Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo

Sharapova, N. E.; Котнова, А. П.; Галушкина, З. М.; Лаврова, Н. В.; Полетаева, Н. Н.; Tukhvatulin, A. E.; Семихин, А. С.; Gromov, A. V.; Soboleva, L. A.; Ershova, Anna S.; Зайцев, В. В.; Сергиенко, О. В.; Лунин, В. Г.; Karyagina, A. S.

doi:10.1134/s0026893310060099

Cited by 31 publications

(22 citation statements)

References 19 publications

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“…In all these works, expressing genetically engineered constructs were obtained using the native bmp-2 gene sequence amplified by PCR. Expression levels in this case did not exceed 25% of the total cell protein [3], and the protein was synthesized in the form of insoluble inclusion bodies. Higher expression MOLECULAR levels can be achieved using a microbiological (exhaustive selection of strains) and molecular-genetic (optimization of codons composition) approaches.…”

Section: Resultsmentioning

confidence: 86%

“…2, rows 3-4) and E. coli BL21(DE3) (see Fig. 2, rows 5-6), expression of the gene with the optimized codon composition results in synthesis of the target protein making up 37.8 and 56.7% of the total cell protein, respectively, which 1.5 and 2.3 times more than expression with the native gene sequence in E. coli BL21 (DE3) of 25% demonstrated previously [3]. It should be noted that the gene with a native sequence was expressed under the control of the T7 promoter in E. coli BL21 (DE3).…”

Section: Characterization Of Gene Expression For Levels Of Synthesis mentioning

confidence: 72%

“…The possibility of heterologous expression of the rhBMP-2 gene in E. coli cells has been demonstrated previously [3][4][5][6]. In all these works, expressing genetically engineered constructs were obtained using the native bmp-2 gene sequence amplified by PCR.…”

Section: Resultsmentioning

confidence: 99%

“…The activity of rhBMP-2 in vitro was determined by measuring the activity of alkaline phosphatase synthesized by C2C12 myoblasts after adding the factor, as described previously [3].…”

Section: Methodsmentioning

confidence: 99%

“…rhBMP-2 obtained by synthesis in Escherichia coli cells is not glycosylated, has a lower solubility, and tends to form aggregates. Nevertheless, there are numerous studies in vitro and on laboratory animals indicating that the osteogenic activity of rhBMP-2 synthesized in E. coli is comparable to the activity of the protein obtained in eukaryotic cells [3][4][5][6]. Moreover, there are examples of successful application of materials containing microbiologically produced rhBMP-2 in surgical practice [7][8][9][10], as well as in dentistry [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches

Karyagina

Boksha

Грунина

et al. 2016

Mol. Genet. Microbiol. Virol.

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Abstract⎯Recombinant bone morphogenetic protein-2 (rhBMP-2) has pronounced osteoinductive properties, as evidenced by the results of experimental and clinical practices. This applies to both the protein produced in eukaryotic cells and the protein synthesized in bacterial cells. In eukaryotic expression systems, production of the protein is extremely low and, consequently, the cost of materials on its basis is very high. Therefore, optimization of heterologous expression systems for rhBMP-2 production represents an important task. In the present work, optimization of codon composition of the rhBMP-2 gene nucleotide sequence and secondary structure of the transcript, as well as strain selection for efficient gene expression, were carried out. The producing strain based on Escherichia coli BL-21(DE3) provides a high level of rhBMP-2 synthesis (about 57% of total cell proteins). Biological activity of rhBMP-2 dimeric forms purified from the obtained producing strain was measured by induction of alkaline phosphatase activity in C2C12 cells. It is comparable with that of commercial rhBMP-2 expressed in E. coli (R&D Systems, United States). Purified rhBMP-2 does not contain impurities of E. coli endotoxin and can be used in experimental studies of osteoinduction in laboratory animals.

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Section: Resultsmentioning

confidence: 86%

Section: Characterization Of Gene Expression For Levels Of Synthesis mentioning

confidence: 72%

Section: Resultsmentioning

confidence: 99%

“…The activity of rhBMP-2 in vitro was determined by measuring the activity of alkaline phosphatase synthesized by C2C12 myoblasts after adding the factor, as described previously [3].…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches

Karyagina

Boksha

Грунина

et al. 2016

Mol. Genet. Microbiol. Virol.

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Strategies for humanizing glycosylation pathways and producing recombinant glycoproteins in microbial expression systems

Khan

Noordin

2018

Biotechnology Progress

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Homogeneously glycosylated proteins are essential for analyzing the structure of N‐glycans, studying their consequences inside cells, and developing therapeutic glycoproteins. However, the isolation of glycoproteins with homogeneous glycans from human is difficult since glycoforms slightly differ from each other with respect to molecular weight and charge. Microbial expression systems have numerous benefits in expression technology and have gained great attention, because they are more adaptable to the biotechnology industry. While selecting an expression host, the glycosylation pattern must be taken into account, because glycosylation strongly depends on cellular production system and selected production clone. This review discussed the technological developments in glycoengineering of microbial expression systems for humanizing the glycosylation profile and highlighted the expression potential of Leishmania expression system. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2752, 2019.

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Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis

Bayat

Shojaei²,

Bahrami

et al. 2017

J Biomedical Materials Res

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The aim of the present study was to assess the recombinant bonemorphogenetic protein 2 (RHBMP-2) with higher substantively and solubility for use in calcium phosphate scaffolds for better release in differentiation of mesenchymal stem cells to osteoblast cells. Using bioinformatics tools, two mutations (p. L10D and p. S12E) were chosen and applied in BMP2 CDS sequence to increase interaction with calcium derived composite. The new recombinant mutated sequence (BMP2 ) was synthesized and then subcloned to expression vector pBV220. Experimental data regarded functional protein expression in E. coli. Since no modification was made in the active sites of proteins namely β-sheets and α-helixes, not only was there any change in the specific activity occurred in the specific activity of the enzyme in comparison to its commercial counterpart, but also mesenchymal osteogenesis occurred more efficient on biphasic CaP scaffold model. As we hypothesized, use of negatively charged amino acids such as aspartate and glutamate in protein loops increased the interactions of BMP2-Ca and resulted in its slower and more sustained released from CaP scaffolds compare to commercial RHBMP2. Our data suggested that new BMP2 have greater osteoinductive capacity than RHBMP2 in the same time and dose than RHBMP2. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2799-2805, 2017.

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Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo

Cited by 31 publications

References 19 publications

Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches

Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches

Strategies for humanizing glycosylation pathways and producing recombinant glycoproteins in microbial expression systems

Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis

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