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. 1993 Dec;2(12):2198–2205. doi: 10.1002/pro.5560021219

P22 Arc repressor: enhanced expression of unstable mutants by addition of polar C-terminal sequences.

M E Milla 1, B M Brown 1, R T Sauer 1
PMCID: PMC2142328  PMID: 8298465

Abstract

Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C-terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10- to 20-fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, HHHHHH (st6), was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild-type Arc. The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel-chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background. The thermodynamic stability of the EA28 mutant (delta delta Gu approximately -0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (delta delta Gu approximately -3.0 kcal/mol) and SA32 mutant (delta delta Gu approximately -3.3 kcal/mol) are substantially less stable.

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Selected References

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  1. Baker T. A., Grossman A. D., Gross C. A. A gene regulating the heat shock response in Escherichia coli also affects proteolysis. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6779–6783. doi: 10.1073/pnas.81.21.6779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowie J. U., Sauer R. T. Equilibrium dissociation and unfolding of the Arc repressor dimer. Biochemistry. 1989 Sep 5;28(18):7139–7143. doi: 10.1021/bi00444a001. [DOI] [PubMed] [Google Scholar]
  3. Bowie J. U., Sauer R. T. Identification of C-terminal extensions that protect proteins from intracellular proteolysis. J Biol Chem. 1989 May 5;264(13):7596–7602. [PubMed] [Google Scholar]
  4. Bowie J. U., Sauer R. T. Identifying determinants of folding and activity for a protein of unknown structure. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2152–2156. doi: 10.1073/pnas.86.7.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breg J. N., van Opheusden J. H., Burgering M. J., Boelens R., Kaptein R. Structure of Arc repressor in solution: evidence for a family of beta-sheet DNA-binding proteins. Nature. 1990 Aug 9;346(6284):586–589. doi: 10.1038/346586a0. [DOI] [PubMed] [Google Scholar]
  6. Brown B. M., Bowie J. U., Sauer R. T. Arc repressor is tetrameric when bound to operator DNA. Biochemistry. 1990 Dec 25;29(51):11189–11195. doi: 10.1021/bi00503a006. [DOI] [PubMed] [Google Scholar]
  7. Brown B. M., Sauer R. T. Assembly of the Arc repressor-operator complex: cooperative interactions between DNA-bound dimers. Biochemistry. 1993 Feb 9;32(5):1354–1363. doi: 10.1021/bi00056a022. [DOI] [PubMed] [Google Scholar]
  8. Gottesman S., Zipser D. Deg phenotype of Escherichia coli lon mutants. J Bacteriol. 1978 Feb;133(2):844–851. doi: 10.1128/jb.133.2.844-851.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hochuli E., Döbeli H., Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 1987 Dec 18;411:177–184. doi: 10.1016/s0021-9673(00)93969-4. [DOI] [PubMed] [Google Scholar]
  10. Horovitz A., Serrano L., Avron B., Bycroft M., Fersht A. R. Strength and co-operativity of contributions of surface salt bridges to protein stability. J Mol Biol. 1990 Dec 20;216(4):1031–1044. doi: 10.1016/S0022-2836(99)80018-7. [DOI] [PubMed] [Google Scholar]
  11. Knight K. L., Bowie J. U., Vershon A. K., Kelley R. D., Sauer R. T. The Arc and Mnt repressors. A new class of sequence-specific DNA-binding protein. J Biol Chem. 1989 Mar 5;264(7):3639–3642. [PubMed] [Google Scholar]
  12. Kohno T., Roth J. Electrolyte effects on the activity of mutant enzymes in vivo and in vitro. Biochemistry. 1979 Apr 3;18(7):1386–1392. doi: 10.1021/bi00574a041. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Pakula A. A., Sauer R. T. Amino acid substitutions that increase the thermal stability of the lambda Cro protein. Proteins. 1989;5(3):202–210. doi: 10.1002/prot.340050303. [DOI] [PubMed] [Google Scholar]
  15. Parsell D. A., Sauer R. T. The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. J Biol Chem. 1989 May 5;264(13):7590–7595. [PubMed] [Google Scholar]
  16. Parsell D. A., Silber K. R., Sauer R. T. Carboxy-terminal determinants of intracellular protein degradation. Genes Dev. 1990 Feb;4(2):277–286. doi: 10.1101/gad.4.2.277. [DOI] [PubMed] [Google Scholar]
  17. Reidhaar-Olson J. F., Parsell D. A., Sauer R. T. An essential proline in lambda repressor is required for resistance to intracellular proteolysis. Biochemistry. 1990 Aug 21;29(33):7563–7571. doi: 10.1021/bi00485a004. [DOI] [PubMed] [Google Scholar]
  18. Sauer R. T., Krovatin W., DeAnda J., Youderian P., Susskind M. M. Primary structure of the immI immunity region of bacteriophage P22. J Mol Biol. 1983 Aug 25;168(4):699–713. doi: 10.1016/s0022-2836(83)80070-9. [DOI] [PubMed] [Google Scholar]
  19. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  20. Silber K. R., Keiler K. C., Sauer R. T. Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):295–299. doi: 10.1073/pnas.89.1.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vershon A. K., Youderian P., Susskind M. M., Sauer R. T. The bacteriophage P22 arc and mnt repressors. Overproduction, purification, and properties. J Biol Chem. 1985 Oct 5;260(22):12124–12129. [PubMed] [Google Scholar]

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