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Your Position :Home->Past Journals Catalog->2009年46 No.5

Cloning, expression and purification of a recombinant triosephosphate isomerase from Apis mellifera ligustica
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Key Words:triosephosphate isomerase, Apis mellifera ligustica, cloning, protein expression
Abstract:      Total RNA was isolated from the muscles of honeybee, and the cDNA sequence of triosephosphate isomerase gene was then cloned by RTPCR and sequenced (GenBank accession No. EU760983). The gene was 744 bp in length, encoding 247 amino acids. The calculated molecular weight of the mature TPI protein was about 26.89 kD. The similarity between the AmTPI nucleotide sequence and those of Bombyx mori, Blattella germanica, Tenebrio molitor, Nasonia vitripennis and Oryza sativa TPI genes was over 69%, but the similarity between their amino acid sequences was over 59%. The target gene was cloned to the pGEX-4T-2 vector GST fusion expression system. The results indicated that the fusion protein was expressed successfully in the E. coli BL21 (DE3) cells and the amount of the expressed protein accounted for 42.1% of the total proteins after 4 h induction. The recombinant fusion TPI protein was purified and concentrated to test its enzymatic properties. The brighter fluorescence recombinant EGFP vector is constructed for further study of expression in mammalian cells.
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