Studies On Cloning,Site-directed Mutagenesis And Expression Of CDNA Encoding Melittin And Promelittin In Escherichia Coli

Melitttn, a main polypeptide component in the bee poison, is of great importance to medical science. It not only possesses the parmacalogical activity of anti- inflammation, anti-arthritis, anti-bacterial, hut also the toxicologic effect to tumor cells. It also can pies ent and treat radiation sickness, therefore enhance the survival rate of the radiated animals. Moreover, melittin has strong surface activity, it can pass through the lecithin's and the mixed lipid membranes much faster than any other surface-active agents This character has made it an effective tool for bio-membrane research So far, melittin has been mainly obtained by separating and extracting from bee venom or by chemical synthesis. The spreading of melittin was limited because of the following two reasons first, the purification difficulty caused by the phospholipase A2, second, the higher cost of protein chemical synthesis The development of biotechnology provides a new way for melittin production. So in this study the cDNA encoding melittin and its pre-protein were cloned and modified by molecular bio- technique, the basic expression system was established in E. coli. In the work, it was found that 1-18 days of work bees can produce more melittin, so bees at this age were selected as test materials. First, a simple and effective modit5ed uanidine isothiocyanate method was established to extract RNA from poison gland. Then cDNA encoding melittin was cloned, and the initiation codon ATG which is necessary to the initiation of protein translation was added to the cloned sequence, and the eDNA could be expressed to produce melittin under the control of a suitable prornotor The melittin directly expressed in procaryotic or eucaryotic expression systems is deadly to the cells, because it can destroy the bio-membrane and cause the leak of endocvte This has been hindering the melittin production by genetic engineering. Promelittin is the natural fusion protein of melittin. It was supposed theoretically that promelittin was innoxious to the expression cells. This presumption has been proved by experiment In this study, the cDNA encoding promelittin was cloned, and its vector expressed in F ccli was constructed. This proved that promelittin with partial sequence ol P-gdIactoiddse was ilOfl-tetIldl w tile expression cells. After proving the fact, it was possible to express and produce melittin in the tbrrn of fusion protein in the energetic bacteria. In order to obtain the objective protein, the supporter protein has to be excised from the fusion protein For this purpose, the melittin cDNA obtained by cloning was site-directed mutagenized, AAT codon encoding asparamide was arranged before the mehttin sequence, and the mutational protein was expressed in E coli. The induced asparamide residue and the first residueof melittin constitUted the hydroxylamine cleavage site of the ndational prednencoded by the cDN^ and the fiJsion protein could be proceed intO actiVe melittin bythe hydroxylamine cleavne modification.