N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.
The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes.
Centre for Green Chemistry, School of Chemistry, Monash University, Clayton, Victoria, 3800, Australia.
Jane T
JT
Mooney
Centre for Green Chemistry, School of Chemistry, Monash University, Clayton, Victoria, 3800, Australia.
Dale P
DP
Fredericks
Novo Nordisk A/S, Novo Nordisk Park, DK-2760, Måløv, Denmark.
Thorkild
T
Christensen
Novo Nordisk A/S, Novo Nordisk Park, DK-2760, Måløv, Denmark.
Christine
C
Bruun Schiødt
Centre for Green Chemistry, School of Chemistry, Monash University, Clayton, Victoria, 3800, Australia.
Milton T W
MT
Hearn
D000626:Aminopeptidases / Q000737:chemistry; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags / Q000737:chemistry*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006571:Heterocyclic Compounds / Q000737:chemistry; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012549:Schistosoma japonicum; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006571:Heterocyclic Compounds; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D019382:Human Growth Hormone; C075712:1,4,7-triazacyclononane; D005982:Glutathione Transferase; D000626:Aminopeptidases
IMAC; macrocyclic chelating ligands; purification; recombinant proteins; tag design and removal