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Zhong X.,Hubei University | Zhong X.,Hubei Key Laboratory of Industrial Biotechnology | Zhai C.,Hubei University | Chen L.,Hubei University | And 7 more authors.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2013

Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression. © 2013 Chin J Biotech, All rights reserved. Source

Yu X.,Hubei University | Yu X.,Hubei Key Laboratory of Industrial Biotechnology | Yu X.,Hubei Collaborative Innovation Center for Green Transformation of Bio Resources | Wang X.,Hubei University | And 13 more authors.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2014

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6×His tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75℃ and pH 7.5|, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future. © 2014 Chin J Biotech, All rights reserved. Source

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