Clone of soybean asparagine synthetase B gene and its expression in E.coli
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.coli 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· L-1 IPTG for 14 h.The recombinant AS-B was purified by Ni-NTA affinity chromatography and Sephadex G50 chromatogrephy 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 μmol · min-1 · mg-1 using L-glutamine and NH4Cl as nitrogen donor,respecitively.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.
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