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 ß-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.

Role of serum VEGF-A biomarker for early diagnosis of ovarian cancer instead of CA-125.

OBJECTIVE: To assess the significance of serum vascular endothelial growth factor-A (VEGF-A) as a potential biomarker for the diagnosis of ovarian cancer instead of cancer antigen-125 (CA-125). METHODS: The case-control study was conducted at the obstetrics departments of Sir Ganga Ram Hospital and Jinnah Hospital, Lahore, Pakistan, from April to September 2018, and comprised cases of epithelial ovarian tumour and healthy female controls. Serum VEGF-A and CA-125 levels were evaluated using Luminex multi-analyte profiling technology and enzyme immunoassays technique. Age, stage, grading, metastasis and ascites of ovarian cancer patients were investigated and compared with serum VEGF-A and CA-125 levels. Data was analysed using SPSS 20. RESULTS: Of the 89 subjects, 44(49.4%) were cases and 45(49.6%) were controls. Among the cases, 13(29.5%) were benign and 31(70.5%) were malignant. The mean serum VEGF-A values were inversely proportional to the stages of ovarian cancer i.e. stage I, II, III and IV showed 762.2pg/ml, 267.3pg/ml, 233.1pg/ml and 125.5pg/ml VEGF-A levels respectively. A steady increase in the mean serum CA-125 values with the progression of the disease was observed i.e. in stage I, II, III and IV the levels of CA-125 were 146.2U/ml, 268.5U/ml and 477.2U/ml and 844.4U/ml respectively. CONCLUSIONS: The detection of high concentrations of serum VEGF-A level supported its use as one of the diagnostic parameters in the timely investigation of ovarian cancer.

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in Escherichia coli.

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon + and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~ 25.5% and ~ 6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.

Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli.

Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.

Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis.

Recombinant human Bone Morphogenetic Protein 2 (rhBMP2) has important applications in the spine fusion and ortho/maxillofacial surgeries. Here we first report the secretory expression of biological active dimerized rhBMP2 from Bacillus subtilis system. The mature domain of BMP2 gene was amplified from pTz57R/BMP2 plasmid. By using pHT43 expression vector two constructs, pHT43-BMP2-M (single BMP2 gene) and pHT43-BMP2-D (two BMP2 genes coupled with a linker to produce a dimer), were designed. After primary cloning (DH5α strain) and sequence analysis, constructs were transformed into Bacillus subtilis for secretory expression. Expression conditions like media (2xYT) and temperature (30°C) were optimized. Maximum 35% and 25% secretory expression of monomer (~13 kDa) and dimer (~25 kDa), respectively, were observed on SDS-PAGE in SCK6 strain. The expression and dimeric nature of rhBMP2 were confirmed by western blot and native PAGE analysis. For rhBMP2 purification, 200 ml culture supernatant was freeze dried to 10 ml and dialyzed (Tris-Cl, pH 8.5) and Fast Protein Liquid Chromatography (6 ml, Resource Q column) was performed. The rhBMP2 monomer and dimer were eluted at 0.9 M and 0.6 M NaCl, respectively. The alkaline phosphatase assay of rhBMP2 (0, 50, 100, 200, and 400 ng/ml) was analyzed on C2C12 cells and maximum 200 ng/ml activity was observed in dose dependent manner.