Title: Cloning and expression of heat-resistant serine protease gene of Bacillus subtilis
Abstract:Objective To clone and express the heat-resistant serine protease gene of Bacillus subtilis and determine the enzymological property of expressed product.Methods The heat-resistant serine protease gen...Objective To clone and express the heat-resistant serine protease gene of Bacillus subtilis and determine the enzymological property of expressed product.Methods The heat-resistant serine protease gene Sapr of B.subtilis was amplified from chromosome DNA of B.subtilis by PCR and inserted into vector pET-28a(+).The constructed recombinant secretory expression vector pET-28a-Sapr was transformed to E.coli BL21(DE3)by heat shock,and induced with IPTG at 25 and 37℃.The expressed recombinant protein was analyzed by SDS-PAGE and determined for activity by Folin method.Results Both restriction analysis and PCR proved that recombinant plasmid pET-28a-Sapr was constructed correctly.Sequencing result proved that the homology of nucleotide sequence of target gene was 100% to that reported in GenBank.The optimal temperature for induction was 25 ℃.The expressed recombinant protein,with a relative molecular mass of about 39 600,mainly existed in a soluble form and contained about 12% of total somatic protein.The enzyme activity of fermentation product of recombinant E.coli was 322 U / ml.Conclusion The specific protein successfully expressed in BL21 / pET-Sapr showed biology activity,which laid a foundation of further study on function and property of modified protein isolate.Read More
Publication Year: 2013
Publication Date: 2013-01-01
Language: en
Type: article
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