Waheed A, Pham T, Won M, Okuyama T, Sly W S
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University, School of Medicine, Missouri 63104, USA.
Protein Expr Purif. 1997 Mar;9(2):279-87. doi: 10.1006/prep.1996.0691.
Human carbonic anhydrase IV (CA IV) expressed in Escherichia coli was refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione. The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones. Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4 degrees C in the presence of 2 mM oxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37 degrees C. The enzyme purified from the E. coli cell extracts following activation in vitro showed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line. These studies suggest that the soluble truncated form of human CA IV expressed in E. coli, which is disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activation in vitro. Furthermore, the procedure described for recovery of CA IV following expression in E. coli may be useful for in vitro activation and subsequent purification of other disulfide-containing proteins.
在大肠杆菌中表达的人碳酸酐酶IV（CA IV），在氧化型谷胱甘肽存在的情况下，借助内周质蛋白二硫键异构酶DsbA在细胞提取物中进行重折叠并激活。重折叠和激活受到杆菌肽的抑制，但不受其他伴侣蛋白已知的辅因子或激活剂的影响。尽管在2 mM氧化型谷胱甘肽存在下于4℃激活时，从细胞提取物中回收的纯化CA IV产量最高，但在25℃和37℃时重折叠和激活的速率要快得多。体外激活后从大肠杆菌细胞提取物中纯化的酶，与从稳定转染的CHO细胞系分泌培养基中纯化的CA IV具有相似的结构稳定性和功能特性。这些研究表明，在大肠杆菌中表达的人CA IV的可溶性截短形式，即二硫键连接的锌金属酶，可为体外蛋白质折叠和酶激活研究提供有用的模型酶。此外，所述在大肠杆菌中表达后回收CA IV的方法，可能对其他含二硫键蛋白质的体外激活及后续纯化有用。
