Manuscript accepted on : February 17, 2009
Published online on: 28-06-2009
Study on the Extraction and Charecterization of Outer Membrane Proteins of E. Coli From Poultry
M. Suresh1, Y. Hari Babu1, S. Venkateshwar2 and M. Narsi Reddy2*
1Department of Veterinary Microbiology, Veterinary College, Bidar - 585 401 India.
2Centre for Biotechnology, University College of Technology, Osmania University, Hyderabad - 500 007 India.
Corresponding Author E_mail: mnreddydr@gmail.com
ABSTRACT: The outer membrane proteins of avian E. coli play significant role in the pathogenesis of avian colibacillosis and have been identified as potent immunogen. Outer membrane proteins may be pivotal to the virulence of the organism and given their prominence on the cell surface may prove to be suitable candidate for vaccine development.The E. coli cultures grown under the above conditions were pelleted at 4000 rpm for 10 minute at 4°C. The pellet was washed three times in 10mM HEPES buffer and finally re-suspended in 10 mM HEPES buffer. Outer membrane proteins were extracted as per the method described by Bolin and Jensen (1987).The protein profiles of outer membrane proteins were analyzed by SDS-PAGE in vertical slab gel unit.Analysis of the OMP profiles of E.coli serotype O6 revealed three bands approximately of 34, 38 and 44 kDa molecular weight. Serotype O20 revealed the presence of two thick bands of 34 and 38 kDa molecular weight and faint bands of 21 kDa and 58 kDa molecular weight. Similarly O60 serotype shows the presence of two major OMP bands of approximately 38, 43 kDa molecular weight. The serotype O109 had two thick bands of 34 and 38 kDa molecular weight and 21 kDa, 29 kDa and 58 kDa molecular weight faint OMP bands.
KEYWORDS: OMP; E.coli; colibacillosis; SDS-PAGE
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| Copy the following to cite this article: Suresh M, Babu Y. H, Venkateshwar S, Reddy M. N. Study on the Extraction and Charecterization of Outer Membrane Proteins of E. Coli From Poultry. Biosci Biotechnol Res Asia 2009;6(1) |
| Copy the following to cite this URL: Suresh M, Babu Y. H, Venkateshwar S, Reddy M. N. Study on the Extraction and Charecterization of Outer Membrane Proteins of E. Coli From Poultry. Biosci Biotechnol Res Asia 2009;6(1). Available from: https://www.biotech-asia.org/?p=8441 |
Introduction
Therapeutic agents against complex enteropathy are of little value, because not only of high cost involved but also drug resistance problem and difficulty in achieving effective tissue concentrations besides altering, the normal gut flora.
Outer membrane proteins of pathogenic Gram negative bacteria and are often implicated as virulence factors. They are surface exposed and therefore suitable for vaccine development. Outer membrane proteins are indispensable components of bacterial cells and participate in relevant functions of the microorganisms. Changes in the outer membrane protein composition might alter antibiotic sensitivity and pathogenicity.
Material and Methods
Bacterial strains
Extraction of outer membrane proteins
SDS-PAGE analysis
The following serotypes O6, O20, O60 and O109 isolated from the cases of colibacillosis used in the present study for extraction of outer membrane proteins.
E. coli cultures grown in laboratory were pelleted at 4000 rpm for 10 min at 4o the pellet was then washed thrice in 10mM HEPES buffer. Finally resuspended in 10 mM HEPES buffer. The extraction of outer membrane proteins done as per the method described by Bolin and Jenson 1987.
SDS-PAGE analysis of outer membrane proteins
The outer membrane proteins profiles were analyzed by SDS-PAGE in vertical slab gel according to the method described by Anbalagan 1985 and Hari babu 1995.
Results and Discussion
A total of seven E. coli isolates were recovered from fifteen processed samples, the serotype O20 and O109 were most prevalent (two each), O6 and O60 (one each).
Analysis of outer membrane protein was done by SDS-PAGE. The protein profiles of OMPs revealed that the serotypes O20 and O109 had two thick bands of 34 and 38 kDa molecular weight. The 29 kDa molecular weight OMP band present only in the serotype O109. These findings correlated with the findings of Overbeeke and Lungtenberg (1980) reported that E. coli K12 had 2-5 OMP bands in 30-42 kDa regions.
They also contain protein bands of 21 and 58 kDa which were faint. The results are in accordance with Zadeh et al (2004) who observed that O2, O78 and O6 had 50, 58 and 68 kDa low density bands.
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Figure 1: SDS PAGE analysis of outer membrane proteins of serotype O109 and O20.
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The serotype O6 revealed three bands with 34, 38 and 44 kDa. The results are in accordance with Inouye and Yee (1973) who showed the presence of 38, 44, 58 kDa by E. coli. Overbeeke and Lugtenberg (1980) showed that E. coli K12 had 2-5 OMP bands in 30-42 kDa regions.
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Figure 2: SDS PAGE analysis of outer membrane protein of serotype O6.
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The serotype O60 had two bands with 38 and 43 kDa. In the present study the results were correlated with the findings of Inouye and Yee (1973) who reported the presence of 38, and 43 kDa protein bands in E. coli.
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Figure 3: SDS PAGE analysis of outer membrane protein of serotype O60.
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References
- ANBALAGAN, K., 1985, Electrophoresis a practical approach, First edition. Life Sciences Book House, Madurai, pp: 86-91.
- BOLIN, C. A. AND JENSEN, A. E., 1987, Passive immunization with antibodies against iron regulated outer membrane proteins protects turkeys from Escherichia coli Infect. Immun., 55:1239-1242.
- HARIBABU, Y., 1995, Production of monoclonal antibodies against Newcastle Disease virus for characterization of field strain. Ph.D. thesis submitted to Jawaharlal Nehru Technological University, Hyderabad.
- INOUYE, M. AND YEE, M.L., 1973, Homogeneity of enveloped proteins of Escherichia coli separated by gel elctrophoresis in sodium dodecyl sulphate. Bacteriol., 113(1): 304-312.
- OVERBEEKE, N. AND LUGTENBERG, B., 1980, Major outer membrane proteins of Escherichia coli strains of human origin. Gen. Microbiol., 121(2):373-380.
- ZADEH, F.V., ESMIALY, F. AND YAZDI, M.K.S., 2004, Protective immune responses induced in chickens by outer membrane proteins extracted from different trains of Escherichia coli. Iranian Journal of Allergy, Asthma and Immunology. 3(3): 133-137.
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