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A genetic system yields self-cleaving inteins for bioseparations


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.


  1. 1

    Perler, F.B. et al. Protein splicing elements: inteins and exteins—a definition of terms and recommended nomenclature. Nucleic Acids Res. 22, 1125–1127 (1994).

    CAS  Article  Google Scholar 

  2. 2

    Gimble, F.S. Putting protein splicing to work. Chem. Biol. 5, R251–R256 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Chong, S. et al. Single-column purification of free recombinant proteins using a self-cleavage affinity tag derived from a protein splicing element. Gene 192, 271–281 (1997).

    CAS  Article  Google Scholar 

  4. 4

    Chong, S. et al. Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 26, 5109–5115 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Dalgaard, J.Z. et al. Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the H-N-H family. Nucleic Acids Res. 25, 4626–4638 (1997).

    CAS  Article  Google Scholar 

  6. 6

    Duan, X., Gimble, F.S. & Quiocho, F.A. Crystal structure of PI-SceI, a homing endonuclease with protein splicing activity. Cell 89, 555–564 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Derbyshire, V. et al. Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution. Proc. Natl. Acad. Sci. USA 94, 11466–11471 (1997).

    CAS  Article  Google Scholar 

  8. 8

    Chong, S & Xu, M.-Q. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J. Biol. Chem. 272, 15587–15590 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Shingledecker, K., Jiang, S. & Paulus, H. Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments. Gene 207, 187–195 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Chong, S. et al. Protein splicing involving the Saccharomyces cerevisiae VMA intein: the steps in the splicing pathway, side reactions leading to protein cleavage and establishment of an in vitro splicing system. J. Biol. Chem. 271, 22159–22168 (1996).

    CAS  Article  Google Scholar 

  11. 11

    Xu, M. & Perler, F.B. The mechanisms of protein splicing and its modulation by mutation. EMBO J. 15, 5146–5153 (1996).

    CAS  Article  Google Scholar 

  12. 12

    Stoddard, B.L. & Pietrokovski, S. Breaking up is hard to do. Nat. Struct. Biol. 5, 3–5 (1998).

    CAS  Article  Google Scholar 

  13. 13

    Chong, S., Williams, K.S., Wotkowicz, C. & Xu, M. Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273, 10567–10577 (1998).

    Article  Google Scholar 

  14. 14

    Shao, Z. & Arnold, F.H. Engineering new functions and altering existing functions. Curr. Opin. Struct. Biol. 6, 513–518 (1996).

    CAS  Article  Google Scholar 

  15. 15

    Belfort, M. & Pedersen-Lane, J. A genetic system for analyzing E. coli thymidylate synthase. J. Bacteriol. 160, 371–378 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Davis, E.O., Jenner, P.J., Brooks, P.C., Colston, M.J. & Sedgwick, S.G. Protein splicing in the maturation of the M. tuberculosis RecA protein: a mechanism for tolerating a novel class of intervening sequence. Cell 71, 201–210 (1992).

    CAS  Article  Google Scholar 

  17. 17

    Derbyshire, V., Kowalski, J.C., Dansereau, J.T., Hauer, C.R. & Belfort, M. Two-domain structure of the td intron-encoded endonuclease I-TevI correlates with the two-domain configuration of the homing site. J. Mol. Biol 265, 494–506 (1997).

    CAS  Article  Google Scholar 

  18. 18

    Klabunde, T., Sharma, S., Telenti, A., Jacobs, W.R. & Sacchettini, J.C. Crystal structure of gyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nat. Struct. Biol. 5, 31–36 (1998).

    CAS  Article  Google Scholar 

  19. 19

    Pietrokovski, S. Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins. Protein Sci. 3, 2340–2350 (1994).

    CAS  Article  Google Scholar 

  20. 20

    Dalgaard, J.Z., Moser, M.J., Hughey, R. & Mian, I.S. Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins. J. Comput. Biol. 4, 193–214 (1997).

    CAS  Article  Google Scholar 

  21. 21

    Horton, R.M., Cai, Z., Ho, S.N. & Pease, L.R. Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction. Biotechniques 8, 528–536 (1990).

    CAS  Google Scholar 

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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).

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