重组精氨酸脱亚胺酶的制备工艺

Study of Preparation Technology of Recombinant Arginine Deiminase

研究了重组精氨酸脱亚胺酶(r ADIES)的30 L规模发酵、制备工艺及其生物学活性。将构建好的工程菌先进行摇瓶活化获得种子液,转入30 L发酵罐进行培养,用IPTG进行诱导表达;表达产物进行高压匀浆破菌、包涵体洗涤、溶解稀释复性后经DEAE阴离子交换层析柱和Butyl疏水层析柱纯化;通过SDS-PAGE和RP-HPLC等方法检测表达和纯度;用体外测活法检测活性。将工程菌进行放大培养并诱导表达,获得了相对分子质量在46 k Da的r ADIES。重组蛋白表达量占总蛋白的25%以上,经制备工艺获得的r ADIES纯度高于95%,酶活性为42IU。实验结果表明:该工艺具有可行性,可以获得较高活性的重组精氨酸脱亚胺酶。

The 30 L scale fermentation, preparation technology and biological activities of recombinant arginine deiminase （rADI-ES） were investigated. The seed solution was obtained by shaking flask activation of the constructed engineering bacteria, and then transferred it to 30 L fermenter for enlarging cultivation, and IFFG was used for its induction expression. The expression products broke bacterium by high-pressure homogenizer, and inclusion bodies were washed by buffer, and after dissolved refolding, the product was purified with DEAE anion exchange chromatography and Butyl hydrophobic interaction chromatography column, and we detected its expression and purity by SDS-PAGE and RP-HPLC analysis. The activity of rADI-ES was determined in vitro. We successfully amplified and induced expression of the engineering bacteria, and obtained the rADIES with the molecular weight of 46 kDa. The expression quantity of recombinant protein counts about more than 25 % total protein, and the purity of the purified rADI-ES is more than 95 % and its enzymatic activity is 42 IU. The expeiment show that the process is feasible to obtain a higher purity and activity of recombinant arginine deiminase.