多聚磷酸盐激酶的原核表达、纯化及酶活性测定

Prokaryotic expression,purification and enzyme activity identification of polyphosphate kinase

目的原核表达、纯化多聚磷酸盐激酶(polyphosphate kinase,PPK),并检测其酶活性。方法采用PCR法从尿路致病性E.coli CFT073中扩增ppk基因,克隆至原核表达载体pET-28a中,构建重组表达质粒pET-28a-ppk,转化感受态E.coli BL21(DE3),IPTG诱导表达。重组蛋白经His-镍亲和层析柱分离纯化后,采用4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)染色法测定纯化蛋白的酶活性。结果重组表达质粒pET-28a-ppk经双酶切及测序鉴定,证明构建正确。重组蛋白的相对分子质量约80000,纯化后蛋白浓度为518.9μg/mL。在加入10μg PPK酶,37℃孵育30 min的条件下测得的PPK酶活性最大,为(645.16±32.98)U/mg。结论成功在E.coli中表达了重组PPK,纯化产物纯度较高,具有PPK酶活性,为后续以PPK为靶点新型抗菌药物的研发奠定了基础。

Objective To express polyphosphate kinase(PPK)in prokaryotic cells,purify the expressed product and determine its enzyme activity.Methods The ppk gene was amplified from uropathogenic E.coli CFT073 by PCR and cloned into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-ppk was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by His-nickel ion affinity chromatography and determined for enzyme activity by using DAPI(4',6-diamidino-2-phenylindole)staining.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a-ppk was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 80000,reached a concentration of 518.9μg/mL.After 10μg of PPK was added into catalytic system which was incubated at 37℃for 30 min,the measured enzyme activity reached a maximum of(645.16±32.98)U/mg.Conclusion Recombinant PPK was successfully expressed in E.coli,which reached a high purity after purification and showed PPK enzyme activity.It laid a foundation of subsequent development of new antibacterial drugs targeting PPK.