核苷二磷酸激酶A的定点突变及C4S突变体的制备和活性研究

Point Mutation of Nucleoside Diphosphate Kinase A and Preparation and Bioactivity Determination of its C4S Mutant

目的:对核苷二磷酸激酶A(NDPK-A)二硫键异构的关键残基C4进行定点突变,构建、表达并纯化C4S突变体,测定其磷酸转移酶活性和DNase活性,研究二硫键异构对NDPK-A活性的影响。方法:以pBV220-nm23-H1质粒作为模板,通过设计合适的引物对NDPK-A进行定点突变,将第4位半胱氨酸突变为丝氨酸,构建NDPK-AC4S突变体;在大肠杆菌BL21中表达,DEAE-sepharose Fas tFlow与Cibacron Blue 3GA Sepharose CL-4B纯化目的蛋白,获得均一重组蛋白,纯度达到98%;DNA序列测定及重组蛋白的肽质量指纹图谱(PMF)分析均证明构建正确突变体;高效液相色谱法(HPLC)与DNA消化法分别测定野生型NDPK-A与C4S突变体的磷酸转移酶活性与DNase活性差异。结果:NDPK-AC4S突变体的磷酸基转移酶与DNase酶活性均高于野生型NDPK-A。结论:NDPK-A缺失二硫键后,活性增高。NDPK-A形成链内二硫键可能是其活性负调控模式之一。

Objective： To construct nucleoside diphosphate kinase A mutant （NDPK-A C4S）, and detect its phosphotransferase activity and DNase activity to study the influence of the disulfide bond on the bioactivities of NDPK-A. Methods： A synthetic oligonucleotide primer for site-directed mutagenesis was designed with the template pBV220-nm23-H1, where the codon for the residue cysteine-4 was replaced with that of serine. Expressed by BL21 host cell and purified by DEAE-sepharose Fast Flow and Cibacron Blue 3GA Sepharose CL-4B, the homogeneous recombination protein was obtained with the purity of 98%. The DNA sequencing and MALDI-TOF mass spectrography analysis demonstrated the success of constructed NDPK-A C4S mutant. The diversities of the phosphotransferase activity and DNase activity from NDPK-A C4S mutant and wild type NDPKA were measured by reverse high performance liquid chromatography （HPLC） and DNA cleavage （DNase） analysis respectively. Results： After site-specifically mutating the critical residue cysteine-4 contributing to the disulfide bond isomerism, not only the phosphotransferase activity but also the DNase activity of NDPK-A C4S are higher than that of wild type NDPK-A. Conclusion： Forming the intrachain disulfide bond in NDPK-A is possibly one of the negative functional regulation modes, which put a solid foundation for the research of NDPKA’s bioactivity, and it will be an alternative practical application for the more active function of NDPK-A C4S.