HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 35, 25734-25744, September 1, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/35/25734    most recent M604343200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jones, L. L. Articles by Kranz, D. M. Search for Related Content PubMed PubMed Citation Articles by Jones, L. L. Articles by Kranz, D. M. Social Bookmarking                      What's this?

Engineering and Characterization of a Stabilized 1/2 Module of the Class I Major Histocompatibility Complex Product L^d*

Lindsay L. Jones¹, Susan E. Brophy¹², Alexander J. Bankovich, Leremy A. Colf, Nicole A. Hanick, K. Christopher Garcia, and David M. Kranz³

From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 and the Department of Microbiology and Immunology, Stanford University, Stanford, California 94305

The major histocompatibility complex (MHC) is the most polymorphic locus known, with thousands of allelic variants. There is considerable interest in understanding the diversity of structures and peptide-binding features represented by this class of proteins. Although many MHC proteins have been crystallized, others have not been amenable to structural or biochemical studies due to problems with expression or stability. In the present study, yeast display was used to engineer stabilizing mutations into the class I MHC molecule, L^d. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability. To engineer a more stable L^d, we selected L^d mutants with increased surface expression from randomly mutated yeast display libraries using anti-L^d antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable L^d mutant, L^d-m31, consisted of a single-chain MHC module containing only the1 and2 domains. The enhanced stability was in part due to a single mutation (Trp-97 Arg), shown previously to be present in the allele L^q. Mutant L^d-m31 could bind to L^d peptides, and the specific peptide·L^d-m31 complex (QL9·L^d-m31) was recognized by alloreactive TCR 2C. A soluble form of the L^d-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide·L^d-m31 complexes with affinities similar to those of native full-length L^d. The TCR and QL9·L^d-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized 1/2 class I platforms may enable various structural studies.

Received for publication, May 8, 2006

^* This work was supported by National Institutes of Health (NIH) Grant GM55767 (to D. M. K.), by NIH Grant AI048540 (to K. C. G.), by a Stanford Immunology Program Training Grant (to A. J. B. and N. A. H.), and by the National Science Foundation (to L. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Current address: Abbott Laboratories, 100 Abbott Park Rd., Abbott Park, IL 60064.

³ To whom correspondence should be addressed: Dept. of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-244-2821; Fax: 217-244-5858; E-mail: d-kranz{at}uiuc.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. S. Chervin, J. D. Stone, P. D. Holler, A. Bai, J. Chen, H. N. Eisen, and D. M. Kranz The Impact of TCR-Binding Properties and Antigen Presentation Format on T Cell Responsiveness J. Immunol., July 15, 2009; 183(2): 1166 - 1178. [Abstract] [Full Text] [PDF]

				C. Kiss, J. Temirov, L. Chasteen, G. S. Waldo, and A. R.M. Bradbury Directed evolution of an extremely stable fluorescent protein Protein Eng. Des. Sel., May 1, 2009; 22(5): 313 - 323. [Abstract] [Full Text] [PDF]

				L. L. Jones, L. A. Colf, J. D. Stone, K. C. Garcia, and D. M. Kranz Distinct CDR3 Conformations in TCRs Determine the Level of Cross-Reactivity for Diverse Antigens, but Not the Docking Orientation J. Immunol., November 1, 2008; 181(9): 6255 - 6264. [Abstract] [Full Text] [PDF]

				L. Varani, A. J. Bankovich, C. W. Liu, L. A. Colf, L. L. Jones, D. M. Kranz, J. D. Puglisi, and K. C. Garcia Solution mapping of T cell receptor docking footprints on peptide-MHC PNAS, August 7, 2007; 104(32): 13080 - 13085. [Abstract] [Full Text] [PDF]