Cloning, Sequence Analysis, Prokaryotic Expression And Purification Of A Recombination Triosephosphate Isomerase From Apis Mellifera Ligustica

Honeybee is not only an economic insect with agelong breeding history but also a model animal with great value in theoretical research field. The homodimeric enzyme triosephosphate isomerase, which has an important role in glycolysis, gluconeogenesis, fatty acid synthesis, and the pentose shunt, converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate. TPI has been found in nearly every organism searched for the enzyme, including animals such as mammals and insects as well as in fungi, plants and bacteria. In order to study this enzyme at the molecular level, total RNA was isolated from muscles of honeybee, and then the cDNA sequence of triosephosphate isomerase gene was cloned by RT-PCR with specific primers. The cloning was confirmed for cleavage with restriction enzymes, PCR and DNA sequencing (GenBank accession number is EU760983). To analyze and predict the physical and chemical characteristics of Apis mellifera ligustica triosephosphate isomerase (AMTPI), several intergrated bioinformatics databases and local programs were used frequently. AMTPI is 744 bp in length with an open reading frame encoding a 247 amino acid protein. The alignment analysis showed high positional identities in several conserved regions. TPI is a useful model for studying the enzyme degradation and a credible phylogenetic tree was constructed based on ME method in MEGA 4. The results provide an important theoretical reference for further study of TPI enzyme characteristics. The similarity of AMTPI nucleotide sequence was over 69% compared with Bombyx mori, Blattella germanica, Tenebrio molitor, Nasonia vitripennis and Oryza sativa TPI genes, and of amino acids was over 59%. E.coli BL21 (DE3) was transformed with pGEX-4T-2 vector for expression of GST fusion recombinant protein. It indicated that the fusion protein was expressed successfully and amount to 42.1% of the total proteins after 4 hours induction. The recombinant fusion TPI protein was purified and concentrated to test its following enzymatic properties. The brighter fluorescence recombinant EGFP vector is constructed for the further study of the expression in mammalian cells.