Hostname: page-component-76fb5796d-zzh7m Total loading time: 0 Render date: 2024-04-27T02:29:22.407Z Has data issue: false hasContentIssue false
  • English
  • Français

Disulfide Bonds in Recombinant Repeat Units From an Aquatic Insect's Silk Protein

Published online by Cambridge University Press:  15 February 2011

Steven T. Case ,
Stanley V. Smith  and
Melyssa R. Bratton
Steven T. Case
Affiliation:
Department of Biochemistry, 2500 North State Street, The University of Mississippi Medical Center, Jackson, MS, 39216-4505, USA
Stanley V. Smith
Affiliation:
Department of Biochemistry, 2500 North State Street, The University of Mississippi Medical Center, Jackson, MS, 39216-4505, USA
Melyssa R. Bratton
Affiliation:
Department of Biochemistry, 2500 North State Street, The University of Mississippi Medical Center, Jackson, MS, 39216-4505, USA
Get access

Abstract

rCAS is a recombinant Constant And Subrepeat (rCAS) protein modelled after tandem core repeats found in a 1000-kDa silk protein synthesized by larvae of the midge, Chironomus tentans. rCAS is encoded by a synthetic gene (synCAS) which is expressed in bacteria. Purified rCAS has four cysteine residues that participate in formation of two intramolecular disulfide bonds. Here we report the results of amino acid sequencing and electrospray ionization mass spectroscopic analyses of tryptic fragments of native and reduced rCAS which suggest that these disulfide bonds are heterogeneous. To assist in studying the formation of disulfide bonds in reduced and refolded rCAS, a series of synCAS mutants were constructed with cysteine to alanine substitutions. In comparison to wild-type rCAS and a Cys79→Ala mutant, the refolding of Cys9→Ala appears to be partially inhibited.

Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 330: Symposium S – Biomolecular Materials by Design , 1993 , 31
Copyright
Copyright © Materials Research Society 1994

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

1. Case, S.T., Powers, J., Hamilton, R., Burton, M.J., in Silk Polymers: Materials Science and Biotechnology, edited by Kaplan, D., Adams, W.W., Farmer, B. and Viney, C. (American Chemical Society Symposium Series 544, Washington, DC, 1994), p. 80–90.CrossRefGoogle Scholar
2. Case, S.T. and Wieslander, L., in Structure, Cellular Synthesis and Assembly of Biopolymers, edited by Case, S.T. (Results and Problems in Cell Differentiation, 19,Springer-Verlag, Berlin Heidelberg, 1992) p. 187–226.CrossRefGoogle Scholar
3. Wieslander, L. and Paulsson, G., Proc. Natl. Acad. Sci. USA 89, 4578–4582 (1992).CrossRefGoogle Scholar
4. Paulsson, G., Bemholm, K., and Wieslander, L., J. Mol. Evol. 35, 205–216 (1992).Google Scholar
5. Paulsson, G., Hoog, C., Bemholm, K., and Wieslander, L., J. Mol. Biol. 225, 349–361 (1992).CrossRefGoogle Scholar
6. Wellman, S.E. and Case, S.T., J. Biol. Chem. 264, 10878–10883 (1989).CrossRefGoogle Scholar
7. Smith, S.V. and Case, S.T., in Biomolecular Materials, edited by Viney, C., Case, S.T., and Waite, J.H. (Mater. Res. Soc. Proc. 292, Pittsburgh, PA, 1992) pp. 93–98.Google Scholar
8. Case, S.T. and Smith, S.V., in Silk Polymers: Materials Science and Biotechnology, edited by Kaplan, D., Adams, W.W., Farmer, B. and Viney, C. (American Chemical Society Symposium Series 544, Washington, DC, 1994), p. 91–97.CrossRefGoogle Scholar
9. Hunkapiller, M.W., Hewick, R.M., Dreyer, W.J., and Hood, L.E., Methods Enzymol. 91, 399–413 (1983).Google Scholar
10. Edmonds, C.G. and Smith, R.D., Methods Enzymol. 193, 412–431 (1990).Google Scholar
11. Vandeyar, M., Weiner, M., Hutton, C., and Batt, C., Gene 65, 129–133 (1988).CrossRefGoogle Scholar
12. Studier, F.W., Rosenberg, A.H., Dunn, J.D., and Dubendorff, J.W., Methods Enzymol. 185, 60–89 (1990).CrossRefGoogle Scholar