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.

Institute of Microbiology of the Academy of Sciences of the Czech Republic CZ 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

Arnau, J., Lauritzen, C., Petersen, G.E., Pedersen, J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr. Purif. 2006;48:1–13. PubMed

Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene. 1997;192:271–281. PubMed

Chu, L.H., Choy, W.Y., Tsai, S.N., Rao, Z., Ngai, S.M. Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein Sci. 2006;15:699–709. PubMed PMC

Ford, C.F., Suominen, I., Glatz, C.E. Fusion tails for the recovery and purification of recombinant proteins. Protein Expr. Purif. 1991;2:95–107. PubMed

Graslund, S., Nordlund, P., Weigelt, J., Bray, J., Gileadi, O., Knapp, S., Oppermann, U., Arrowsmith, C., Hui, R., Ming, J., et al. Protein production and purification. Nat. Methods. 2008;5:135–146. PubMed PMC

Hartley, J.L. Cloning technologies for protein expression and purification. Curr. Opin. Biotechnol. 2006;17:359–366. PubMed

Hunt, I. From gene to protein: A review of new and enabling technologies for multi-parallel protein expression. Protein Expr. Purif. 2005;40:1–22. PubMed

Jenny, R.J., Mann, K.G., Lundblad, R.L. A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expr. Purif. 2003;31:1–11. PubMed

Ladant, D. Interaction of Bordetella pertussis adenylate cyclase with calmodulin: Identification of two separated calmodulin-binding domains. J. Biol. Chem. 1988;263:2612–2618. PubMed

Mao, H. A self-cleavable sortase fusion for one-step purification of free recombinant proteins. Protein Expr. Purif. 2004;37:253–263. PubMed

Miller, J.H. Experiments in molecular genetics. Cold Spring Harbor Press; Cold Spring Harbor, NY: 1972.

Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M., Nygren, P.A. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 1997;11:1–16. PubMed

Osicka, R., Prochazkova, K., Sulc, M., Linhartova, I., Havlicek, V., Sebo, P. A novel “clip-and-link” activity of repeat in toxin (RTX) proteins from gram-negative pathogens. Covalent protein cross-linking by an Asp-Lys isopeptide bond upon calcium-dependent processing at an Asp-Pro bond. J. Biol. Chem. 2004;279:24944–24956. PubMed

Sambrook, J., Fritsch, E.F., Maniatis, T. Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 1989.

Shaw, W.V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. PubMed

Shuker, S.B., Mariani, V.L., Herger, B.E., Dennison, K.J. Understanding HTLV-I protease. Chem. Biol. 2003;10:373–380. PubMed

Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins

Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif

Structural basis of the interaction between the putative adhesion-involved and iron-regulated FrpD and FrpC proteins of Neisseria meningitidis

General and molecular microbiology and microbial genetics in the IM CAS

RTX proteins: a highly diverse family secreted by a common mechanism