Engineering and characterization of a stabilized α1/α2 module of the class I major histocompatibility complex product Ld

Lindsay L. Jones, Susan E. Brophy, Alexander J. Bankovich, Leremy A. Colf, Nicole A. Hanick, K. Christopher Garcia, David M. Kranz

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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, Ld. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability.Toengineer a more stable Ld, we selected L d mutants with increased surface expression from randomly mutated yeast display libraries using anti-Ld antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable Ld mutant, L d-m31, consisted of a single-chain MHC module containing only the α1 and α2 domains. The enhanced stability was in part due to a single mutation (Trp-97 → Arg), shown previously to be present in the allele Lq. Mutant Ld-m31 could bind to Ld peptides, and the specific peptide·Ld-m31 complex (QL9·Ld-m31) was recognized by alloreactive TCR 2C. A soluble form of the Ld-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide·Ld-m31 complexes with affinities similar to those of native full-length Ld. The TCR and QL9·Ld-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized α1/α2 class I platforms may enable various structural studies.

Original languageEnglish (US)
Pages (from-to)25734-25744
Number of pages11
JournalJournal of Biological Chemistry
Issue number35
StatePublished - Sep 1 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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