Roosbeek Stein, Caster Hans, Liu Qing Zhou, Berne Pierre-François, Duverger Nicolas, Christiaens Bart, Vandekerckhove Joël, Peelman Frank, Labeur Christine, Rosseneu Maryvonne
Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
Protein Expr Purif. 2004 May;35(1):102-10. doi: 10.1016/j.pep.2003.12.015.
The aim of this study was the expression and production in Escherichia coli of the nucleotide-binding domains (NBDs) of the human ABCA1 transporter, in a soluble, non-denatured form. To increase the protein solubility, and avoid expression in E. coli inclusion bodies, we extended the length of the expressed NBD domains, to include proximal domains. The corresponding cDNA constructs were used to express the N-terminal His-tagged WT and mutant proteins, which were purified by Ni(2+)-affinity chromatography. Optimal expression of soluble proteins was obtained for constructs including the NBD, the downstream 80-residue domain, and about 20 upstream residues. The size homogeneity of WT and mutant NBDs was determined by Dynamic Light Scattering, and ATP-binding constants and ATPase activities were measured. The NBD1 and NBD2 domains bound ATP with comparable affinity. The ATPase activity of WT His-NBD1 was about three times higher than that of NBD2 and amounted to 5913 compared to 1979 nmol Pi/micromol NBD/min for WT His-NBD2. All engineered mutants had comparable ATPase activity to the corresponding WT protein. The optimisation of the length of the expressed proteins, based upon the boundary prediction of NBDs and neighbour domains, enables the expression and purification of soluble ABCA1 NBDs, with high ATPase activity. This approach should prove useful for the study of the structural and functional properties of the NBDs and other domains of the ABC transporters.
本研究的目的是以可溶、非变性形式在大肠杆菌中表达和生产人ABCA1转运蛋白的核苷酸结合结构域（NBDs）。为了提高蛋白质的溶解性并避免在大肠杆菌包涵体中表达，我们延长了所表达的NBD结构域的长度，使其包含近端结构域。相应的cDNA构建体用于表达N端带有His标签的野生型和突变型蛋白，这些蛋白通过Ni(2+)亲和层析进行纯化。对于包含NBD、下游80个残基的结构域和约20个上游残基的构建体，可获得可溶性蛋白的最佳表达。通过动态光散射测定野生型和突变型NBDs的大小均一性，并测量ATP结合常数和ATP酶活性。NBD1和NBD2结构域以相当的亲和力结合ATP。野生型His-NBD1的ATP酶活性比NBD2高约三倍，达到5913，而野生型His-NBD2为1979 nmol Pi/μmol NBD/min。所有工程突变体的ATP酶活性与相应的野生型蛋白相当。基于NBDs和相邻结构域的边界预测对所表达蛋白的长度进行优化，能够表达和纯化具有高ATP酶活性的可溶性ABCA1 NBDs。这种方法对于研究ABC转运蛋白的NBDs和其他结构域的结构和功能特性应该是有用的。
