长双歧杆菌NCC2705 ATP结合蛋白BL0034表达与活性检测

Expression and purification of the ATP banding protein BL0034 of ABC transporter system for fructose from Bifidobacterium longum NCC2705

目的克隆长双歧杆菌NCC2705中ATP结合蛋白BL0034的基因,利用大肠杆菌表达并纯化GST-BL0034融合蛋白,测定其结合ATP的活性。方法以长双歧杆菌NCC2705基因组为模板PCR扩增BL0034基因,双酶切后连接到pGEX-4T-1表达载体上,转入大肠杆菌BL21中表达;用谷胱甘肽-Sepharose 4B树脂对可溶性的GST-BL0034融合蛋白进行纯化,并用Western印迹验证。结果将BL0034基因克隆至质粒pGEX-4T-1,构建表达载体pGEX-4T-1-BL0034。BL0034经0.05 mmoL/L IPTG诱导,16℃过夜表达,SDS-PAGE可见相对分子质量约为82×103的可溶性表达条带;亲和纯化GST-BL0034,Western印迹结果为阳性。对纯化后的融合BL0033蛋白结合ATP的能力进行了定量测定,每毫克BL0034蛋白能结合约40 nmol/L ATP。结论成功克隆了BL0034蛋白的基因,表达并纯化GST-BL0034蛋白,证实了BL0034与ATP具有较强的结合能力,为进一步研究长双歧杆菌NCC2705 BL0034蛋白的功能奠定了基础,为阐明其在果糖ABC转运系统中如何通过水解ATP产生能量提供了前提。

Objective To clone BL0034 of Bifidobacterium longum NCC2705,express and purify GST-BL0034 fusion protein in E.coli BL21,then to detect its ATP binding activity with purified protein.Methods BL0034 was amplified from the genomic DNA of B.longum NCC2705 by PCR.The product was cloned into the expression vector pGEX-4T-1 following enzyme digestion.After being sequenced,the positive recombinant plasmid constructed was transferred to BL21 and induced to express the fusion protein.The expression condition was optimized.The fusion protein GST-BL0034 was detected by SDS-PAGE and purified by GST-sepharose 4B beads.The purification was detected by Western blot.Results SDS-PAGE analysis revealed a specific and soluble expressing protein band with a relative molecular mass of nearly 82×103 with IPTG 0.05 mmoL/L at 16℃ over night.With affinity beads,the high purity GST-BL0034 was achieved.The function of binding ATP was demonstrated by the detection of the purified protein binding ATP activity quantitatively.Conclusion The successful expression of fused protein GST-BL0034 and binding assay can contribute to further functional studies on B.longum NCC2705,shedding light on how BL0034 supplies energy by hydrolysis ATP in fructose ABC transport system.