title: Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC.
abstract: Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp-Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, beta-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.
authors.affiliation: Institute of Microbiology of the Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Sadilkova
authors.forename: Radim
authors.initials: R
authors.lastname: Osicka
authors.forename: Miroslav
authors.initials: M
authors.lastname: Sulc
authors.forename: Irena
authors.initials: I
authors.lastname: Linhartova
authors.forename: Petr
authors.initials: P
authors.lastname: Novak
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C083530:frpC protein, Neisseria meningitidis; D001224:Aspartic Acid; D002118:Calcium