关键词: PurR；PurL；植物乳杆菌；细菌素；凝胶阻滞；生物膜干涉技术

Abstract: 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 puri?ed by Ni af?nity 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.

Key words: PurR; PurL; Lactobacillus plantarum; bacteriocin; gel retardation assay; biolayer interferometry