Search results for: Recombinant Sheep GHBP Proteins
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2022/11/27
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Molecular Cloning, Expression, Sequence Characterization and Structural Insight of Bubalus bubalis Growth Hormone-Receptor.
The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl β-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species.Roquyya Gul, Muhammad Umair Hanif, Faiza Gul, Hafiz Muzzammel Rehman, Mahjabeen Saleem, Muhammad Sarfaraz Ahmad, Muhammad Usman Mirza
1095 related Products with: Molecular Cloning, Expression, Sequence Characterization and Structural Insight of Bubalus bubalis Growth Hormone-Receptor.
0.2 mg100.00 ug200ug500 ìg100ug1000 1 mg500 μg100ul50 ug96 wells (1 kit)
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Generation of epitope-specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site-directed mutagenesis of ovine GHBP.
Site-directed antibodies to the growth hormone receptor could be potentially useful as growth hormone mimics but, in previous attempts, we found that antisera generated using peptides derived from growth hormone receptor sequences failed to recognize the intact protein. As an alternative approach to this problem, we have now adopted a strategy of epitope-switching between rat and ovine growth hormone receptors to produce rat epitopes in the correct structural context. Using site-directed mutagenesis, we altered the two dominant linear epitopes in the ovine growth hormone binding protein to the analogous sequences in rat growth hormone binding protein. Site A, between Thr28 and Leu34, is equivalent to epitope 1 in ovine growth hormone binding protein and site B, between Ser121 and Asp124, corresponds to epitope 5. The wild-type ovine growth hormone binding protein and the two mutant proteins were bacterially expressed, refolded and, following purification by metal-chelate affinity chromatography, used to raise antisera in sheep. We showed using RIA, in which wild-type ovine growth hormone binding protein acted as a competitor for the binding of rat growth hormone binding protein, that only the site A mutant protein elicited a specific anti-rat growth hormone binding protein response. This was confirmed in subsequent RIA studies using the antiserum to the site A mutant protein in which only peptides corresponding to the site A sequences in mutant ovine growth hormone binding protein and rat growth hormone binding protein, but not that in wild-type ovine growth hormone binding protein, were able to act as competitors for rat growth hormone binding protein. Antibodies specific for rat growth hormone binding protein could be separated from the antiserum to the site A mutant protein by means of affinity chromatography using immobilized wild-type ovine growth hormone binding protein to remove antibodies which cross-reacted with the ovine protein. The work lays the foundations for further studies in which the biological effects of these antibody fractions will be investigated and demonstrates an approach with general applicability in the production of antibodies directed towards specific epitopes on protein molecules.J H Shand, G J Allan, J Beattie, D J Flint
2289 related Products with: Generation of epitope-specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site-directed mutagenesis of ovine GHBP.
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Serum growth hormone-binding protein (GHBP) in domestic animals as measured by ELISA.
This research was conducted to develop and characterize a competitive ELISA for bovine serum growth hormone-binding protein (GHBP) using recombinant bovine GHBP and a polyclonal rabbit antiserum. In addition to bovine, however, the assay was found to measure some activity in equine, chicken, porcine, ovine, and human sera. The reference standard curve had an effective range of 3 to 200 ng/mL. Recovery of increasing amounts of GHBP added to ovine serum was 103% but seemed to overestimate the amount of GHBP at low concentrations (intercept = 2.5 ng/mL). Recovery from bovine and porcine serum was near ideal but seems to be overestimated at concentrations higher than 50 ng/microL. Within and between assay coefficients of variation were 12.1 and 18.9%, respectively, for a sheep serum pool. Neither exogenous GH (20 ng/mL) nor prolactin (100 ng/mL) interfered with the measurement of GHBP in serum. The GHBP activity measured in increasing doses of serum from ovine, porcine, and bovine inhibited the assay in a parallel manner. This observation suggests that the GHBP antiserum contains antibodies that are directed toward epitopes of GHBP, which are common among these species. Serum GHBP concentrations were similar among samples from a line of miniature Brahman and normal stature Brahman and Angus cattle. In mature ewes, there were no differences in serum GHBP among three different breed types. An increase (P < .0001) in serum GHBP was observed in pigs between 1 and 6 mo of age but no sex effect was observed.(ABSTRACT TRUNCATED AT 250 WORDS)S L Davis, N B Wehr, D M Laird, A C Hammond