Abstract
This report describes the strategy for overexpression of caprine growth hormone (cGH) gene of beetal goat in E. coli through introducing silent mutations in the 5'-end of the coding sequence. The silent mutations introduced were aimed at minimizing translation-inhibiting secondary structures in the mRNA. Free energies of the resultant mRNAs were calculated from the ribosomal binding site of mRNA to +24 base using the Mfold web server. The construct with native sequence did not show any expression, whereas introduction of the silent mutations had strong influence on the expression levels. Some constructs (pETcGH2-7) showed 12-30% expression of total cell proteins while some others (pETcGH8-16) showed 30 to 53% of total cell protein. Any variation in the amount of mRNA transcript for the various constructs, as determined by quantitative PCR, was not enough to suggest that the variable level of the gene expression was due to variation in the transcription levels. It appears that the expression levels are not always correlated with free-energy values of the secondary structures in the 5'-end region of the mRNA; instead some key silent nucleotide alterations at certain sites of 5'-end of the sequence reorganize the secondary structure in such a way that it has positive impact on translation without considerably altering the free-energy values. An empirical approach for determining the optimum 5'-end substitutions for hyperexpression of a recombinant protein thus seems necessary.