Abstract
BackgroundAlthough Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris.ResultsThe results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.ConclusionsThese results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0315-4) contains supplementary material, which is available to authorized users.
Highlights
Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies
Evaluation of single factors on the secretion in P. pastoris To investigate the effects of protein N-terminal sequence, endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and acidic stability of the protein on the secretory expression of methyl parathion hydrolase (MPH) from P. pastoris, pPIC9-based yeast expression constructs were generated
One hundred His+ transformants from the wild type and each mutant were analyzed for MPH production and the secretion using the standard enzyme assay
Summary
Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. The methylotrophic Pichia pastoris has been used extensively and successfully for the secretion of expressed recombinant proteins, because of its high expression level, stability, heredity, and mature fermentation process [1, 2]. Strain engineering by genetic modification has become the most useful and effective method to overcome the drawbacks of yeast secretion pathways [17]. These methods can effectively enhance the expression of some proteins, the efficiency is variable and it is difficult for some foreign proteins to achieve optimal secretory expression in P. pastoris. We hypothesized that some factors affecting secretion exist in the internal regions of proteins [18]
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