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

ABSTRACT: 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 (DH5? 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.

SUBMITTER: Hanif MU

PROVIDER: S-EPMC5733156 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in <i>Bacillus subtilis</i>.

Hanif Muhammad Umair MU Gul Roquyya R Hanif Muhammad Imran MI Hashmi Abid Ali AA

BioMed research international 20171123

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 <i>Bacillus subtilis</i> 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 designe ...[more]

PMID: 29333457

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Project description:5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.

Dataset Information

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

Publications

Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in <i>Bacillus subtilis</i>.

Similar Datasets

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