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根据GenBank登陆大豆天冬酰胺合成酶(AS-B)基因序列(登录号:GMU55874)设计特异引物,通过PCR扩增从大豆(Glycine max)cDNA中克隆出AS-B基因。将该基因克隆到pET30a(+)载体,构建重组表达载体pET30a-AS,转化大肠杆菌Rosetta(DE3)pLysS,并进行表达条件优化。结果表明,在16℃条件下,经0.5mmol·L-1IPTG诱导14 h,大部分重组AS-B以可溶性蛋白的形式存在。经Ni-NTA亲和层析和Sephadex G50脱盐后,重组蛋白产率为23.4 mg·L-1,纯度>90%,以L-谷氨酰胺和NH4Cl为氮源供体时,其比活力分别为2.32和2.60μmol·min-1.mg-1。研究结果为研究大豆AS-B结构与功能和酶催化动力学机制及建立AS-B抑制剂的高通量筛选平台奠定基础。
Specific primers were designed according to the ASB sequence (GenBank accession number: GMU55874), and AS-B gene was cloned from Glycine max cDNA by PCR. The gene was cloned into pET30a (+) vector, and the recombinant expression vector pET30a-AS was constructed. The recombinant plasmid was transformed into E. coli Rosetta (DE3) pLysS and the expression conditions were optimized. The results showed that most recombinant AS-B was soluble in the form of soluble protein induced by 0.5 mmol·L-1 IPTG at 16 ℃ for 14 h. After Ni-NTA affinity chromatography and Sephadex G50 desalting, the specific activity of the recombinant protein was 23.4 mg · L-1 with a purity of> 90%. When L-glutamine and NH4Cl were used as nitrogen sources, their specific activities were respectively 2.32 and 2.60 μmol · min-1.mg-1. The results lay the foundation for studying the structure and function of soybean AS-B and the mechanism of enzyme catalytic kinetics and establishing a high-throughput screening platform for AS-B inhibitors.