Wang C, Castro A F, Wilkes D M, Altenberg G A
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
Biochem J. 1999 Feb 15;338 ( Pt 1)(Pt 1):77-81.
Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.
许多属于ATP结合盒（ABC）超家族的膜蛋白具有重要临床意义，包括囊性纤维化跨膜传导调节因子、磺脲类受体和P-糖蛋白（多药耐药基因产物；MDR1）。这些蛋白含有两个多次跨膜结构域，每个结构域后接一个核苷酸结合结构域（NBD）以及第一个NBD远端的连接区。NBD进行的ATP水解对于ABC蛋白的功能至关重要；连接区似乎具有调节作用。此前尝试在不使用去污剂溶解的情况下表达可溶性NBD和/或连接区，或者以高产量纯化作为可溶性融合蛋白的NBD，但均未成功。在此，我们展示了一种在大肠杆菌中表达MDR1的第一个NBD及其连接区（NBD1MLD）的系统。对与谷胱甘肽S-转移酶、硫氧还蛋白和麦芽糖结合蛋白（MBP）融合的NBD1MLD的表达情况进行比较，结果表明，只有MBP-NBD1MLD能够在可溶性部分实现高水平表达（约占大肠杆菌总蛋白的8%）。在NBD1MLD N端紧邻处添加一个凝血酶蛋白酶切位点，可将NBD1MLD从MBP上切割下来，MBP易于纯化且能保留其ATP酶活性。总之，只有使用在MBP和NBD1MLD之间含有凝血酶蛋白酶切位点的MBP融合蛋白载体才能取得成功。本文所述方法可能普遍适用于解决ABC蛋白NBD/连接区的表达和纯化问题。
