Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.
In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.
Department of Chemistry and Biochemistry, Miami University, 112 Hughes Hall, Oxford, OH 45056, USA.
Tara K
TK
Sigdel
Renee
R
Cilliers
Priya R
PR
Gursahaney
Michael W
MW
Crowder
D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002847:Chromatography, Agarose / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins