Browse > Article

Purification of Streptomyces Phospholipase D by Immunoaffinity Chromatoghraphy using Peptide Antibodies  

Park, In-Sun (Division of Biological Sciences, Chonbuk National University)
Kim, Young-Ah (Division of Biological Sciences, Chonbuk National University)
Jeong, Su-Jin (Division of Biological Sciences, Chonbuk National University)
Uhm, Tai-Boong (Division of Biological Sciences, Chonbuk National University)
Publication Information
Korean Journal of Microbiology / v.42, no.4, 2006 , pp. 294-298 More about this Journal
Abstract
An immunoaffinity chromatography for the specific binding of Streptomyces somaliensis phospholipase D (PLD) that is considered as an industrially potential enzyme was developed. By using the protein structure prediction programs and the X-ray crystal structure of a Streptomyces PLD, 5 different epitopes with high antigenicity that are predicted to locate on the surface of the S. somaliensis PLD were selected and then synthesized for the preparation of antipeptide antibodies. Each purified rabbit IgG was coupled with NHS-activated Sepharose to prepare the immunoaffinity resins. After one-step purification of the culture concentrate on the antipeptide IgG-coupled Sepharose column, SDS-PAGE and the Western blot analysis of the purified samples showed that purification of PLD on the affinity columns was satisfactory, indicating that the peptide design using the structural information of Streptomyces PLDs was rational. However, the purified PLD in the solution aggregated rapidly, which resulted in poor specific activity and low purification yield.
Keywords
immunoaffinity; phospholipase D; purification; Streptomyces;
Citations & Related Records
연도 인용수 순위
  • Reference
1 정수진, 이선희, 엄태붕. 2004. Streptomyces somaliensis가 생산하는 세포외 phospholipase D의 유전자 서열분석과 transphosphatidylation활성 특성. 미생물학회지 40, 211-216
2 Iwasaki, Y., H. Nakano, and T. Yamane. 1994. Phospholipase D from Streptomyces antibioticus: cloning, sequencing, expression, and relationship to other phospholipases. Appl. Microbiol. Biotechnol. 42, 290-299
3 Kolaskar, A.S. and P.C. Tongaonkar. 1990. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett. 276, 172-174   DOI   ScienceOn
4 Thompson J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680   DOI
5 Yang, H. and M.F. Roberts. 2002. Cloning, overexpression, and characterization of a Bacteriol $Ca^{2+}$-dependent phospholipase D. Protein Sci. 11, 2958-2968   DOI   ScienceOn
6 Cuff, J.A., M.E. Clamp, A.S. Siddiqui, M. Finlay, and G.J. Barton. 1998. Jpred: A Consensus Secondary Structure Prediction Server. Bioinformatics 14, 892-893   DOI
7 Uhm, T.B. and S.H. Lee. 2003. A peptide antibody for rapid screening of Streptomyces species producing phospholipase D. Biotechnol. Lett. 25, 883-886   DOI   ScienceOn
8 Leiros, I., F. Secundo, C. Zambonelli, S. Servi, and E. Hough. 2000. The first crystal structure of a phospholipase: D. Structure 8, 655-667   DOI   ScienceOn
9 Jone, K.L. 1949. Fresh isolation of Actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. J. Bacteriol. 57, 141-145
10 Emini, E.A., J.V. Hughes, D.S. Perlow, and J. Boger. 1985. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J. Virol. 55, 836-839
11 Hatanaka, T., T. Negish, M. Kubota-Akizawa, and T. Hagishita. 2002. Purification, characterization, cloning and sequencing of phospholipase D from Streptomyces septatus TH-2. Enzyme Microb. Thechnol. 31, 233-241   DOI   ScienceOn
12 Rost, B. 1996. PHD: predicting one-dimensional protein structure by profile based neural networks. Method Enzymol. 266,525-539   DOI
13 Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254   DOI   ScienceOn
14 Ogino, C., Y. Negi, T. Matsumiya, K. Nakaoka, A. Kondo, S. Kuroda, S. Tokuyarna, S. kikkawa, T. Yamane, and H. Fukuda. 1999. Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum. J. Biochem. 125, 263-269   DOI
15 Hasegawa, M. and N. Ota. 1992. Production of phospholipase DK. Japan Patent. JPI992088981-A
16 Hatanaka, T., M. Kubota-Akizawa, T. Negish, and T. Hagishita, 2002. Study on thermostability of phospholipase D from Streptomyces sp. Biochim. Biophys. Acta 1598, 156-164   DOI
17 Imamura, S. and Y. Horiuti. 1978. Enzymatic determination of phospholipase D activity with choline oxidase. J. Biochem. 83, 677-680   DOI