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- Expression, Purification, and Characterization of Recombinant Fibulin-5 in a Prokaryote Expression System
- Myoung Seok Jeong , Chang Soo Kang , Yeon Soo Han , In Seok Bang
- J. Microbiol. 2010;48(5):695-700. Published online November 3, 2010
- DOI: https://doi.org/10.1007/s12275-010-0320-6
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Abstract
- Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N2+-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.
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- Impact of UV pre-treatment on the Longissimus thoracis et lumborum muscle proteomes of dry-aged beef cuts: A characterisation within two sampling locations
Sara Álvarez, Carlos Álvarez, Anne Maria Mullen, Eileen O'Neill, Mohammed Gagaoua
Meat Science.2025; 221: 109729. CrossRef - Genome-wide association study on reproductive traits in Jinghai Yellow Chicken
G.X. Zhang, Q.C. Fan, J.Y. Wang, T. Zhang, Q. Xue, H.Q. Shi
Animal Reproduction Science.2015; 163: 30. CrossRef - Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli
Sun Kwon Bang, Young Sik Kim, Byung Soo Chang, Cheol Beom Park, In Seok Bang
Biotechnology and Bioprocess Engineering.2013; 18(5): 835. CrossRef
- Impact of UV pre-treatment on the Longissimus thoracis et lumborum muscle proteomes of dry-aged beef cuts: A characterisation within two sampling locations
- Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes
- Boyun Jang , Yuna Jung , Dongbin Lim
- J. Microbiol. 2007;45(6):593-596.
- DOI: https://doi.org/2602 [pii]
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Abstract
- In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.
- Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography
- Tau Chuan Ling , Chee Kin Loong , Wen Siang Tan , Beng Ti Tey , Wan Mohammad Wan Abdullah , Arbakariya Ariff
- J. Microbiol. 2004;42(3):228-232.
- DOI: https://doi.org/2084 [pii]
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Abstract
- In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine^TM 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (H_o=15cm) of STREAMLINE DEAE (r=1.2 g/cm^3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.
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