Journal of Biological Chemistry vol:268 issue:33 pages:24555-24558
Chloramphenicol acetyltransferase (CAT, EC 18.104.22.168) is a bacterial chloramphenicol resistance marker that is commonly used as a reporter enzyme in gene expression studies and as a carrier protein for the production of fused peptides. The latter can be done by insertion of target sequences into the native ScaI site near the 3'-end of the Tn9 cat gene. CAT activity in the resulting fusion proteins is retained. We observed that creation of a stop codon at this ScaI, which causes a COOH-terminal 9-amino acid deletion, results in loss of chloramphenicol resistance and total deposition of the mutant protein in inclusion bodies in Escherichia coli. Cytoplasmic solubility and enzyme activity are completely regained by elongation of this mutant with only 2 residues. Apparently, terminal residues of the alpha5-helix play a crucial role in achieving the native conformation of nascent CAT molecules. Thus, CAT provides an interesting model system for mutational analysis of protein folding in vivo.