proteins with a simple cloning work and cheap culture. In this study, codon optimization of gene encoding human p53 protein was performed for suitable expression in Pichia pastoris yeast. It was designed as construct of p53 fused with TAT and His-tag sequences (TAT-p53-His). The construct was cloned into pPICZαA expression vector by using EcoRI and XbaI enzymes to make pPICZαA-TAT-p53-His. Yeast strains containing genes coding for TAT-p53-His was obtained. The integration of TAT-p53-His construct into yeast genome was verified by using PCR with AOX1 primers. The expression of the recombinant TAT-p53-His in the culture of P. pastoris X33 was confirmed by SDS-PAGE.
Keywords: Pichia pastoris, gene expression, p53, codon optimization.References
[1] Isobe M., Emanuel B. S., Givol D., Oren M., Croce C. M. (1986) Localization of gene for human p53 tumour antigen to band 17p13. Nature, 320(6057): 84-85.
[2] May P., May E. (1999) Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene, 18: 7621-7636.
[3] Goh A. M., Coffill C. R., Lane D. P. (2011) The role of mutant p53 in human cancer. J. Pathol., 223(2): 116-26.
[4] Collot-Teixeira S., Bass J., Denis F., Ranger-Rogez S. (2004) Human tumor suppressor p53 and DNA viruses. Rev. Med. Virol., 14: 301-319.
[5] Arnold J. L., Morshe O. (2009) The first 30 years of p53: growing ever more complex. Nature Reviews Cancer, 9(10): 749-758.
[6] Cereghino J. L., Cregg J. M., (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev., 24: 45-66.
[7] Macauley-Patrick S., Fazenda M. L., McNeil B., Harvey L. M. (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast, 22: 249-270.
[8] Anumanthan A., Li P., Gao X. G., Ilangovan K., Suzara V.V., Düzgüneş N., Renugopalakrishnan V. (2007) Expression of recombinant proteins in Pichia pastoris. Appl. Biochem. Biotechnol., 142(2): 105-124.
[9] Cereghino G. P., Cereghino J. L., Ilgen C., Cregg J. M. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol., 13: 329-332.
[10] Bretthauer R. K., Castellino F.J. (1999) Glycosylation of Pichia pastoris derived proteins. Biotechnol. Appl. Biochem., 30: 193-200.
[11] Leader B., Baca Q. J., Golan D. E. (2008) Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug. Discov., 7(1): 21-39.
[12] Ryu J, Lee HJ, Kim KA, Lee JY, Lee KS, Park J, Choi SY (2004) Intracellular Delivery of p53 Fused to the Basic Domain of HIV-1 Tat. Mol. Cells, 17(2): 353-359.
[13] Jiang L, Ma Y, Wang J, Tao X, Wei D, (2008) The transduction of His-TAT-p53 fusion protein into the human osteogenic sarcoma cell line (Saos-2) and its influence on cell cycle arrest and apoptosis. Mol. Biol. Rep., 35(1): 1-8.
Haowei Y., N. Liu, Z. Zhao, X. Zhang, Hao X., B. Shao and W. Yan (2012), Expression and purification of human TAT-p53 fusion protein in Pichia pastoris and its influence on HepG2 cell apoptosis. Biotechnol. Lett., 34(7): 1217-1233.