Main Article Content

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

Roquyya Gul
Saima Sadaf
Muhammad Waheed Akhtar


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 BbGHBP cDNA with Bos taurus, Ovis aries and Capra hircus. An expression plasmid was constructed for the production of BbGHBP in Escherichia coli BL21 (RIPL) CodonPlus under the control of T7lac promoter. On induction with isopropyl β-D thiogalactopyranoside, the BbGHBP 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 BbGHBP showed a single peak of 30,756 Da. A radioprotein assay evaluated the binding affinity of recombinant BbGHBP with iodinated bovine growth hormone (bGH) which demonstrated active conformation of BbGHBP. These results demonstrate high expression and sequence characterization of BbGHBP in Nili-Ravi buffaloes and provide the basis for the assessment of BbGHBP in other breeds of buffalo.

Keywords: Liver, Nili-Ravi buffalo, GHBP, MALDI-TOF mass spectrometry, radioprotein binding assay, refolding

Journal Identifiers

eISSN: 1684-5315