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Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC

Institute of Microbiology of the Academy of Sciences of the Czech Republic

(RECEIVED April 9, 2008; ACCEPTED July 20, 2008)

Purification of recombinant proteins is often a challenging process involving several chromatographic steps to 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 Neisseria meningitidis FrpC protein with a C-terminal affinity tag. 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 of 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 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, such as glutathione-S-transferase, maltose-binding protein, β-galactosidase, chloramphenicol acetyltransferase, or 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 self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.

Keywords: Purification methods, general; Asp-Pro bond; FrpC; self-cleavage; self-excising; self-processing

¹ E-mail: osicka{at}biomed.cas.cz

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