Cloning, characterization, and expression studies in Escherichia coli of growth hormone cDNAs from Indian zebu cattle, reverine buffalo, and beetal goat.
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.
Mukhopadhyay
U K
UK
Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India. ukmukh@ndri.hry.nic.in
Sahni
G
G
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes / Q000235:genetics*; D002417:Cattle / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005982:Glutathione Transferase; D006041:Goats / Q000235:genetics*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D005982:Glutathione Transferase