Synthesis of Recombinant Gamma Interferons Resistant to Proteolysis in the Yeast Pichia pastoris

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.

Section: получение штаммовunclassified

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

Tsygankov

Александрович²,

Падкина

et al. 2017

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mentioning

confidence: 99%

“…Methylotrophic yeast Komagataella рhaffii (also known as Pichia pastoris) is widely applied in biotechnology for recombinant protein production. K. рhaffii particularly proved to be a successful host system for the synthesis of immunomodulators such as interferons [1].…”

mentioning

confidence: 99%

Methylotrophic yeast Komagataella phaffii as Neoleukin producer

Makeeva

Shubert²,

Shanaa³

et al. 2022

Methylotrophic yeast Komagataella рhaffii (also known as Pichia pastoris) is widely applied in biotechnology for recombinant protein production. K. рhaffii particularly proved to be a successful host system for the synthesis of immunomodulators such as interferons [1]. In this study, we engineered K. рhaffii strains capable of producing the immunomodulatory protein Neoleukin (Neo-2/15). Neo-2/15 is an interleukin-2 mimetic, designed by in silico methods [2]. In preclinical studies on murine cancer models, Neo-2/15 showed superior therapeutic effect to inerleukin-2 with reduced toxicity. In this work, we show that K. рhaffii can successfully synthesize and secrete Neo-2/15. We have obtained a number of K. phaffii strains, including MutS and Mut+, with different Neo-2/15 expression cassettes integrated into the genome, carrying up to five copies of Neo-2/15 gene. In fact, the higher number of Neo-2/15 gene copies in K. рhaffii genome allowed a higher protein yield. In this study, we further developed a split marker approach [3] for yeast transformation using two DNA fragments, comprising of the expression cassette and the overlapping fragments of the marker gene. This allowed us to generate MutS strains with two copies using pPICZB vector, which is not originally intended for MutS strain generation. As a result, we demonstrated that K. phaffii is a perspective producer of Neo-2/15, providing wide opportunities to increase the production of this therapeutic protein.

Transgenic plants as bioreactors for the production of substances of medicinal and veterinary importance

Savel’eva¹,

Burlakovskiy

Yemelyanov

et al. 2015

The use of plants as bioreactors has become of a great importance in the modern biotechnology. The transgenic plants are capable of synthesizing of many substances, including valuable pharmaceuticals. Plants possess a number of advantages compared to conventional bioreactors - microorganisms and animal cell cultures. The product safety and lower production costs are among them. One of the promising directions in plant biotechnology is the creation of “edible vaccines, plantibodies and adjuvants” based on recombinant antigens, immunoglobulins and immunoregulatory cytokines. Edible bioreactor plants can be administered as food and feed additives in medicine and veterinary avoiding expensive purification procedures. Interferons have antiviral, antibacterial, antitumor and immunomodulatory activity, and are implicated in the prophylaxis and therapy of diseases of different etiologies. Investigations concerning with obtaining of bioreactor plants synthesizing γ-interferons of mammals and birds are carried out in the laboratory of genetic and cellular engineering of plants St. Petersburg State University. Our recent achievements in the creation of inbreed tobacco line producing bovine γ-interferon are described.