Guo Wanhua, Cao Lin, Jia Zhijun, Wu Gang, Li Teng, Lu Fengxia, Lu Zhaoxin
Department of Nuclear Medicine, Nanjing University, Nanjing 210008, People's Republic of China.
Protein Expr Purif. 2011 Jun;77(2):185-92. doi: 10.1016/j.pep.2011.01.015. Epub 2011 Feb 1.
Ribonuclease inhibitor (RI) is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases which inhibits ribonucleolytic activity. Expression of recombinant RI is extremely difficult to reach high levels in soluble form in the cytoplasm of Escherichia coli. Here, we utilized five N-terminal fusion partners to improve the soluble expression of RI. Among these five fusion partners which have been screened, maltose-binding protein (MBP), N-utilization substance A (NusA) and translation initiation factor 2 domain I (IF2) have greatly improved the soluble expression level of recombinant murine RI under the drive of T7 promoter, while glutathione S-transferase (GST) and small ubiquitin modifying protein (SUMO) were much less efficient. All these RI-fusion proteins remained to be highly active in inhibiting RNase A activity. Furthermore, all fusion tags can be efficiently removed by enterokinase digestion to generate native RI which results the highest yield to date (>30mg of native RI per liter culture). And a convenient two-step immobilized metal affinity chromatography (IMAC) method has been implemented in our study, comparing with the traditional RNase A affinity chromatography method.
核糖核酸酶抑制剂（RI）是一种50 kDa的胞质型胰腺型核糖核酸酶清除剂，可抑制核糖核酸酶活性。重组RI在大肠杆菌细胞质中以可溶形式表达很难达到高水平。在此，我们利用了五个N端融合伴侣来提高RI的可溶性表达。在筛选出的这五个融合伴侣中，麦芽糖结合蛋白（MBP）、氮利用物质A（NusA）和翻译起始因子2结构域I（IF2）在T7启动子驱动下极大地提高了重组鼠RI的可溶性表达水平，而谷胱甘肽S-转移酶（GST）和小泛素修饰蛋白（SUMO）的效率则低得多。所有这些RI融合蛋白在抑制核糖核酸酶A活性方面仍保持高活性。此外，所有融合标签都可以通过肠激酶消化有效去除，从而产生天然RI，这是迄今为止产量最高的（每升培养物>30mg天然RI）。并且在我们的研究中实施了一种便捷的两步固定化金属亲和色谱（IMAC）方法，与传统的核糖核酸酶A亲和色谱方法进行了比较。
