Visit our Expo - Redox and Inflammation signaling 2012

Session I : protein domains Poster I, 2
Preparation and characterization of recombinant chicken growth hormone (chGH) and
its putative antagonist chGH G119R mutein
Helena E. Paczoska-Eliasiewicz1, Gili Salomon2, Shay Reicher2, Eugene E.
Gussakowsky3, Anna Hrabia1 and Arieh Gertler2
1Department of Animal Physiology, University of Agriculture, Kraków, Poland,
2Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew
University of Jerusalem, Israel and 3Department of Botany, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada.
Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized according to
published sequence but optimized for expression in E. coli. The respective cDNAs were
inserted into pMON expression vector and transformed into Mon 105 E. coli strain. The
proteins expressed upon induction with nalidixic acid were 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 liters of
fermentation. Both proteins were > 98% pure as evidenced by SDS-PAGE and contained at
least 95% monomers as documented by gel-filtration chromatography on a SuperdexTM75
column under not 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 oGHR-
ECD (oGH receptor extracellular domain) though its affinity to ECD as determined by RRA
was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed
using PDF-P1 3B9 cells stably transfected with rabbit GHR was 20-25 fold lower, whereas
chGH G119R mutant did not bind to oGHR-ECD and was devoid any biological activity in
PDF-P1 3B9 cells. In contrast, in binding experiments carried-out using chicken liver
membranes both oGH and chGH showed similar EC50 values in competition with 125I-oGH.
These EC50 and were 5-9 fold lower than that of G119R mutein. These results emphasize the
importance of species specificity and indicate the possibility of antagonistic activity of chGH
G119R.
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