Gene prokaryotic expression and specific activity determination of β-glucan synthase KRE9 in Candida tropicalis

Objectiveβ-glucan synthase KRE9 of Candida tropicalis MYA-3404 was expressed and purified,and its specific activity was analyzed,in order to provide reference for the study on the structure and biological function of β-glu-can synthase KRE9.MethodThe DNA of C.tropicalis was extracted,gene KRE9 was amplified by PCR,and prokary-otic expression vector pET28a-KRE9 was constructed.Competent cell of Escherichia coli BL21(DE3)was transfered.Fu-sion protein KRE9 was obtained by induction of IPTG as well as histidine(His)tag purificationof resin affinity chromatog-raphy,and its specific activity was determined by 3,5-dinitrosalicylic acid(DNS)colorimetricmethod.ResultThe cloned gene KRE9 was 816 bp in total length,encoding 271 amino acids.The sequencing results showed that gene KRE9 was highly similar to reference genes(GenBank accession number XM_002547984),which reached 99.75%.The prokaryotic expression vector pET28a-KRE9 was transformed into competent cell of E.coli BL21(DE3).Then the recombinant pro-tein KRE9 was obtained by induction and purification.SDS-PAGE detection results showed that the recombinant proteins was 35 kDa with high purity.Western blotting identification indicated that there was specific binding between purified re-combinant protein KRE9 and His antibody.The specific activity of KRE9 was 9.169×104U/mg.ConclusionRecombi-nant protein vegetable proteinis of fine specificity and high β-glucan synthase activity,and it can be used for its structural and biological function research.