Hinchman S K, Schuster S M
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610.
Protein Eng. 1992 Apr;5(3):279-83. doi: 10.1093/protein/5.3.279.
In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.
为了探究大肠杆菌天冬酰胺合成酶A的结构-功能关系，有必要设计一个系统来实现该基因的过表达以及基因产物的纯化。将大肠杆菌天冬酰胺合成酶A的结构基因与人碳酸酐酶II的结构基因的3'端融合，并在大肠杆菌中进行过表达。该基因产物是一种66 kDa的融合蛋白，具有天冬酰胺合成酶活性，通过亲和层析一步纯化，用作制备单克隆抗体的抗原。通过酶联免疫吸附测定法（ELISA）筛选单克隆抗体。选择对纯化的融合蛋白呈阳性而对纯化的人碳酸酐酶II呈阴性的菌落。然后使大肠杆菌天冬酰胺合成酶A基因过表达，其基因产物未经纯化用于最终筛选。所选择的抗体用于免疫亲和层析以纯化重组过表达的大肠杆菌天冬酰胺合成酶A。因此，现在有了一个程序，使得天冬酰胺合成酶A能够一步纯化至同质状态。
