title: High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.
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.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Hu
authors.initials: H
authors.lastname: Yang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhai
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China; Institute of Molecular Medicine, Peking University, Beijing 100871, People's Republic of China.
authors.forename: Xianhong
authors.initials: X
authors.lastname: Yu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Zhezhe
authors.initials: Z
authors.lastname: Li
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Bioengineering, Zhixing College of Hubei University, Wuhan, People's Republic of China.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhong
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Guolong
authors.initials: G
authors.lastname: Li
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China. Electronic address: malixing@hubu.edu.cn.
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
mesh_terms: D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D019286:Endopeptidase K / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005285:Fermentation; D018628:Gene Dosage; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D011994:Recombinant Proteins; D019286:Endopeptidase K
keywords: Multi-copy expression; Pichia pastoris; Proteinase K