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 codons (12 AGG, 2 AGA) in Escherichia coli genes. To generate sufficient quantity of TnT protein for antibody production, we cloned the corresponding cDNA and expressed it in E. coli. A low-level expression of TnT that comprised only about 1% of total cell protein was initially observed with the use of the native cDNA. The existence of two pairs of consecutive AGG codons AGG(165) AGG(166) and AGG(215) AGG(216) in the cDNA was suspected to be the main cause for this low-level expression. These two pairs of consecutive AGG codons were successively replaced with the major synonymous codon CGT by site-directed mutagenesis. As suspected, a 10-fold increase in TnT expression was obtained when one pair 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 suppression of high-level expression of heterologous proteins in E. coli and suggests that in order to maximize protein expression, a similar approach may be taken with other genes which contain consecutive rare codons.
Hu
X
X
Spectral Diagnostics Inc., Toronto, Ontario, Canada.
Shi
Q
Q
Yang
T
T
Jackowski
G
G
D000328:Adult; D001120:Arginine / Q000737:chemistry*; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014170:Transformation, Genetic; D014336:Troponin / Q000096:biosynthesis / Q000235:genetics*; D020107:Troponin T
D003062:Codon; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014336:Troponin; D020107:Troponin T; D001120:Arginine