Southworth M W, Fuhrman J A, Robbins P W, Beauregard K, Perler F B
New England Biolabs, Inc., Beverly, MA 01915, USA.
Gene. 1996 Oct 24;177(1-2):55-8. doi: 10.1016/0378-1119(96)00270-3.
Canlas and coworkers [Canlas et al. (1984) Am. J. Trop. Med. Hyg. 33, 420-424] isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds. The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1548-1552). This paper describes the production of enzymatically active Bm Cht in Escherichia coli. Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold. The specific activity of the soluble MBP::Cht isolated from the E. coli cytoplasm was low. Exporting MBP::Cht into the E. coli periplasmic space increased the specific activity by 12-fold. This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.
坎拉斯及其同事[坎拉斯等人（1984年），《美国热带医学与卫生杂志》33卷，第420 - 424页]分离出一种单克隆抗体（MF1），经被动转移后，该抗体可使感染的沙鼠体内的马来布鲁线虫（Bm）微丝蚴（mf）清除。MF1的靶标是一种发育调控型的mf几丁质酶（Cht）（福尔曼等人（1992年），《美国国家科学院院刊》89卷，第1548 - 1552页）。本文描述了在大肠杆菌中产生具有酶活性的Bm Cht的过程。标准表达条件下产生了一种不溶性的麦芽糖结合蛋白（MBP）::Cht融合蛋白，但通过优化表达条件，可溶性MBP::Cht的产量增加了25倍。从大肠杆菌细胞质中分离出的可溶性MBP::Cht的比活性较低。将MBP::Cht输出到大肠杆菌周质空间可使比活性提高12倍。这表明通过膜分泌和/或周质空间的环境可使重组Bm Cht的折叠得到改善。
