Müller H N, Skerra A
Max-Planck-Institut f. Biophysik, Frankfurt, Germany.
J Mol Biol. 1993 Apr 5;230(3):725-32. doi: 10.1006/jmbi.1993.1194.
The serum retinol-binding protein solubilizes the lipophilic vitamin A alcohol and plays an important physiological role in the transport of this compound. The monomeric single-domain protein, the three-dimensional structure of which is known, constitutes a well-characterized member of the lipocalin family of proteins. We report here the functional expression of the apo-protein in Escherichia coli by secretion to the periplasm. The recombinant protein, purified in a single step by metal chelate affinity chromatography, exhibits the same ligand binding characteristics as described for the natural protein. Guanidinium chloride-induced unfolding and refolding experiments suggest that the recombinant retinol-binding protein adopts a stable conformation despite being expressed and purified in the absence of the large hydrophobic ligand. The expression system described here should also be useful for the recombinant production of other lipocalin proteins, thus permitting the elucidation of the structure-function relationships of ligand binding by protein engineering.
血清视黄醇结合蛋白可溶解亲脂性维生素A醇，并在该化合物的运输中发挥重要的生理作用。这种单体单结构域蛋白的三维结构已知，是脂质运载蛋白家族中一个特征明确的成员。我们在此报告了脱辅基蛋白在大肠杆菌中通过分泌到周质进行功能性表达的情况。通过金属螯合亲和层析一步纯化得到的重组蛋白，表现出与天然蛋白相同的配体结合特性。氯化胍诱导的去折叠和重折叠实验表明，尽管重组视黄醇结合蛋白是在没有大的疏水配体的情况下表达和纯化的，但它仍采用稳定的构象。这里描述的表达系统对于其他脂质运载蛋白的重组生产也应该是有用的，从而能够通过蛋白质工程阐明配体结合的结构-功能关系。
