Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.

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Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode

Wang

Liang

Liu

et al. 2017

Journal of Asian Natural Products Research

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Purification of Recombinant Mouse C-Reactive Protein fromPichia PastorisGS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography andP-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

Bin

et al. 2021

Journal of Chromatographic Science

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C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

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Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

Cited by 2 publications

References 8 publications

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode

Purification of Recombinant Mouse C-Reactive Protein fromPichia PastorisGS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography andP-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

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