大豆天冬酰胺合成酶B基因的克隆及在大肠杆菌中的表达

Clone of soybean asparagine synthetase B gene and its expression in E.coli

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。

According to the sequence of asparagine synthetase （AS-B） gene in soybean （GenBank accession number： GMU55874）, a pair of specific primers was designed to obtain the AS-B gene from Glycine max by PCR amplification using cDNA as template. The gene of AS-B was then cloned into prokaryotic expression vector pET30a （＋）, constructing a recombinant expression vector pET30a-AS, which was introduced into E. co//Rosetta （DE3） pLysS. After the optimization of induced conditions, recombinant AS-B was expressed in a soluble form when the host harboring pET30a-AS was cultured at 16 ℃ and induced by 0.5 mmol. L1 IPTG for 14 h. The recombinant AS-B was purified by Ni-NTA affinity chromatography and Sephadex G50 chromatography to give a yield of 23.4 mg. L-1 and a purity of 〉90%. The specific activity of recombinant AS-B was measured to be 2.32 and 2.60 IJmol . min^-1. mg^-1 using L-glutamine and NH4CI as nitrogen donor, respectively. These results were believed to pave the way for the investigation of structure, function and kinetic mechanism of AS-B, as well as the development of high throughput screening platform for AS-B inhibitors.