Production of recombinant buffalo (Bubalus bubalis) and goat (Capra hircus) growth hormones from genetically modified E. coli strains.
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.
Mukhopadhyay
Utpal Kumar
UK
Institute of Microbial Technology, Sector 39-A, Chandigarh, 160 036, India. ukmukh@ndri.hry.nic.in
Sahni
Girish
G
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002999:Clone Cells; D004269:DNA, Bacterial / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006041:Goats; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008099:Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010865:Pilot Projects; D010902:Pituitary Gland / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013006:Growth Hormone