Abstract

Finding the best method of cell lysis and extraction of protein from the lysed cells is the key step in detection and identification of extra- and intra-cellular proteins in all applications of proteomics. To develop an optimized protein extraction protocol,Enterococcus faecalis V583, Lactococcus lactis NIZO 0900 and Pediococcus pentosaceus OZF strains, respectively, belonging to each genus of Enterococcus, Lactococcus and Pediococcus were used as a representative cells in a study of lactic acid bacteria (LAB). This report covers the use and comparison of threedifferent protein extraction methods including sonication, centrifugation and rupture by glass beads (FastPrep) to get a better understanding about which methods give better extract quality and higher amount of proteins when applied to one dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and for subsequent analysis by two dimensional (2D)-PAGE. The resultsclearly showed that, all methods can be used to lyse LAB strains. However, a six fold greater amount of protein was obtained when FastPrep was applied to lyse LAB cells. Our results also indicate that, this fast and easy extraction method allows more spot-abundant polyacrylamide gels. More clear and consistent strips were detected by SDS-PAGE when proteins were extracted by FastPrep. These results testify to the suitability of FastPrep protein extraction protocols for 2D proteomicstudies of representative strains of LAB.

Key words: FastPrep, sonication, centrifugation, lactic acid bacteria (LAB).

Abbreviation

LAB, Lactic acid bacteria; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MS, mass spectrometry; MRS, de Man, Rogosa and Sharpe; DTT, dithiothreitol; BSA, bovine serum albumine; IPG,immobilized pH gradient; TCA, trichloroacetic acid.