ArticlePublisher preview available

Molecular Cloning, Expression, Sequence Characterization and Structural Insight of Bubalus bubalis Growth Hormone-Receptor

November 2022
Molecular Biotechnology 65(5)

November 2022
65(5)

DOI:10.1007/s12033-022-00612-y

Authors:

Roquyya Gul

University of Lahore

Muhammad Umair Hanif

Gulab Devi Educational Complex

Hafiz Muzzammel Rehman

University of the Punjab

Show all 7 authorsHide

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.

A Construction of pET22b-BbGHR plasmid. Ligation of GHR1 (0.992 kb) & GHR2 (0.912 kb) fragments in pET22b ( +) expression vector, B Construction of pET22b-BbGHBP plasmid, C 12% SDS showing expression of BbGHR (~ 70kD) in E. coli BL21 codon Plus with IPTG induction. M: protein marker; Lane1: un-induced; Lane2: post-induced sample at 6 h; Lane3: soluble protein; Lane4: insoluble protein, D SDS-PAGE (12%) showing expression of BbGHBP (~ 30kD) in E. coli BL21 codon Plus with IPTG induction. Lane1: un-induced; Lane2: post-induced samples at 8 h; Lane 3: soluble protein; Lane 4: insoluble protein; Lane 5: Western blot analysis of the expressed BbGHBP using primary polyclonal rabbit anti-BbGHR/GHBP antibody and secondary antibody (Polyclonal Goat anti-rabbit IgG-HRP)

…

The interaction plot of oGH-BbGHBP complex. The oGH and BbGHBP residues are labeled pink and green, carbons are white, side chain nitrogen and oxygens are blue and red, respectively. Hydrogen bonds are represented in green dash lines with distance in Angstrom (Å)

…

Structural analysis of hormone-receptor complexes (oGH-BbGHR; BbGH-BbGHR; hGH-BbGHR). Molecular surface representation of each hormone (light gray) with its receptor (Pink) in a complex followed by close up in ribbon representation. Residues in the interface between the hormone and receptor are colored as yellow and cyan in stick representation, respectively. Conserve residues are represented in cyan and Glu62 which is similar binding residue in oGH-BbGHR/BP and hGH-hGHR (determined by De-Vos) is shown in brown. A Complex of oGH-BbGHR, B Complex of BbGH-BbGHR, C Complex of hGH-BbGHR

…

A, B, C, D, E & F Biological activity of the purified BbGHBP on HeLa cell lines. A HeLa cells in the presence of DMEM (Dulbecco’s Modified Eagle Medium), B HeLa cells in the presence of BSA (control), C HeLa cells in the presence of ovine GH, D HeLa cells in the presence of purified GHBP; E HeLa cells in the presence of ovine GH + purified BbGHBP, F Graph showing effect of inducers on HeLa cell proliferation. DMEM, BSA (control), ovine GH, BbGHBP and ovine GH + BbGHBP 10 μg/ml concentration, respectively, used on HeLa cell lines. After 24 h of incubation, maximum cell counts (129,733) seen in case of ovine GH + BbGHBP (asterisk indicating significant difference < 0.05)

…

Three-dimensional structure and Ramachandran plot of the BbGHBP. A Three-dimensional structure of BbGHBP, brown indicates alpha helix, blue indicates β-strands and coil is shown in white color. B Ramachandran plot of the BbGHBP after structural refinement

…

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Vol:.(1234567890)

Molecular Biotechnology (2023) 65:1062–1075

https://doi.org/10.1007/s12033-022-00612-y

1 3

ORIGINAL PAPER

Molecular Cloning, Expression, Sequence Characterization

andStructural Insight ofBubalus bubalis Growth Hormone‑Receptor

RoquyyaGul1,2 · MuhammadUmairHanif1· FaizaGul2· HazMuzzammelRehman3,4· MahjabeenSaleem5·

MuhammadSarfarazAhmad6· MuhammadUsmanMirza7

Received: 3 July 2022 / Accepted: 14 November 2022 / Published online: 27 November 2022

Abstract

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 prolif-

eration 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 inter-

actions 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.

Keywords Nili-Ravi buﬀalo· GHR· GHBP· HeLa cell lines· Molecular Dynamics Simulation

Introduction

The growth hormone (GH) system consists of 22kD GH,

70kD growth hormone-receptor (GHR) and 30kD growth

hormone binding protein (GHBP) [1]. GH also known as

somatotropin, produced by somatotroph cells of the ante-

rior pituitary gland and binds to transmembrane protein

i.e. GHR. GHR belongs to a family of cytokine receptor

and is mainly expressed in the liver [2]. It is believed to

play an important role in the normal somatic growth and

* Roquyya Gul

roquyya@yahoo.com; roquyya.gul@gdec.edu.pk

Muhammad Umair Hanif

umair.hanif@gdec.edu.pk

Mahjabeen Saleem

mahjabeen.mlt@mul.edu.pk

1 Gulab Devi Educational Complex, Ferozepur Road Lahore,

Lahore, Pakistan

2 School ofBiological Sciences, University ofthePunjab,

Lahore, Pakistan

3 Alnoorians Group ofInstitutes, 55-Elahi Bukhsh Park, Amir

Road, Shad Bagh, Lahore, Pakistan

4 School ofBiochemistry andBiotechnology, University

ofthePunjab, Lahore, Punjab, Pakistan

5 School ofMedical Laboratory Technology, Minhaj

University Lahore, Lahore, Pakistan

6 Drug Design andDevelopment Research Group (DDDRG),

Department ofChemistry, Faculty ofScience, University

ofMalaya, 50603KualaLumpur, Malaysia

7 Department ofChemistry andBiochemistry, University

ofWindsor, ON, Canada

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