Carboxyl terminus is essential for intracellular folding of chloramphenicol acetyltransferase

• Published in: The Journal of biological chemistry 1993-11, Vol.268 (33), p.24555-24558
• Main Authors: Robben, J, Van der Schueren, J, Volckaert, G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Chloramphenicol O-Acetyltransferase - chemistry; DNA Mutational Analysis; DNA, Bacterial; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; Molecular Sequence Data; Protein Folding; Sequence Homology, Amino Acid; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74502-5

Chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) 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 alpha 5-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.