IJCRR - Vol 05 Issue 16, August, 2013
A METHOD FOR EFFICIENT EXTRACTION OF CELL WALL PROTEINS FROM CANDIDA ALBICANS
Author: Pavithra Amrath Jain, R. D. Kulkarni, B.M. Swamy, Gopala Mugeraya, Srinikethan G., Sharanbasu K.A.
Category: General Sciences
Candida cell wall proteomics is a challenging area of research. Extraction of cell wall proteins needs to be done carefully to obtain reproducible results. We used two different methods for extraction of candida cell wall proteins and documented the modifications required. In the course of extraction, we also evaluated efficacy of cell wall disruption by mechanical procedure of homogenization with glass beads to increase the protein yield and purity. For extraction of candida cell wall proteins, we utilized detergents like Sodium dodecyl sulphate (SDS), dithiothreitol (DTT), tris base and chitinase. This method was further optimized using homogenization of candida cells with glass beads over different time intervals. Amount of cell disruption at each time interval of bead beating using two varieties of beads was evaluated by gram stain, culture, protein estimation and SDS PAGE subsequently. We observed that method using detergents and Chitinase yielded better quality proteins, less contaminated with salts. For homogenization, bead beating with glass beads of 0.5 mm diameter over 18 minutes was found to be superior. Our initial experiments revealed limits of both the methods in candida cell wall protein extraction. Mechanical disruption of the cells could be further optimized using routinely available vortex mixer to increase final protein yield and gave consistent and reproducible results.
Keywords: Candida albicans, Cell wall proteins, glass bead homogenization, SDS PAGE.
Pavithra Amrath Jain, R. D. Kulkarni, B.M. Swamy, Gopala Mugeraya, Srinikethan G., Sharanbasu K.A.. A METHOD FOR EFFICIENT EXTRACTION OF CELL WALL PROTEINS FROM CANDIDA ALBICANS International Journal of Current Research and Review. Vol 05 Issue 16, August, 01-07
1. Scully C, El-Kabir M, Samaranayake LP. Candida and oral candidosis: a review. Crit Rev Oral Biol Med 1994; 5: 125-57.
2. Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP. Cell wall and secreted proteins of Candida albicans; identification, function, and expression. Microbiol Mol Biol Rev. 1998; 62(1): 130-80.
3. de Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ, de Koster C, Klis FM. Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryotic Cell. 2004; 3(4): 955-65.
4. Marcilla A, Valentín E, Sentandreu R. The cell wall structure; developments in diagnosis and treatment of candidiasis. Int Microbiol. 1998; 1(2): 107-16.
5. Casanova M, Lopez-Ribot JL, Martinez JP, Sentandreu R.. Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin; comparison with other techniques. Infect Immun. 1992; 60(11): 4898-906.
6. Pitarch A, Sánchez M, Nombela C, Gil C. Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome. Mol Cell Proteomics. 2002; 1(12): 967-82.
7. Feiz L, Irshad M, Pont-Lezica RF, Canut H, Jamet E. Evaluation of cell wall preparations for proteomics; a new procedure for purifying cell walls from Arabidopsis hypocotyls. Plant Methods. 2006; 27: 2-10.
8. Kapteyn JC., Dijkgraaf GJ., Montijin RC., and Klis FM. Glucosylation of cell wall proteins in regenerating spheroplasts of Candida albicans. FEMS Microbiol Lett. 1995; 128: 271-7.
9. Elorza MV., Rico H., Gozalbo D., and Sentandreu R. Cell wall composition and protoplast regeneration in Candida albicans. Antonie Leeuwenhoek 1983; 49: 457-69.
10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1): 265-75.
11. Casanova M, Chaffin WL. Cell wall glycoproteins of Candida albicans as released by different methods. J Gen Microbiol. 1991; 137(5): 1045-51.
12. Mackie and McCartney, Practical Medical Microbiology. 14th edition. 2006 Edited by J.G.Collee, A.G.Fraser, B.P.Marmion, A.Simmons.”Centrifuges, colorimeters and bacterial counts “ by R.Brown, I.R.Poxton. P 845-52. Churchhill Livingstone Elsevier
13. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259): 680-5.
14. Martínez JP, Gil ML, López-Ribot JL, Chaffin WL,. Serologic response to cell wall mannoproteins and proteins of Candida albicans. Clin Microbiol Rev. 1998; 11(1): 121-41.
15. Ruiz-Herrera J, Mormeneo S, Vanaclocha P, Font-de-Mora J, Iranzo M, Puertes I, Sentandreu R. Structural organization of the components of the cell wall from Candida albicans. Microbiology. 1994; 140: 1513-23.
16. Protein gel electrophoresis tips and troubleshooting guide, aldrin.tripod.com/index-3.html.
17. Klimek-Ochab M, Brzezi?ska-Rodak M, Zyma?czyk-Duda E, Lejczak B, Kafarski P. Comparative study of fungal cell disruption-- scope and limitations of the methods. Folia Microbiol (Praha). 2011; 56(5): 469-75.
18. F.I. Okungbowa, A.K. Ghosh, R. Chowdhury, P. Chaudhuri, A. Basu and K. Pa. Mechanical Lysis of Candida Cells for Crude Protein and Enzymatic Activity Estimation; Comparison of Three Methods. World Journal of Medical Sciences. 2007; 2 (2): 101-104.
19. Kessler G, Nickerson WJ. Glucomannanprotein complexes from cell walls of yeasts. J Biol Chem. 1959; 234: 2281-5.
20. Klis FM, de Jong M, Brul S, de Groot PW. Extraction of cell surface-associated proteins from living yeast cells. Yeast. 2007; 24(4): 253.