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Acyl carrier protein (ACP) is a central cofactor for de novo fatty acid synthesis, acyl chain modification and chain-length termination during lipid biosynthesis in living organisms. Although the structural and functional organization of the ACPs in bacteria and plant are highly conserved, the individual ACP is engaged in the generation of sets of signature fatty acids required for specific purpose in bacterial cells and plant tissues. Realizing the fact that the bacterial ACP being originated early in molecular evolution is characteristically different from the plant's counterpart, we explored the property of an ACP from Azospirillum brasilense (Ab), a plant-associative aerobic bacterium, to find its role in changing the fatty acid profile in heterologous systems. Functional expression of Ab-ACP in Escherichia coli, an enteric bacterium, and Brassica juncea, an oil-seed crop plant, altered the fatty acid composition having predominantly 18-carbon acyl pool, reflecting the intrinsic nature of the ACP from A. brasilense which usually has C18:1 rich membrane lipid. In transgenic Brassica the prime increment was found for C18:3 in leaves; and C18:1 and C8:2 in seeds. Interestingly, the seed oil quality of the transgenic Brassica potentially improved for edible purposes, particularly with respect to the enhancement in the ratio of monounsaturated (C18:1)/saturated fatty acids, increment in the ratio of linoleic (C18:2)/linolenic (C18:3) and reduction of erucic acid (C22:1).

The growth hormone cDNAs from three different economically important animal species of indian origin viz., indian zebu cattle (Bos indicus), indian reverine buffalo (Bubalus bubalis), and beetal goat (Capra hircus) were isolated by the RT-PCR technique. The amplified product was then cloned into phagemid pBluescriptIIKS- and the nucleotide sequence of the entire 573 base coding region for each product was determined. The genetic sequences as well as the translated protein sequence of these ruminant species were compared to that of closely related species like taurine cattle (Bos taurus) and sheep (Ovis aries). A very high degree of nucleotide sequence homology, ranging between 97-98%, was observed. Subsequently, the buffalo and goat cDNAs were used for expression studies in Escherichia coli. Very low levels of expression resulted when the growth hormone cDNAs were directly placed under the strong E. coli (trc) or phage (T7) promoters with the approximate level being less than 0.1% and 1% of the intracellular E. coli proteins, respectively. The nearly 10-fold enhancement of the level of expression as observed was attributable to the nature of the untranslated leader sequence donated by the individual expression element. High level (about 20% of soluble E. coli protein) expression of buffalo/goat growth hormone was achieved as a fusion protein with glutathione-s-transferase (GST) in pGEX-KT. Further, although attempts at converting the GST-GH fusion protein system to a two-cistronic gene expression system were unsuccessful, the utilization of a short synthetic first cistron in the two-cistronic mode of expression resulted in high levels (approximately 30% of soluble protein cell fraction) of GH polypeptide with a native N-terminus in E. coli for all three cDNAs.

The growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique. Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence. The high level expression of both the polypeptide hormones was achieved in E. coli (> or =30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system. The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out. A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones. A moderate yield ( approximately 40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed. The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones. The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone.