SPECIFIC REPLACEMENT OF CONSECUTIVE AGG CODONS RESULTS IN HIGH-LEVEL EXPRESSION OF HUMAN CARDIAC TROPONIN-T IN ESCHERICHIA-COLI

The adult isoform of human cardiac troponin T (TnT) contains 288 amino acids, 14 of which (4.9%) are encoded by the rarely used arginine cod ons (12 AGG, 2 AGA) in Escherichia coli genes, To generate sufficient quantity of TnT protein for antibody production, we cloned the corresp onding cDNA and expressed it in E. coli. A low-level expression of TnT that comprised only about 1% of total cell protein was initially obse rved with the use of the native cDNA. The existence of two pairs of co nsecutive AGG codons (AGG(165)AGG(166) and AGG(215)AGG(216)) in the cD NA was suspected to be the main cause for this low-level expression. T hese two pairs of consecutive AGG codons were successively replaced wi th the major synonymous codon CGT by site-directed mutagenesis. As sus pected, a 10-fold increase in TnT expression was obtained when one pai r of the rare arginine codons was replaced and a 40-fold increase was achieved when both pairs of the rare codons were replaced. Our finding demonstrates the importance of consecutive rare codons in the suppres sion of high-level expression of heterologous proteins in E. coli and suggests that in order to maximize protein expression, a similar appro ach may be taken with other genes which contain consecutive rare codon s. (C) 1996 Academic Press, Inc.