Influence of PDI gene overexpression on heterological proteins production in yeast Pichia pastoris

Abstract

Background. The yeast Pichia pastoris is used for synthesis of recombinant secretory proteins. Overexpression of assistant genes, coding proteins involved in secretion, is one of approaches to improve the production of target protein. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study was to evaluate the effect of Pdi overproduction on recombinant interferons (human interferon-alfa16 and chicken interferon-gamma) production. Materials and Methods. PpPDI gene was cloned under the control of the AOX1 gene promoter in plasmid pPICZαA. Primers for AJ302014.1 nucleotide sequence of NCBI data base were used for PpPDI gene cloning. The chromosomal DNA of the GS115 strain was used as a template. To generate strains with PpPDI gene overexpression we used a previously obtained strains producing human interferon-alfa16 and chicken interferon-gamma. Yeast transformation was performed by electroporation. Cultivation was performed using single and two-stage strategies in standard media containing methanol as the sole carbon source to induce the AOX1 gene promoter. Results. We obtained interferon-producing strains with PpPDI gene overexpression. Over-expression of the PpPDI gene in yeast P. pastoris increases the production of interferon-alfa16, a protein containing disulfide bonds, regardless of the mode of cultivation. Effect of PpPDI gene over-expression on the production of interferon-gamma - the protein without disulfide bonds, depends on cultivation mode. Conclusion. PpPDI gene overexpression can be used to enhance the production of interferons and other proteins that contain disulfide bonds. Effect of PpPDI gene overexpression on recombinant proteins without disulfide bonds may depend on cultivation procedure.

Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

“…При синтезе гетерологичных белков повышается уровень экспрессии как гена PpKar2, так и гена PpPdI1 [12]. С другой стороны, известно, что сверхэкспрессия этих генов может повышать продукцию гетерологичных белков [17,18], в частности интерферонов [19].…”

“…Для получения дрожжевых штаммов нужного генотипа применяли штамм P. pastoris GS115 (his4) (Invitrogen) (his4 -мутация, приводящая к потребности в гистидине). Также использовали ранее полученные штаммы P. pastoris -GS-AOX1PHO5 [20], GS-AOX1PDI1 [19], в которых гены PHo5 и PpPdI1 находятся под контролем промотора PaoX1.…”