Browse > Article
http://dx.doi.org/10.7841/ksbbj.2015.30.6.291

Simple Purification of BA-RGD Protein Based on CaCl2/EDTA Treatment and Inclusion Body Washing  

Song, Wooho (Department of Chemical Engineering and Applied Chemistry, Chungnam National University)
Byun, Chang Woo (Department of Chemical Engineering and Applied Chemistry, Chungnam National University)
Yoon, Minho (Department of Chemical Engineering and Applied Chemistry, Chungnam National University)
Eom, Ji Hoon (Department of Chemical Engineering and Applied Chemistry, Chungnam National University)
Choi, Yoo Seong (Department of Chemical Engineering and Applied Chemistry, Chungnam National University)
Publication Information
KSBB Journal / v.30, no.6, 2015 , pp. 291-295 More about this Journal
Abstract
The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.
Keywords
Recombinant shell matrix protein; Protein purification; Lipopolysaccharide; Inclusion body;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Leive, L. (1974) The barrier function of the gram-negative envelope. Ann. N. Y. Acad. Sci. 235: 109-129.   DOI
2 Lezin, G., M. R. Kuehn and L. Brunelli (2011) Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents. Biotech. Bioeng. 108: 1872-1882.   DOI
3 Lim, S., Y. S. Choi, D. G. Kang, Y. H. Song and H. J. Cha (2010) The adhesive properties of coacervated recombinant hybrid mussel adhesive proteins. Biomaterials. 31: 3715-3722.   DOI
4 Shirai, A., A. Matsuyama, Y. Yashiroda, A. Hashimoto, Y. Kawamura, R. Arai, Y. Komatsu, S. Horinouchi and M. Yoshida (2008) Global analysis of gel mobility of proteins and its use in target identification. J. Biol. Chem. 283: 10745-10752.   DOI
5 Dhami, N. K., M. S. Reddy and A. Mukherjee (2013) Biomineralization of calcium carbonates and their engineered applications: a review. Front. Microbiol. 4: 31.
6 Feng, Q. (2011) Principles of calcium-based biomineralization. pp. 113-140. In: W. E. G. Muller (ed.). Molecular Biomineralization: Aquatic Organisms Forming Extraordinary Materials. Springer- Verlag Berlin Heidelberg.
7 Wang, X. H., H. C. Schroder and W. E. Muller (2014) Enzymebased biosilica and biocalcite: biomaterials for the future in regenerative medicine. Trends Biotechnol. 32: 441-447.   DOI
8 Bahn, S. Y., B. H. Jo, B. H. Hwang, Y. S. Choi and H. J. Cha (2015) Role of Pif97 in nacre biomineralization: In vitro characterization of recombinant Pif97 as a framework protein for the association of organic-inorganic layers in nacre. Cryst. Growth Des. 15: 3666-3673.   DOI
9 Falini, G., S. Albeck, S. Weiner and L. Addadi (1996) Control of aragonite or calcite polymorphism by mollusk shell macromolecules. Science 271: 67-69.   DOI
10 Belcher, A. M., X. H. Wu, R. J. Christensen, P. K. Hansma, G. D. Stucky and D. E. Morse (1996) Control of crystal phase switching and orientation by soluble mollusc-shell proteins. Nature 381: 56-58.   DOI
11 Ponce, C. B. and J. S. Evans (2011) Polymorph crystal selection by n16, an intrinsically disordered nacre framework protein. Cryst. Growth Des. 11: 4690-4696.   DOI
12 Furuhashi, T., C. Schwarzinger, I. Miksik, M. Smrz and A. Beran (2009) Molluscan shell evolution with review of shell calcification hypothesis. Comp. Biochem. Phys. B 154: 351-371.   DOI
13 Marin, F., G. Luquet, B. Marie and D. Medakovic (2008) Molluscan shell proteins: Primary structure, origin, and evolution. Curr. Top. Dev. Biol. 80: 209-276.
14 Joubert, C., D. Piquemal, B. Marie, L. Manchon, F. Pierrat, I. Zanella-Cleon, N. Cochennec-Laureau, Y. Gueguen, and C. Montagnani (2010) Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: Focus on biomineralization. BMC Genomics 11: 613.   DOI
15 Miyamoto, H., H. Endo, N. Hashimoto, K. Iimura, Y. Isowa, S. Kinoshita, T. Kotaki, T. Masaoka, T. Miki, S. Nakayama, C. Nogawa, A. Notazawa, F. Ohmori, I. Sarashina, M. Suzuki, R. Takagi, J. Takahashi, T. Takeuchi, N. Yokoo, N. Satoh, H. Toyohara, T. Miyashita, H. Wada, T. Samata, K. Endo, H. Nagasawa, S. Asakawa and S. Watabe (2013) The diversity of shell matrix proteins: Genome-wide investigation of the pearl oyster, Pinctada fucata. Zool. Sci. 30: 801-816.   DOI
16 Picker, A., M. Kellermeier, J. Seto, D. Gebauer and H. Colfen (2012) The multiple effects of amino acids on the early stages of calcium carbonate crystallization. Z. Kristallogr. 227: 744-757.
17 Kumar, A., S. Tiwari, D. Thavaselvam, K. Sathyaseelan, A. Prakash, A. Barua, S. Arora and M. K. Rao (2012) Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and beta-mercaptoethanol, Protein Expres. Purif. 83: 226-232.   DOI
18 Magdalena, W., P. Dobryszycki and A. Ozyhar (2012) Intrinsically disordered proteins in biomineralization. pp 3-32. In: J. Seto (ed.), Advanced Topics in Biominerlization. InTech.
19 Song, A., S. Y. Bahn, H. J. Cha and Y. S. Choi (2014) Recombinant Calcium Binding Proteins and Nanofibrous Web Containing the Same. Korea Patent 10-2014-0053450.
20 Choi, B-H., H. Cheong, Y. K. Jo, S. Y. Bahn, J. H. Seo and H. J. Cha (2014) Highly purified mussel adhesive protein to secure biosafety for in vivo applications. Microb. Cell Fact. 13: 52.   DOI
21 Frisch, S. M. and H. Francis (1994) Disruption of epithelial cellmatrix interactions induces apoptosis. J. Cell Biol. 124: 619-626.   DOI
22 Giancotti, F. G. and E. Ruoslahti (1999) Integrin signaling. Science 285: 1028-1032.   DOI
23 Wang, X. and P. J. Quinn (2010) Endotoxins: Lipopolysaccharides of gram-negative bacteria. Subcell Biochem. 53: 3-25.   DOI
24 Magalhaes, P. O., A. M. Lopes, P. G. Mazzola, C. Rangel-Yagui, T. C. V. Penna and A. Pessoa (2007) Methods of endotoxin removal from biological preparations: a review. J. Pharm. Pharm. Sci. 10: 388-404.
25 Hedhammar, M., H. Bramfeldt, T. Baris, M. Widhe, G. Askarieh, K. Nordling, S. von Aulock and J. Johansson (2010) Sterilized recombinant spider silk fibers of low pyrogenicity, Biomacromolecules 11: 953-959.   DOI