Protein Engineering, Vol. 16, No. 2, 147-156,
February 2003
© 2003 Oxford University Press
Directed evolution of a single-chain class II MHC product by yeast display
1 Department of Biochemistry, University of Illinois, 600 S. Matthews Avenue, Urbana, IL 61801 and 2 The Diabetes Center, University of California–San Francisco, San Francisco, CA 94143, USA
Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific ‘diabetogenic’ peptides bound to a class II MHC allele called I-Ag7. The I-Ag7 molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-Ag7. A library containing mutations at two residues (positions 56 and 57 of the I-Ag7 ß-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-Ag7 followed by selection with an anti-I-Ag7 antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. L. Jones, S. E. Brophy, A. J. Bankovich, L. A. Colf, N. A. Hanick, K. C. Garcia, and D. M. Kranz Engineering and Characterization of a Stabilized {alpha}1/{alpha}2 Module of the Class I Major Histocompatibility Complex Product Ld J. Biol. Chem., September 1, 2006; 281(35): 25734 - 25744. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.A. Richman, S.J. Healan, K.S. Weber, D.L. Donermeyer, M.L. Dossett, P.D. Greenberg, P.M. Allen, and D.M. Kranz Development of a novel strategy for engineering high-affinity proteins by yeast display Protein Eng. Des. Sel., June 1, 2006; 19(6): 255 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Park, Y. Xu, X. F. Stowell, F. Gai, J. G. Saven, and E. T. Boder Limitations of yeast surface display in engineering proteins of high thermostability Protein Eng. Des. Sel., May 1, 2006; 19(5): 211 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wang, A. Rubtsov, R. Heiser, J. White, F. Crawford, P. Marrack, and J. W. Kappler Using a baculovirus display library to identify MHC class I mimotopes PNAS, February 15, 2005; 102(7): 2476 - 2481. [Abstract] [Full Text] [PDF]
|
|