An expression vector tailored for large-scale, high-throughput purification of recombinant proteins.
Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his(6)-tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his(6)-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his(6)-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his(6)-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his(6)-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his(6)-tag.
Donnelly
Mark I
MI
Biosciences Division, Argonne National Laboratory, IL 60439, USA. mdonnelly@anl.gov
Zhou
Min
M
Millard
Cynthia Sanville
CS
Clancy
Shonda
S
Stols
Lucy
L
Eschenfeldt
William H
WH
Collart
Frank R
FR
Joachimiak
Andrzej
A
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D058619:Maltose-Binding Proteins; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins