Design of protein purification pathways: application to the proteome of Haemophilus influenzae using heparin chromatography.
The design of efficient protein purification protocols is a scientific challenge and can be facilitated by the use of master protein enrichment or purification steps. A master purification step is in principle a list including the major proteins expected to be present in the various fractions collected from a particular enrichment process. Consideration of a master step in the design of a purification pathway can reduce the time and effort usually invested in "trial and error" approaches. Moreover, master purification steps will certainly become valuable tools in the isolation of proteins today designated as hypothetical or predicted coding regions, resulting from the sequencing of the various genomes, and for which no information on their purification exists. The construction of such a master step consists of performance of the protein separation by the particular chromatographic method, analysis by two-dimensional polyacrylamide gel electrophoresis, and identification by mass spectrometry of the proteins present in each fraction. This can be efficiently accomplished now thanks to developments in two-dimensional gel technology and large-scale sample throughput mass spectrometry. Here we present an example of construction of a master protein enrichment step using the soluble protein fraction of the bacterium Haemophilus influenzae and applying Heparin chromatography.
Pharmaceutical Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
M
M
Fountoulakis
B
B
Takács
D001426:Bacterial Proteins / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002847:Chromatography, Agarose / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005782:Gels; D006193:Haemophilus influenzae / Q000737:chemistry*; D006493:Heparin; D012685:Sepharose / Q000031:analogs & derivatives; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D005782:Gels; D006493:Heparin; D012685:Sepharose; C071734:actigel