Effect of the codon following the ATG start site on the expression of ovine growth hormone in Escherichia coli

For expression of ovine growth hormone (OGH) in inclusion bodies without an affinity histidine tag at either end of the protein, three clones, differi ng only in the second codon following the ATG start site, were constructed. Their expression was studied by SDS-PAGE followed by immunoblotting. Clone Ala.OGH (clone 1), beginning with Met.Ala.Phe.Pro..., did not show any exp ression. Clone Phe.OGH (clone 3), beginning with Met.Phe.Pro..., gave very high levels of OGH expression following IPTG induction. However, in clone G ly.OGH (clone 2), in which the Ala codon was replaced with a Gly codon at t he second position after the start site, a lower level of expression was ob tained. Northern hybridization analysis showed that upon IPTG induction, OG H mRNA was transcribed from all three clones. These results therefore, impl y that lack of expression in clone 1 and a lower level of expression in clo ne 2 are not due to a failure of transcription; however, they may be due to inefficient initiation of translation. The secondary structure analysis of mRNA predicts inaccessibility of different elements of the RES in the case of Ala.OGH (clone 1). The present study highly underscores the importance of mRNA secondary structure at the start site in regulation of expression o f a cloned gene in Escherichia coli, a prokaryotic expression system. (C) 1 999 Academic Press.