Research On Cloning And Expression Of The Bacteriocin Abc Transpoter Gene From Lactobacillus Plantarum ZJ316

ATP-binding cassette (ABC) transporter proteins are one of the largest protein superfamilies and are present in all organisms from bacteria to human beings. Each member contains two highly conserved ATP-binding domain (ATP binding cassette), They can utilize the energy derived from ATP dimerization hydrolysis to drive substrate translocation across the transmembrane channel. Transhipment of substrates include:inorganic ion, inorganic acids, amino acids, lipids, carbohydrates, peptides, various types of drugs, cellular metabolites. Some ABC transporter protein transport substrates with the high specificity, but the others can transport substances with completely different structure. In this paper, We started from the bacteriocin transporter gene sequence of Lactobacillus plantarum ZJ316, predicted the biological function of the protein PlnH, A obtained expressed protein in the exogenous expression strain, and optimized the conditions of producing exogenous protein. Which will result in a better understanding of the functional mechanism of PlnH, and provide a new way of thinking for the future research and microbial utilization.The research started from the Lactobacillus plantarum ZJ316which produced bacteriocins highly efficiently and separated from infant faeces and saved in our laboratory. We used the modified CTAB method to extract the total genome of Lactobacillus plantarum ZJ316. Taken through the NCBI database and software Primer Premier5.0, We designed the primer of gene plnH, the bacteriocin ABC transporter gene, and cloned it in suitable conditions and reaction systems. The gene sequence composes of1377bp and have a homology of99%with the plnH gene published on the NCBI database, encoding458amino acid residues with the predicted molecular weight of50.0kDa and isoelectric point of9.44, and it is a stable protein. The secondary structure of protein PlnH is about51.97%of the α-helix,11.14%of the β-sheet,36.90%of the random coil; The strong hydrophobicity exists in the first100amino acids of the PlnH protein, and the hydrophobicity and hydrophilicity arranged alternately in the subsequent part; A transmembrane domain presences from the19th AA to the41th AA, with two low-complexity sequences and one coiled-coil region, there may be a signal peptide; There are five N-glycosylation sites, nine protein kinase C phosphorylation sites,11casein kinase Ⅱ phosphorylation sites, one cAMP and cGMP-dependent protein kinase phosphorylation site and three N-myristic sites. To obtain PlnH protein expressed in vitro experiment, We constructed recombinant plasmid pET28a-p/nH with efficient restriction endonuclease sites BamH I and Xho I. The correctness of the recombinant plasmid was verified by colony PCR, single and double digestion of plasmid and gene sequencing. The ABC transporter protein PlnH was expressed in the E.coli system though18℃overnight induction and SDS-PAGE electrophoresis. The induced expression Of gene plnH show that the optimal conditions are temperature of30℃,IPTG concentration of1.0mmol/L, OD600=0.7, time of induced expression for4h. and the protein PlnH exists in the form of inclusion bodies. The result of purification of recombination protein from PlnH by Ni2+affinity chromatography sugguested that a clear single-purpose strips emerged in elution buffer with500mM imidazole, but the refolding yield was zero.