| The plant peptide hormone PA1b /Leginsulin which constituted by 37 amino-acid included 3 disulfide bonds and its molecular weight is about 4 kD. It was paid much attention these years because it can accelerate the growth of the plant. Previous study revealed that bovine insulin, IGF- I or IGF-II is able to bind to Bg, which is the ligand of PA1b. Bg has autophosphorylation activity and protein kinase activity similar to insulin receptor. PA1b participates in signal transduction by activate the protein kinase activation when combines with Bg, so the relation between PA1b and Bg is similar to that of insulin and insulin receptor. NMR study showed that PA1b and insulin possess similar tertiary structure, which did Bg and insulin receptor also. So, we are interesting in the study whether PA1b can combine with insulin receptor and regulate the carbohydrate metabolism similar insulin.PA1b is one of peptide hormone and there is little in wild legume, so it is difficult to purify from wild legume. In this study, we designed its oligo-nucleotide fragment by Oligo 4.0, based on the amino-acid sequence of PA1b. The gene fragment of PA1b was synthesized and amplified by PCR. And then the product of PCR was cloned into prokaryotic expression vector pMAL-p2x. The recombinant expression plasmid pMAL-p2X-PA1b was transformed into E. coli DH5α. Then the conditions of inducement and espression were optimized, the fusion protein MBP-PA1b, was solubly expressed after 2h inducement at 30℃. The MBP-PA1b was produced and purified by Amylose Resin affinity column. The concentration of product was 28.6mg/L culture medium. analysis confirmed that The MBP-PA1b fusion protein was successfully expressed in E. coli.,which analyzed by SDS-PAGE and Western blot. Finally,MBP and PA1b was separated by the cleavage of Factor Xa. In this study we firstly produced PA1b by recombined gene which offered the foundation for further study of the function of PA1b if it can develop into new medicine for diabetes.
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