链球菌表面三磷酸甘油醛脱氢酶及其突变体重组蛋白的表达和纯化

EXPRESSION AND PURIFICATION OF RECOMBINANT STREPTOCOCCAL SURFACE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND C-TERMINAL LYSINE RESIDUES-TRUNCATED VARIANT

表达并纯化M6型GAS表面三磷酸甘油醛脱氢酶以及其敲除C末端赖氨酸残基重组蛋白(rGAPDH和rGAPDHΔ345)。克隆了M6型GAS ATCC32175的GAPDH基因以及GAPDHΔ345基因,与pASK-IBA37载体连接后,表达蛋白并用亲和层析色谱纯化重组蛋白;对重组蛋白进行质谱检测,并用酶切方法进一步纯化目的蛋白,通过酶促反应实验测定了重组蛋白的生物活性。2种基因克隆条件稳定,蛋白表达量大,酶切后纯度高,纯化的重组蛋白具有较高的生物活性。功表达并纯化了rGAPDH和rGAPDHΔ345蛋白。

To express and purify the recombinant streptococcal surface glyceraldehyde-3-phosphate dehydrogenase（rGAPDH） and its C-terminal lysine residues-truncated variant（rGAPDHΔ345）.We cloned GAPDH and GAPDHΔ345 from M6-type GAS ATCC32175,produced rGAPDH and rGAPDHΔ345 in E.coli using the 6×Histag pASK-IBA37 expression vector and purified the recombinant proteins by affinity chromatography with TALON metal affinity resins.Mass spectrometric detection and then enzyme cutting for the recombinant proteins.The enzyme reaction was performed to determine enolase activity.PCR conditions ampifing GAPDH and GAPDHΔ345 were veridical and expression and purity after enzyme cutting of recombinant proteins were profuse.The purified rGAPDH and rGAPDHΔ345 were found to have relatively full enolase activity.We succusfully expressed and purified rGAPDH and rGAPDHΔ345.