番茄(Solanum lycopersicum)果胶酸裂解酶P56在大肠杆菌中的重组表达

果胶酸裂解酶P56在番茄花粉管伸长过程中起着重要的作用,为了制备番茄P56蛋白的抗体,进行番茄花粉管萌发过程中P56蛋白的免疫组织化学研究,对P56基因在大肠杆菌系统的重组表达进行了研究。先采用Overlap-PCR的方法,从番茄基因组DNA中克隆了成熟P56蛋白的cDNA序列(LAT56),再构建重组表达质粒pET28a(+)-LAT56,转化大肠杆菌BL21-CodenPlus(DE3)-RIL,得到了重组表达工程菌pET-28a(+)-LAT56-BL21-Co-denPlus(DE3)-RIL。在0.5 mmol/L IPTG、15℃和180 r/min条件下,经过60 h的诱导培养,重组蛋白表达量为细胞总蛋白的30%左右,主要以包涵体形式存在,重组蛋白经Ni²⁺-nitrilotriacetate-agrose亲和柱层析,得到了SDS-PAGE显示为单一蛋白带的纯化蛋白。

Expression of P56 Pectate Lyase from Solanum lycopersicum in Escherichia coli

P56 pectate lyase play an important role during the extension of pollen tube.In order to obtain the antibody of P56 for the study of action machnism and distribution of P56 along pollen tube,the recombinant expression of P56 was carried out in Escherichia coli.The cDNA coding mature P56 without signal peptide was amplified from Solanum lycopersicum by Overlap-PCR.The amplified fragment was ligated to pET28a(+)expression vector,then transformed into Escherichia coli BL21-CodenPlus(DE3)-RIL.The expression of P56 was applied at 0.5 mmol/L IPTG,15℃ and 180 r/min for 60 h.The recombinant protein reached about 30% of total cell proteins and existed as inclusion body in pET-28a(+)-LAT56-BL21-CodenPlus(DE3)-RIL cell.Through Ni²⁺-nitrilotriacetate-agrose affinity chromatography,the recombinant P56 was purified to homology based on SDS-PAGE.