Recombinant proteins are commonly engineered with a tag located at either the N- or C-termini to facilitate single step affinity purification. These tags include the peptide tags FLAG and myc which are recognized by specific monoclonal antibodies and eluted with free peptide, fusion proteins with Glutathione-S-transferase or maltose binding protein which bind immobilised glutathione or amylose respectively and eluted with excess corresponding free ligand, and histadine tags (hex-His, octa-His, deca-His) which have affinities for nickel or cobalt resins (IMAC) and are subsequently eluted with increasing concentrations of imadazol. There are many different commercial available IMAC resins that utilise different chemistries for coordinating the metal ligands, and different types of matrices for ligand attachment. These matrices should be chemically and physically inert, however sometimes they non-specifically bind host cell proteins (HCPs) which co-purify with these His tagged proteins.

This study investigates different IMAC resins for their ability to purify N or C-terminal His tagged FUN (Function UNknown) proteins expressed using either bacterial or baculovirus/insect cell expression systems to achieve a suitable separation from HCPs, and compare the profiles of HCPs that co-purify with these His tagged FUN proteins. Using a 96 well based filter plate format we have been able to rapidly screen 12 different IMAC resins with 8 different FUN proteins from lactococcal bacteriophage and Salmonella enterica subsp. II serovar Sofia expressed in Escherichia coli BL21 AI. While gravity fed open IMAC columns were used to scout a number of different IMAC resins for the purification of a human FUN protein.