用蛋白质工程的方法改良枯草杆菌蛋白酶E(Subtilisin E)的抗氧化性

Improvement of Antioxidation Property of Subtilisin E by Protein Engineering Method

以含有蛋白酶E基因(aprE)的单链M13mp18-aprE DNA为模板,合成的寡核苷酸5′-3′为诱变引物,用缺口双链法对aprE进行Met-222-Ala点突变。经菌落印迹杂交筛选,选出阳性噬斑。用SaⅡ酶解M13mp18-aprE得到aprE,将它和pPZW103重组,转化中性、碱性蛋白酶缺失宿主菌DB104。经含卡那霉素和脱脂奶粉板筛选和比较aprE限制性内切酶NcoⅠ和SacⅡ水解电泳图谱分析,完成构建一个分泌抗氧化的枯草杆菌蛋白酶E的工程菌PW8888。

Single strand M13mp18-aprE (aprE as gene of subtilisinE) was hybridized with linear M13mp18 to form a gapped duplex DNA (gdDNA) associated withthe mutagenic primer AIa/(5'-CGGAACGTCCGCGGCGACTCC-3')/SacII.The primer hybridizedwith gdDNA was extended by DNA polymerase and ligated with M13mp18 by ligase to get aprE mutant M13mp18-aprE'.By in situ hybridization of the bacteriophage plaques with mutagenic primerprobe, the bacteriophage M13mp18-aprE' (Met-222-Ala site mutant in aprE) was screened.Comparison the hydrolyzed fragments of aprE' and aprE by SacII and NcoI on gel electrophorysis, showed that the construction of Met-222-Ala mutant of M13mp18-aprE' has been completed.AprE' was recombined with pPZW103 and transformed into DB104 to obtain PW8888 mutant, in from which the mutant subtilisinE with antioxidative property has been obtained.