It is not completely clear why these consensus RTX constructs are able to function as bioseparation tags. We do observe a correlation between length and precipitation (Figure 2), so size certainly plays a role. However, there has not been extensive work in studying the role of the number of repeats on RTX behavior. We recently studied the impact of altering the number of native RTX repeats in the block V CyaA RTX domain of B. pertussis but no significant size effect was observed and, furthermore, C-terminal capping was required for calcium-responsiveness (31). As for past efforts to design synthetic RTX domains, the synthetic domains created by Scotter et al. consisted of 4 RTX repeats and those prepared by Lilie et al. consisted of 8 repeats (22, 24). The peptides create by Lilie et al. were weakly calcium-responsive, while those of Scotter et al. were only lanthanum-responsive and formed partially insoluble filaments in the presence of lanthanum. In general, it is fairly well established that β sheets are prone to aggregation and nature uses various strategies to ensure solubility of proteins containing these motifs (32), so perhaps BRTs are a balance between this tendency and the calcium-responsiveness of the β roll. Further investigation will be required to better elucidate the mechanism of BRT functionality, but their use as a tool for protein purification is clear.
The technique described here offers a new stimulus-responsive phase-changing peptide that could be useful in a range of applications similar to those for which ELPs have been used, such as recombinant protein purification or the creation of “smart” biomaterials. This new tag possesses certain advantages over ELPs and annexin B1 since precipitation is simpler to achieve and the BRT peptide is significantly smaller. Additionally, BRT17 precipitates in as little as 25 mM CaCl2 at room temperature, compared to the larger ionic strength and higher temperature increases required for ELP precipitation. Precipitation also occurs instantaneously, whereas annexin B1-based systems require a 2 h incubation period at 4°C. Overall, BRTs offer a new tool for rapid purification of recombinant proteins. The protocol described here can be performed to obtain purified fusion protein from lysate in only a few minutes. Further optimization of the BRT system should enable the use of specific proteases to purify target proteins and further improve the precipitation and resolubilization process, greatly enhancing the ability to rapidly purify recombinant proteins.
The authors gratefully acknowledge funding from the National Science Foundation.
The authors declare no competing interests.
Address correspondence to Scott Banta, Department of Chemical Engineering, Columbia University, New York, New York. E-mail: [email protected]
1.) Przybycien, T.M., N.S. Pujar, and L.M. Steele. 2004. Alternative bioseparation operations: life beyond packed-bed chromatography. Curr. Opin. Biotechnol. 15:469-478. 2.) Balan, S., J. Murphy, I. Galaev, A. Kumar, G.E. Fox, B. Mattiasson, and R.C. Willson. 2003. Metal chelate affinity precipitation of RNA and purification of plasmid DNA. Biotechnol. Lett. 25:1111-1116. 3.) Kumar, A., P.O. Wahlund, C. Kepka, I.Y. Galaev, and B. Mattiasson. 2003. Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers. Biotechnol. Bioeng. 84:494-503. 4.) McPherson, D.T., J. Xu, and D.W. Urry. 1996. Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP. Protein Expr. Purif. 7:51-57. 5.) Meyer, D.E., and A. Chilkoti. 1999. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat. Biotechnol. 17:1112-1115. 6.) Yamaoka, T., T. Tamura, Y. Seto, T. Tada, S. Kunugi, and D.A. Tirrell. 2003. Mechanism for the phase transition of a genetically engineered elastin model peptide (VPGIG)40 in aqueous solution. Biomacromolecules 4:1680-1685. 7.) Wood, D.W., W. Wu, G. Belfort, V. Derbyshire, and M. Belfort. 1999. A genetic system yields self-cleaving inteins for bioseparations. Nat. Biotechnol. 17:889-892. 8.) Banki, M.R., L. Feng, and D.W. Wood. 2005. Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat. Methods 2:659-661. 9.) Fong, B.A., W.Y. Wu, and D.W. Wood. 2009. Optimization of ELP-intein mediated protein purification by salt substitution. Protein Expr. Purif. 66:198-202. 