Prokaryotic Expression And Characterization Of BANLEC1 In Escherichia Coli

Banana Lectin (BanLec) is a plant Lectin, a member of the mJRLs(mannosespecific jacalin-related Lectins)group from the jacalin-related Lectin family. In view of the structure of Banlec, it is a dimeric plant Lectin composed of two identical subunits showing specificity toward mannose or glucose. In view of the function of BanLec, BanLec exhibits some extraordinary carbohydrate binding properties, which is the sign of the difference with other plant protein. The specific sugar binding site decide to bind with sugar receptor on cell surface, which can precipitate and agglutinate cells. With the deeply research on the function of plant Lectins, we found that plant Lectins had many biological functions, such as storage nutrition, plant defence and biological nitrogen fixation. Therefore, the research of plant Lectins is not only in the aspect of basic theory research, also it is a rising attention for researchers in medicine research and agriculture. Based on these findings, banana Lectin could specifically bind to HIV-1 glycosylated envelope protein gp120, so as to prevent HIV infection on cell. This experiment cloned the BanLecl gene and induced the rBanLecl protein. And analyzed that rBanLecl protein had sugar inhibition, stability, bacteriostatic, preventing Tobacco mosaic virus (TMV) infection of tobacco.Using bioinformatics method to analysis, the BanLecl gene has 426bp, encoding 141 amino acids, BanLec protein protomer molecular weight is 15KD. This experiment adopts mature banana pulp as experimental materials, the BanLecl gene was amplified by RT-PCR method, then BanLecl gene was cloned into expression vector pETGST. The recombinant expression vector pETGST-BanLecl was transformed into E. coli C43, expression is induced by the isopropyl-β-D-thiogalactoside (IPTG) and detected by SDS-PAGE electrophoresis. The fusion protein rBanLecl was purified by High Affinity Ni-NTA resin and removed the maltose binding protein label (MBP) to get BanLecl protein with biological activity.Prokaryotic expression of the fusion rBanLecl protein had the agglutination of dog erythrocyte, and had no the agglutination of rabbit erythrocyte. Digested the MBP label from the rBanLec1 protein, the BanLec1 protein and the banana extracts had strong agglutination of rabbit and dog erythrocyte.Sugar inhibition experiments showed that the agglutination of rabbit erythroeytes could be inhibited by D-maltose, glucose, mannose and D-fructose in different level and sucrose, D-galactose, lactose could not inhibit agglutination activity in different level. This indicates that rBanLecl protein belongs to specificity toward mannose or glucose. rBanLec1 protein was incubated under different temperature for 20 minutes, agglutination activity remained almost unchanged under 20℃, 40℃, agglutination activity remained 40% under 60℃, most loss of agglutination activity under 70℃,80℃ and 90℃. The agglutination activity gradually was losed less than pH 1.4 and was stable in a broad pH range from 4 to 12, which indicated that the rBanLecl protein showed stability under the heat and alkali conditions, instability under the strong acid, which is similar to the majority of other Lectins.The fusion rBanLecl protein has no inhibition to the growth of Vibrio Parahemolyticus, Staphyloccocu saureus, Escherichia coli, Bacillus subtilis, Xanthomonas oryzae, Sclerotinia sclerotiorum, which indicated that the fusion rBanLec1 protein showed no inhibition to the growth of bacteria and fungi. TMV, a common plant viruses, is consisted of RNA and protein capsid. We have a mixture of TMV and BanLecl protein to infect tobacco, the experiment result shows that BanLecl protein can effectively prevent TMV infecting tobacco.