植物乳杆菌KLDS1.0391 PurR和PurL蛋白的原核表达、纯化及其与细菌素合成启动子的相互作用

通过体外实验探究purR、purL基因对植物乳杆菌（Lactobacillus plantarum）KLDS1.0391细菌素合成是否具有直接调控作用。基于植物乳杆菌KLDS1.0391全基因组序列，通过聚合酶链式反应扩增得到PurR和PurL蛋白的编码基因，构建原核表达载体，将重组质粒导入大肠杆菌M15中进行诱导表达，采用镍柱亲和层析法纯化得到目的蛋白，透析后超滤浓缩，通过凝胶阻滞实验以及生物膜干涉技术研究两种蛋白与菌株细菌素合成调控区域是否具有结合作用。结果表明，PurR蛋白以可溶性蛋白形式存在，PurL蛋白以包涵体蛋白形式存在，纯化后经聚丙烯酰胺凝胶电泳显示条带单一，达到电泳纯，经过浓缩后PurR蛋白质量浓度为0.74 mg/mL，PurL蛋白质量浓度为3.8 mg/mL。凝胶阻滞实验以及生物膜干涉技术结果表明，2 个重组蛋白与菌株细菌素合成基因的启动子在菌体外无直接的结合作用，对于两种蛋白对细菌素合成的调控是否属于间接调控作用以及在菌体内的调控情况，需进一步研究。

Prokaryotic Expression and Purification of PurR and PurL from Lactobacillus plantarum KLDS1.0391 and Their Interaction with Bacteriocin Synthesis Promoter

The purpose of this study was to investigate whether purR and purL directly regulate the synthesis of bacteriocin by Lactobacillus plantarum KLDS1.0391 through in vitro experiments.Based on the whole genome sequence of L.plantarum KLDS1.0391,we obtained the genes encoding PurR and PurL proteins by PCR amplification.The genes were separately cloned into the pQE-30 vector to construct recombinant expression vectors.Then the recombinant vectors were transformed into Escherichia coli M15 for protein expression.The expressed proteins were purified by Ni affinity chromatography.After dialysis and ultrafiltration concentration,we studied whether the two proteins bind to the bacteriocin synthesis regulatory region of the strain by using the gel retardation assay and biolayer interferometry.The results showed that the recombinant proteins were successfully expressed,PurR was present in a soluble form while PurL was present as an inclusion body.The purified proteins showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).After being concentrated,the PurR concentration was 0.74 mg/mL and the PurL concentration was 3.8 mg/mL.The results of gel retardation assay and biolayer interferometry showed that the two recombinant proteins did not directly bind to the promoter of the bacteriocin synthesis gene of the strain.Further studies are needed to clarify whether and how bacteriocin synthesis is indirectly regulated by the two proteins.