Expression and sequence characterization of growth hormone binding protein of Nili-Ravi buffaloes (Bubalus bubalis)

Abstract

The growth hormone binding protein (GHBP) was isolated from the liver of Nili-Ravi buffaloes ( Bubalus bubalis ), reverse transcriptase-polymerase chain reaction (RT-PCR) amplified and sequence characterized. RT-PCR analysis demonstrated high degree sequence identities (97.3 to 99.6%) of Bb GHBP cDNA with Bos taurus , Ovis aries and Capra hircus . An expression plasmid was constructed for the production of Bb GHBP in Escherichia coli BL21 (RIPL) CodonPlus under the control of T7lac promoter. On induction with isopropyl β-D thiogalactopyranoside, the Bb GHBP was expressed at levels >30% of the total E. coli proteins. The target protein expressed as inclusion bodies was solubilized in denaturing solution and refolded by step/pulsatile dilution method using cysteine and cystine redox potential. Purification to near homogeniety (>98%) was achieved by ion-exchange chromatography with a recovery yield of 64%. Mass spectrometric analysis of the purified Bb GHBP showed a single peak of 30,756 Da. A radioprotein assay evaluated the binding affinity of recombinant Bb GHBP with iodinated bovine growth hormone (bGH) which demonstrated active conformation of Bb GHBP. These results demonstrate high expression and sequence characterization of Bb GHBP in Nili-Ravi buffaloes and provide the basis for the assessment of Bb GHBP in other breeds of buffalo. Keywords: Liver, Nili-Ravi buffalo, GHBP, MALDI-TOF mass spectrometry, radioprotein binding assay, refolding