10.) Ding, F.X., H.L. Yan, Q. Mei, G. Xue, Y.Z. Wang, Y.J. Gao, and S.H. Sun. 2007. A novel, cheap and effective fusion expression system for the production of recombinant proteins. Appl. Microbiol. Biotechnol. 77:483-488. 11.) Courtemanche, N., and D. Barrick. 2008. The leucine-rich repeat domain of Internalin B folds along a polarized N-terminal pathway. Structure 16:705-714. 12.) Grove, T.Z., A.L. Cortajarena, and L. Regan. 2008. Ligand binding by repeat proteins: natural and designed. Curr. Opin. Struct. Biol. 18:507-515. 13.) Binz, H.K., P. Amstutz, A. Kohl, M.T. Stumpp, C. Briand, P. Forrer, M.G. Grutter, and A. Pluckthun. 2004. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22:575-582. 14.) Mosavi, L.K., T.J. Cammett, D.C. Desrosiers, and Z.Y. Peng. 2004. The ankyrin repeat as molecular architecture for protein recognition. Protein Sci. 13:1435-1448. 15.) Main, E.R., Y. Xiong, M.J. Cocco, L. D'Andrea, and L. Regan. 2003. Design of stable alpha-helical arrays from an idealized TPR motif. Structure 11:497-508. 16.) Parmeggiani, F., R. Pellarin, A.P. Larsen, G. Varadamsetty, M.T. Stumpp, O. Zerbe, A. Caflisch, and A. Pluckthun. 2008. Designed armadillo repeat proteins as general peptide-binding scaffolds: Consensus design and computational optimization of the hydrophobic core. J. Mol. Biol. 376:1282-1304. 17.) Binz, H.K., M. Stumpp, P. Forrer, P. Amstutz, and A. Plückthun. 2003. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332:489-503. 18.) Holland, I.B., L. Schmitt, and J. Young. 2005. Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway review. Mol. Membr. Biol. 22:29-39. 19.) Chenal, A., J.I. Guijarro, B. Raynal, M. Delepierre, and D. Ladant. 2009. RTX calcium binding motifs are intrinsically disordered in the absence of calcium: implication for protein secretion. J. Biol. Chem. 284:1781-1789. 20.) Bauche, C., A. Chenal, O. Knapp, C. Bodenreider, R. Benz, A. Chaffotte, and D. Ladant. 2006. Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin. J. Biol. Chem. 281:16914-16926. 21.) Blenner, M.A., O. Shur, G.R. Szilvay, D.M. Cropek, and S. Banta. 2010. Calcium-induced folding of a beta roll motif requires C-terminal entropic stabilization. J. Mol. Biol. 400:244-256. 22.) Lilie, H., W. Haehnel, R. Rudolph, and U. Baumann. 2000. Folding of a synthetic parallel beta-roll protein. FEBS Lett. 470:173-177. 23.) Ringler, P., and G.E. Schulz. 2003. Self-assembly of proteins into designed networks. Science 302:106-109. 24.) Scotter, A.J., M. Guo, M.M. Tomczak, M.E. Daley, R.L. Campbell, R.J. Oko, D.A. Bateman, A. Chakrabartty. 2007. Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides. BMC Struct. Biol. 7:63. 25.) Dooley, K., Y.H. Kim, H.D. Lu, R. Tu, and S. Banta. 2012. Engineering of an Environmentally Responsive Beta Roll Peptide for Use As a Calcium-Dependent Cross-Linking Domain for Peptide Hydrogel Formation. Biomacromolecules 13:1758-1764. 26.) Meyer, D.E., and A. Chilkoti. 2002. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: Examples from the elastin-like polypeptide system. Biomacromolecules 3:357-367. 27.) McDaniel, J.R., J.A. MacKay, F.G. Quiroz, and A. Chilkoti. 2010. Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes. Biomacromolecules 11:944-952. 28.) Campbell, E., I.R. Wheeldon, and S. Banta. 2010. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior. Biotechnol. Bioeng. 107:763-774. 29.) Szilvay, G.R., M.A. Blenner, O. Shur, D.M. Cropek, and S. Banta. 2009. A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase. Biochemistry 48:11273-11282. 30.) Gill, S.C., and P.H. Vonhippel. 1989. Calculation of Protein Extinction Coefficients from Amino-Acid Sequence Data. Anal. Biochem. 182:319-326. 31.) Shur, O., and S. Banta. 2012. Rearranging and concatenating a native RTX domain to understand sequence modularity. Protein Engineering Design and Selection.. 32.) Richardson, J.S., and D.C. Richardson. 2002. Natural beta-sheet proteins use negative design to avoid edge-to-edge aggregation. Proc. Natl. Acad. Sci. USA 99:2754-2759. 33.) Crooks, G.E., G. Hon, J.M. Chandonia, and S.E. Brenner. 2004. WebLogo: A sequence logo generator. Genome Res. 14:1188-1190. 34.) Schneider, T.D., and R.M. Stephens. 1990. Sequence Logos - a New Way to Display Consensus Sequences. Nucleic Acids Res. 18:6097-6100.