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A designed, phase changing RTX-based peptide for efficient bioseparations
Oren Shur1, Kevin Dooley1, Mark Blenner2, Matthew Baltimore1, and Scott Banta1
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Supplementary Material

Figure 5.  SDS-PAGE results for purification and cleavage of AdhD. (Click to enlarge)

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.

Competing interests

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]

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