Short cut of protein purification by integration of cell-disrupture and affinity extraction.
Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5' end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 microns diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 microliters yeast cell suspension and 100 microliters glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70-80%.
Schuster
M
M
Novartis-Forschungsinstitut, Vienna, Austria.
Wasserbauer
E
E
Ortner
C
C
Graumann
K
K
Jungbauer
A
A
Hammerschmid
F
F
Werner
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D006801:Humans; D018189:Immunomagnetic Separation; D051379:Mice; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012709:Serum Albumin / Q000235:genetics / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C085706:FLAG peptide