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A genetic system yields self-cleaving inteins for bioseparations

Abstract

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

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Figure 1: Intein–thymidylate synthase (TS) fusions and fusion phenotypes.
Figure 2: Temperature and pH effects on intein cleavage.
Figure 3: Structure/function analysis of mutations.

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Acknowledgements

This work was supported by NIH grants GM39422 and GM44844 to M.B., a Howard P. Isermann fellowship through the Department of Chemical Engineering, Rensselaer Polytechnic Institute to D.W., and a gift from Baxter Healthcare to G.B. The authors acknowledge the contributions of the Wadsworth Center Molecular Genetics Core and thank Drs. Richard Lease for many valuable discussions, David Shub for reading the MS, and Monica Parker for sound advice.

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Correspondence to Marlene Belfort.

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Wood, D., Wu, W., Belfort, G. et al. A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol 17, 889–892 (1999). https://doi.org/10.1038/12879

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