β-内酰胺酶E166C突变型的原核表达、纯化及鉴定  

Protein Expression,Purification and Identification ofβ-Lactamase Pen P-E166C Wildtype and Mutants

作　　者：肖美芳[1] 王昌富[1] 周义正[1] 邱晓燕[1] 

机构地区：[1]华中科技大学附属荆州医院,湖北荆州434020

出　　处：《四川兵工学报》2015年第6期153-156,160,共5页Journal of Sichuan Ordnance

摘　　要：目的本研究旨在构建β-内酰胺酶E166C突变体并于大肠杆菌中原核表达,纯化及鉴定该蛋白。方法利用体外PCR扩增获得β-内酰胺酶pen P突变质粒并通过DNA测序鉴定,将重组质粒转化至BL21(DE3)中,以IPTG诱导蛋白表达。菌液超声裂解后首先以镍柱纯化融合蛋白,蛋白水解酶HRV3C除去融合标签,然后用凝胶过滤层析色谱进一步纯化得到目标蛋白,最后应用SDS-PAGE和电喷雾质谱法鉴定纯化的目标蛋白。结果成功构建了β-内酰胺酶pen P-E166C突变质粒,并且先后通过亲和镍柱和凝胶过滤层析柱纯化获得了纯度非常高的单一蛋白,SDSPAGE和电喷雾质谱法确定蛋白的分子量与理论值一致。结论本课题获得的pen P的活性位点突变体E166C蛋白将用于以后进一步的研究,可以有助于我们从分子水平上去研究抗生素和β-内酰胺酶的相互作用。Objective This study was aimed to construct β-lactamase penP-E166C mutant and over-express it in E. coli, followed by purification and characterization. Method The plasmid of mutated β-lactamase penP was successfully amplified by PCR-mediated mutagenesis, which was then confirmed by DNA se- quencing. The confirmed plasmids were firstly transformed into competent BL21 （DE3 ） cells and the posi- tive colonies were selected by antibiotics kanamycin, which were then induced by the addition of IPTG. The bacterial cells were firstly liaised by sonication, followed by Ni2＋ -affinity column purification, protease 3C digestion and gel filtration column purification to obtain target protein. The proteins were characterized by SDS-PAGE and ESI-mass spectrometry. Results We successively constructed penP-E166C mutant and purified this protein by Ni2＋ -affinity column and gel filtration column. SDS-PAGE and ESI-mass spectrom- etry further identified the protein molecular weight was consistent with theoretical value. Conclusion The obtained penP-E166C protein with mutation at the active site will be used in later study, which will help us to understand the interaction between antibiotics and β-1aetamase better.

关 键 词：β-内酰胺酶E166C突变体 PCR-介导的定点突变 蛋白纯化及鉴定 

分 类 号：R961[医药卫生—药理学]