Overproduction, preparation of monoclonal antibodies and purification of E.coli asparagine synthetase A

In order to explore the structure-function relationship of theEscherichia coli asparagine synthetase A it was necessary todevise a system for overexpression of the gene and purificationof the gene product. The E.coli asparagine synthetase A structuralgene was fused to the 3' end of the human carbonic anhydraseII structural gene and overexpressed in E.coli. The gene product,a 66 kDa fusion protein, which exhibited asparagine synthetaseactivity, was purified in a single step by affinity chromatographyand used as the antigen for the production of monoclonal antibodies.The monoclonal antibodies were screened by ELISA. Colonies werechosen which were positive for purified fusion protein and negativefor purified human carbonic anhydrase II. The E.coli asparaginesynthetase A gene was then overexpressed and the gene productwas used without purification for the final screen. The antibodiesselected were used for immunoaffinity chromatography to purifythe recombinant overexpressed E.coli asparagine synthetase A.Thus, a procedure is now available so that asparagine synthetaseA can be purified to homogeneity in a single step.