Preparation and characterization of recombinant chicken growth hormone (chGH) and its putative antagonist chGH G119R mutein.
Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized after being optimized for expression in E. coli. The respective cDNAs were inserted into expression vector, expressed and found almost entirely in the insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded, and purified to homogeneity by anion-exchange chromatography on Q-Sepharose. The overall yields were 400 to 500 mg from 5 L of fermentation. Both proteins were > 98% pure, as evidenced by SDS-PAGE, and contained at least 95% monomers, as documented by gel-filtration chromatography under non-denaturing conditions. Circular dichroism analysis revealed that both proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGH receptor extracellular domain, but its affinity, as determined by RRA, was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed using FDC-P1 3B9 cells stably transfected with rabbit GHR, was 30-40-fold lower, whereas chGH G119R mutant did not bind to oGHR-ECD and was devoid of any biological activity in FDC-P1 3B9 cells. However, in binding experiments that were carried out using chicken liver membranes, both oGH and chGH showed similar IC(50) values in competition with (125)I-oGH, while the IC(50) of G119R mutein was 10-fold higher. These results emphasize the importance of species specificity and indicate the possibility of antagonistic activity of chGH G119R.
Paczoska-Eliasiewicz
Helena E
HE
Department of Animal Physiology, University of Agriculture, al. Mickiewicza 24/28, 30-059 Kraków, Poland.
Salomon
Gili
G
Reicher
Shay
S
Gussakowsky
Eugene E
EE
Hrabia
Anna
A
Gertler
Arieh
A
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000235:genetics; D002645:Chickens; D005998:Glycine / Q000235:genetics; D013006:Growth Hormone / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology
D011994:Recombinant Proteins; D013006:Growth Hormone; D001120:Arginine; D005998:Glycine