Abstract
Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein–ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.
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Acknowledgments
The authors thank Daniel Knight for the critical reading of the manuscript. This work was partially supported by the grants from the Chinese National High Technology Research and Development Program 863 (no. 2006AA03Z451), the Chinese National Natural Science Foundation of China (no. 31070698), and the Shanghai Key Projects of Basic Research (no. 10JC1400300) to Q.M.; Army Research Office Grant W911NF-04-1-0056 to D.W., and by a grant to Ch-H. Shi (12D11320) from Donghua University, China.
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Shi, C., Meng, Q. & Wood, D.W. A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification. Appl Microbiol Biotechnol 97, 829–835 (2013). https://doi.org/10.1007/s00253-012-4601-3
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DOI: https://doi.org/10.1007/s00253-012-4601-3