Production, purification and characterization of two recombinant DNA-derived N-terminal ovine growth hormone variants: oGH3 and oGH5

Growth hormone (GH) variants have been studied for the structure-function relationship of the molecule. The presence of a potential alternate splicing point in mRNA in bGH gene at exon 3, similar to hGH has been reported by workers. Early investigation on the characteristics of the chemistry of 20k oGH showed that the molecule was produced by site-directed mutagenesis by deleting amino acid residues 33-46 and the resultant DNA was expressed in E. coli under the control of lac promoter in pUC based plasmid. The mutant protein remained insoluble and did not refold. To investigate the effect of deletion on the chemistry of the molecule, computational biology tools were employed. The mutant with the deletion of amino acid residues 33-46, was designed and the model was visualized on computer. The structure of 20k bGH was compared with bGH and dissected for hydrogen bonds and hydrophobicity. Computational biology tools were helpful in elucidating the role of 33-46 amino acid residues domain in the chemistry of the molecule. Furthermore, it was revealed that removal of amino acid residues 33-46 which formed the hydrogen bonds involving Glu 33, Gln 46, Pro 38, Arg 42, Tyr 43,Ala 51, Thr 48, Asn 47, led to the formation of new hydrogen bonds between Thr 33, Tyr 144, Asn 32, Asn 32 and Ser and Asp 153. The removal of the amino acids 33-46 decreased the hydro-phobicity of the first helix of bGH molecule, as compared to 20k hGH, thus altering the solubility of the molecule, confirming the earlier reported results for ovine growth hormone with same deletion.

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Binding properties of beetal recombinant caprine growth hormone to Bovidae liver microsomal membranes

Gul¹,

Alam²,

Saleem³

et al. 2014

Afr. J. Biotechnol.

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The aim of the study was to illustrate the radio-receptor assay of beetal recombinant caprine growth hormone (rcGH). Tracer (125 I-rcGH) was prepared by iodinating beetal rcGH with iodine-125 and its biological activity was analyzed by rabbit anti-rcGH antibodies. Liver microsomal membranes of the Bovidae species (caprine, ovine and bovine) were prepared to study the binding properties of beetal rcGH. The tracer binding was dependent on receptor protein concentration, tracer counts, temperature, time of incubation and assay pH. The maximum percentage specific binding of the tracer to the microsomal membrane was 45% in 32 h at 4°C. In cross-reactive study, human GH competed and displaced the 125 I-rcGH by binding effectively to the cGH receptor. Scatchard analysis of the rcGH binding suggested a single class of binding site to the caprine microsomal membrane with an affinity of 337.1 ± 82.94 × 10 9 M-1 and capacity of 57.61 ± 4.23 fmol mg-1 while rcGH binding affinity and capacity to the ovine and bovine receptor protein showed 365.4 ± 66.82 × 10 9 M-1 , 57.69 ± 3.23 (fmol mg-1) and 392.6 ± 56.25 × 10 9 M-1 , 56.8 ± 2.56 (fmol mg-1) respectively. This study provides data for beetal rcGH interaction with microsomal membrane that shall be beneficial to study hormone receptor interactions of other Bovidae species.

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Production, purification and characterization of two recombinant DNA-derived N-terminal ovine growth hormone variants: oGH3 and oGH5

Cited by 3 publications

References 21 publications

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Deletion of amino acid residues 33-46 in growth hormone alters the hydrophobicity of the molecule

Binding properties of beetal recombinant caprine growth hormone to Bovidae liver microsomal membranes

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