High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration |
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| Affiliation: | 1. Graduate School of Science and Engineering Research, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan;2. Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-8634, Japan;1. State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China;2. Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China;3. Tianjin Children''s Hospital, 300074 Tianjin, China;1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, China;2. School of Medicine and Life Sciences, Xinyu University, Xinyu, Jiangxi, 338004, China;1. Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, People''s Republic of China;2. National Biopesticide Engineering Research Center, Hubei Academy of Agricultural, Wuhan, Hubei Province 430064, People''s Republic of China |
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| Abstract: | A novel serine protease from Trichoderma koningii (SPTK) was synthesized and expressed in Pichia pastoris. The recombinant SPTK was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF), suggesting that SPTK belonged to the subgroup of serine proteases. The optimum pH and temperature for the recombinant SPTK reaction were 6.0 and 55 °C, respectively. SPTK performed a tolerance to most organic solvents and metal ions, and the addition of Triton X-100 exhibited an activation of SPTK up to 243% of its initial activity but SDS strongly inhibited. Moreover, our study showed that a portion of SPTK was N-glycosylated during fermentation. The activity and thermal stability of the recombinant SPTK were improved after the removal of glycosylation, and the N-glycosylation of SPTK could be efficiently removed through co-culture with P. pastoris strains expressing Endo-β-N-acetylglucosaminidase H. We constructed expression vectors harboring from one to four repeats of Sptk-expressing cassettes via an in vitro BioBrick assembly approach. And the result of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the genome of P. pastoris through a single recombination event. These strains were used to study the correlation between the gene copy number and the expression level of SPTK. The results of qPCR and enzyme activity assays indicated that the copy number variation of Sptk gene generally had a positive effect on the expression level of SPTK, while an increase in integration of target gene did not guarantee its high expression. The maximum yield and specific activity of SPTK in P. pastoris were obtained from the recombinant yeast strain harboring two-copy tandem Sptk-expressing cassettes, the yield reached 0.48 g/l after a 6-d induction using menthol in shake flasks and 3.2 g/l in high-density fermentation with specific activity of 5200 U/mg. In addition, the recombinant SPTK could efficiently degrade chicken feather and hydrolyzed the gelatin layer of photographic film. These properties made the recombinant SPTK a suitable candidate for industrial applications and for eliminating the pollution of keratin. |
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| Keywords: | Serine protease Biobrick assembly Multi-copy gene expression Feather degradation |
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