Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris

Abstract

Mammalian peptidoglycan recognition proteins (PGLYRPs) are highly conserved pattern-recognition molecules of the innate immune system with considerable bactericidal activity, which manifest their potential values for the application to food and pharmaceutical industry. However, the effective expression of porcine PGLYRP-1 in Pichia pastoris has not been reported so far. In this study, expression in P. pastoris was explored as an efficient way to produce functional porcine PGLYRP-1. Cooperation of chaperones co-expression and gene dosage (including protein disulfide isomerase (PDI)/binding protein (BiP) and pglyrp-1) were used to enhance functional expression of antimicrobial protein in P. pastoris. Overexpression of PDI was certainly able to increase secretion level of PGLYRP-1 protein because the increase in secreted PGLYRP-1 secretion was correlated with the copy numbers of PDI in high copy pglyrp-1 clones. However, co-expression of BiP was proved to be detrimental to PGLYRP-1 secretion. In addition, we also found that excessive expression of PDI and/or BiP could decrease the mRNA expression of pglyrp-1 gene. This showed that PDI and BiP as the target genes of unfolded protein response (UPR) might regulate the transcription of the target protein. These data demonstrated for the first time that the combination of chaperones and gene dosages could improve the yield of PGLYRP-1, which could facilitate the application to food and pharmaceutical industry.

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

“…These results are in agreement with several previously reported cases where such a plateau-like correlation was observed22454950. Nevertheless, this pattern does not seem to be a general rule for P AOX1 -based systems, as other studies pointed at a positive correlation between gene copy number and mRNA level when increasing gene dosage2051. Strikingly, normalization of mRNA levels with the number of DNA copies of ROL (i.e.…”

Co-expressing GroEL–GroES, Ssa1–Sis1 and Bip–PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone

“…Bip can prevent the accumulation of errors in the peptide chain. PDI can identify disulfide bond to help the protein fold correctly, as supported by our previous observations [11]. And E. coli GroE (including chaperones GroEL and GroES) could form complexes with nonnative polypeptides to avoid intermolecular aggregation formation of newly synthesized proteins [12].…”

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

“…There have been some attempts at circumventing these constraints and enhancing production of various recombinant proteins. For instance, some authors have used increased gene dosages of target genes involved in product folding and secretion such as HAC1 , PDI1 and/or KAR2 (Bankefa et al , ; Guan et al , ; Liu et al , ; Yang et al , ). Other authors (Barrero et.al., ) have engineered the secretion signal in order to improve translocation into the endoplasmic reticulum (ER).…”