An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography.
BACKGROUND
Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner.


RESULTS
We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins.


CONCLUSIONS
A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies.His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.
Scheich
Christoph
C
Protein Structure Factory, Max Planck Institute of Molecular Genetics, Heubnerweg 6, 14059 Berlin, Germany. scheich@molgen.mpg.de
Sievert
Volker
V
Büssow
Konrad
K
D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results
D011993:Recombinant Fusion Proteins; D006639:Histidine; D005982:Glutathione Transferase