Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2020264 | Protein Expression and Purification | 2016 | 7 Pages |
Abstract
Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.
Keywords
qPCRSDSTCABMMYBMGYBSMUPRcycle thresholdtrichloroacetic acidsodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGEdissolved oxygenbuffered methanol-complex mediumminimal dextrose mediumsodium dodecyl sulfateGAPbuffered glycerol-complex mediumquantitative polymerase chain reactionUnfolded protein responseProteinase KPichia pastorisglyceraldehydes-3-phosphate dehydrogenase
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Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Hu Yang, Chao Zhai, Xianhong Yu, Zhezhe Li, Wei Tang, Yunyun Liu, Xiaojian Ma, Xing Zhong, Guolong Li, Di Wu, Lixin Ma,