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.

Removal of cleavage slow points from affinity tags used in the IMAC purification of recombinant proteins.

The complete enzymatic removal of affinity tags from tagged recombinant proteins is often required but can be challenging when slow points for cleavage exist. This study documents a general approach to remove N-terminal tags from recombinant proteins specifically designed to be efficiently captured by IMAC resins. In particular, site-directed mutagenesis procedures have been used to modify the amino acid sequence of metal binding tags useful in IMAC purifications of recombinant proteins with the objective to increase cleavage efficiency with the exopeptidase, dipeptidyl aminopeptidase 1. These tags were specifically developed for application with borderline metal ions, such as Ni(2+) or Cu(2+) ions, chelated to the immobilized ligands, 1,4,7-triazacyclononane (tacn) and its analogs. Due to the ability to control cleavage site structure and accessibility via site directed mutagenesis methods, these procedures offer considerable scope to obtain recombinant proteins with authentic native N-termini, thus avoiding any impact on structural stability, humoral and cellular immune responses, or other biological functions. Collectively, these IMAC-based methods provide a practical alternative to other procedures for the purification of recombinant proteins with tag removal. Overall, this approach is essentially operating as an integrated down-stream purification capability.

Purification of a recombinant human growth hormone by an integrated IMAC procedure.

In this study, integration of three discrete process aspects of the IMAC purification of Escherichia coli expressed recombinant proteins has been investigated. To this end, novel N-terminally tagged human growth hormone variants (tagged-vhGHs) have been expressed in E. coli by tank fermentation and captured directly from the cell lysate by a new IMAC approach. The chelating ligands used were 1,4,7-triaza-cyclononane (tacn) and bis(1,4,7-triazacyclononyl)-propane (dtnp) with copper(II) as the immobilised metal ion. The N-terminal tags were specifically selected for their potential to bind to these immobilised complexes and also for their ease of removal from the tagged protein by the dipeptidyl peptidase, DAP-1. Low levels of detergents in the binding buffer did not dramatically affect the purification, but increased concentrations of NaCl in the loading buffer improved the binding performance. The same IMAC systems, operated in the 'negative' adsorption chromatographic mode, could be used to obtain the purified mature human growth hormone variant, as assessed by MALDI-TOF and N-terminal sequencing studies, following removal of the affinity tag by the dipeptidyl peptidase 1. Western immunoblot analysis of the eluted fractions of both the tagged and de-tagged vhGH demonstrated significant clearance of E. coli host cell proteins (HCPs). Further, these IMAC resins can be used multiple times without the need for metal ion re-charging between runs. This study thus documents an integrated approach for the purification of specifically tagged recombinant proteins expressed in genetically modified E. coli.

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography.

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni²+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.