Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review.

Recombinant protein subunit vaccines are formulated using defined protein antigens that can be produced in heterologous expression systems. The methylotrophic yeast Pichia pastoris has become an important host system for the production of recombinant subunit vaccines. Although many basic elements of P. pastoris expression system are now well developed, there is still room for further optimization of protein production. Codon bias, gene dosage, endoplasmic reticulum protein folding and culture condition are important considerations for improved production of recombinant vaccine antigens. Here we comment on current advances in the application of P. pastoris for the synthesis of recombinant subunit vaccines.

https://www.tandfonline.com/doi/pdf/10.1080/21655979.2016.1191707

Recent advances in the production of recombinant subunit vaccines in Pichia pastoris

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P pastoris under the control of the AOX1 promoter A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621% However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 682% Moreover, Gs4j-amyA was glycosylated in P pastoris which generated higher enzyme activity than that in E coli Generally, regulating HAC1p expression by different strategies enhanced amylase production by 111 folds, indicating a reference for expression of other proteins in P pastoris

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris

A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.

Chinese sacbrood virus (CSBV) is a small RNA virus family belonging to the genus Iflavirus that causes larval death, and even the collapse of entire bee colonies. The virus particle is spherical, non-enveloped, and its viral capsid is composed of four proteins, although the functions of the structural proteins are unclear. In this study, we used codon recoding to express the recombinant proteins VP1, VP2, and VP3 in Escherichia coli. SDS-PAGE analysis and Western blotting revealed that the target genes were expressed at high levels. Mice were then immunized with the purified, recombinant proteins, and antibody levels and lymphocyte proliferation were analyzed by ELISA and the MTT assay, respectively. The results show that the recombinant proteins induced high antibody levels and promoted lymphocyte proliferation. Polyclonal antibodies directed against these proteins will aid future studies of the molecular pathogenesis of CSBV.

/pdf/codon-optimization-expression-in-escherichia-coli-and-4k0rsxrrkp.pdf

Codon Optimization, Expression in Escherichia coli, and Immunogenicity of Recombinant Chinese Sacbrood Virus (CSBV) Structural Proteins VP1, VP2, and VP3

Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.

/pdf/codon-optimization-expression-purification-and-functional-4jv1td6cpo.pdf

Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris

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Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris