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Overexpression and mutagenesis of Escherichia coli asnA andasnB
Abstract
The structure/function relationship of E. coli asparagine synthetase A (AS A) was explored after devising a system for overexpression of the gene and purification of the gene product. E. coli asnA was fused to the 3$\sp\prime$ end the gene for human carbonic anhydrase II and overexpressed in E. coli. The gene product was purified in a single step and used as antigen for the production of monoclonal antibodies. E. coli AS A was overexpressed and the antibodies selected were used for immunoaffinity chromatography to purify the recombinant E. coli AS A. A region of E. coli AS A was found to align with a highly conserved protein motif, characteristic of Class II aminoacyl tRNA synthetases. Site directed mutagenesis of Arg298 of E. coli asnA, an invariant arginine in Class II aminoacyl tRNA synthetases, always resulted in a loss of AS activity. Directed random mutagenesis was used to create a variety of amino acid substitutions within the conserved motif. Thirteen of the 15 clones with amino acid substitutions yielding soluble enzyme had no activity. These results are consistent with the theory that E. coli AS A evolved from an ancestral aminoacyl tRNA synthetase. To obtain large quantities of E. coli AS B, the coding region of asnB was cloned into a pET vector. Overexpression of the target gene, which yielded soluble gene product (40% of the total cell weight) provided sufficient amounts of protein to permit enzyme characterization. The assay conditions for the ammonia and glutamine dependent AS activities were optimized, and substrate Km's determined. To determine which of the evolutionarily conserved amino acids between human AS and E coli AS B were important for catalytic activity, directed random mutagenesis was performed on three regions of E. coli asnB. The nucleotide sequence was determined for the mutated plasmids coding for soluble inactive enzyme. Six amino acids were located which were critical for E. coli AS B activity.
Subject Area
Biochemistry|Molecular biology
Recommended Citation
Hinchman, Susan Kim, "Overexpression and mutagenesis of Escherichia coli asnA andasnB" (1991). ETD collection for University of Nebraska-Lincoln. AAI9200139.
https://digitalcommons.unl.edu/dissertations/AAI9200139