The Fusion Expression Of Melittin And Its Effect On Centrosome Duplication In Cells

Melittin is a small linear peptide composed of26amino acids residues. Melittin has multiple effects that include anti-bacterial, anti-inflammatory, anti-radiation, anti-tumor and anti-AIDS. Many studies have shown that melittin has a considerable influence on the nervous, digestive and endocrine system, too. In recent years, the study of its function and mechanism in anti-tumor has become the hot research field.Due to the hemolytic characteristic of melittin, it is relatively difficult to acquire melittin with high activity. In this study, the fusion protein of melittin and GST were expressed in E.coli. Melittin gene was amplified and inserted into the prokaryotic expression vector pGEX-4T-2, the recombinant expression vector of pGEX-4T-2-mel has been constructed successfully. The human enterokinase cleavage site has been designed between melittin gene and GST-tag gene. Fusion protein GST-Melittin has overexpression at30℃, with IPTG (0.2mmol/L) induced for5h in soluble form. GST affinity chromatography was amplified to purify the target protein and fusion protein GST-Melittin protein was obtained with a purity of more than97%. Fusion protein GST-Melittin was digested by human enterokinase and the GST was removed by ultrafiltration concentration, then through desalination and vacuum freeze-dry, we acquired about0.5mg Melittin powder from1L fermentation broth. Through Oxford cup method, we found melittin could significantly inhibit the growth of white rot fung SQ01.Recent studies have shown that melittin can induce cell cycle arrest, growth inhibition, and apoptosis in various tumor cells. The strong interaction of melittin and centrin in vitro has also been demonstrated. In this study, IC50of melittin on Hela cells was acquired by the MTT assay, and add melittin to Hela cells with IC50, deal with0,24,48and72h, respectively. Immunofluorescent analysis using a-tubulin and y-tubulin antibody was applied to observe the changes of the number of centrioles in Hela cells by the Delta Vision. The results showed that with the effect of melittin, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Abnormal cell division results in daughter cells containing either one or no centrioles at all. Cells are failed to undergo cytokinesis in subsequent cell cycles, and finally die. This study demonstrates that flowing the treatment of melittin, the quantity of cells showing two centrioles has a significant reduction; however the quantities of cells with one or no centriole have gradually increased in a strict time-dependent manner. It completely shows that melittin could kill tumor by inhibiting centrosome duplication.There are many studies reported that enterokinase, such as bovine, mouse enterokinase, have been main cleavage enzyme to express and purify the fusion protein. In this study, the fragment of human enterokinase light chain (hEKL) gene was amplified by PCR and cloned into plasmid pMAL-s downstream to the gene of fusion partner MBP-tag. The recombinant plasmid pMAL-s-hEK1. was transformed into E. coli BL21(DE3). Fusion protein MBP-hEKL has overexpression at37℃, with IPTG (0.2mmol/L) induced for5h in soluble form. After induced expression, Amylose affinity chromatogra-phy was amplified to purify the target protein.40mg fusion protein was obtained with a purity of more than97%from1L fermentation broth. Activity analysis showed that MBP-hEKL could cleavage the fusion protein with enterokinase recognition site specially and the specific activity was reached to6.0×1O5U/mmol.