Role of Silent Gene Mutations in the Expression of Caprine Growth Hormone in Escherchia coli M. Altaf Khan, Saima Sadaf, and M. Waheed Akhtar* School of Biological Sciences, University of the Punjab, Lahore-54590, Pakistan 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. Introduction High level expression of a foreign gene in Escherichia coli is always desirable for cost-effective product formation. How- ever, the level of foreign gene expression in E. coli varies extensively for different eukaryotic genes (Kim et al., 2004; Kim et al., 2005). It has been reported that the codon downstream from the initiation codon affects the gene expression at the translational level (Puri et al., 1999). Growth hormone (GH), a protein of 22 kDa, promotes somatic growth and increases both milk (Bauman, 1999; Walli and Samanta, 2000) and meat production (Bonneau et al., 1999) in animals. GH consists of 190 or 191 amino acids with two disulfide bridges. The production of recombinant growth hormone from various species including bovine (Klein et al., 1991), porcine (Seeburg et al., 1983), and sheep (Rao et al., 1997) has been reported. However, expression levels of unmodified cDNA of these hormone variants in E. coli have in general been poor regardless of the promoter strength, the SD sequence, host strains, and culture conditions. These intricacies show that GH from various animals have inherent properties owing to sequence homologies that hamper a high level expression in E. coli, which may be due to formation of secondary structures in the region of mRNA close to the translational initiation site (Tomich et al., 1989). Therefore, a number of strategies including change of an amino acid following the start codon (Wallis, 1995), using bi-cistronic constructs to enhance the expression through mRNA stability (Schoner et al., 1984; Mukhopadhyay and Sahni, 2002), or expression of the variant containing a short N-terminal extension (Wallis and Wallis, 1989), have been used to overcome this problem. It is, however, preferable to obtain a high level of expression without any change in the amino acid sequence of the expressed protein. In this paper we report the effect of different silent mutations in the 5′-end region of growth hormone gene of a local caprine breed “Beetal” on expression in E. coli. Materials and Methods Bacterial Strains, Plasmids, and Growth Media. E. coli BL21 CodonPlus (DE3) RIPL (Stratagene, CA) was used for expression studies. pET22b expression plasmid was obtained from Novagen Inc. Trizol reagent for RNA isolation from pituitary gland was purchased from Invitrogen. QIAquick gel extraction for DNA extraction from agarose gels, RNeasy mini for RNA preparation from E. coli cells, and QIAprep Spin Miniprep kit for plasmid preparation were procured from Qiagen. InsT/A clone PCR product cloning kit, MMLV-RTase, Taq DNA polymerase, restriction enzymes, IPTG and T4 DNA ligase were purchased from MBI Fermentas. LB medium (Oxoid, England) was used for growth of E. coli cells. Total RNA Isolation and cDNA Cloning. Total RNA was isolated from pituitary gland of local caprine breed “Beetal” which was transported in liquid nitrogen immediately after removal from freshly slaughtered animals, using Trizol reagent according to the procedure described by the manufacturers. RT- PCR was performed using total RNA as template and the primers designed on the basis of cGH sequence reported previously (accession no. AY940163). MMLV-RTase and reverse primer RP-1 was used for the reverse transcription reaction. cDNA thus synthesized was amplified using primer pair FP-1/RP-1 as shown in Table 1. The amplified product * To whom correspondence should be addressed. E-mail: mwapu@brain.net.pk. Address: School of Biological Sciences,University of the Punjab, Lahore-Pakistan. Phone: +92-42-9230970. Fax: +92-42- 9230980. 1049 Biotechnol. Prog. 2007, 23, 1049-1052 10.1021/bp070167x CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical Engineers Published on Web 08/11/2007