Directed evolution of soluble single-chain human class II MHC molecules

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Major histocompatibility complex (MHC) class II molecules are crucial for T cell recognition of antigenic peptides.
• Expressing soluble MHC class II molecules is challenging due to their size, heterodimeric nature, and disulfide bonds.

Purpose of the Study:

• To engineer stable, soluble single-chain human lymphocyte antigen (HLA) class II MHC DR1 molecules (scDR1alphabeta) without covalently attached peptides.
• To develop a yeast surface display platform for efficient expression, characterization, and engineering of MHC class II molecules.

Main Methods:

• Directed evolution and yeast surface display were employed to generate and screen mutant scDR1alphabeta libraries.
• Fluorescence-activated cell sorting (FACS) with conformation-sensitive antibodies identified well-expressed and properly folded variants.
• Molecular modeling was used to investigate the structural basis for improved protein folding.

Main Results:

• Three scDR1alphabeta variants with improved expression and folding were successfully engineered and displayed on yeast.
• Specific amino acid substitutions in the beta1 domain were identified as critical for enhanced protein folding yield.
• Engineered scDR1alphabeta mutants demonstrated remarkable stability and specific, high-affinity binding to the HA(306-318) peptide.

Conclusions:

• The engineered scDR1alphabeta molecules provide a stable and functional soluble form of MHC class II DR1.
• Yeast surface display eliminates the need for complex purification and refolding steps, streamlining the process.
• This technology platform is applicable for developing improved DR1-based therapeutics and diagnostics and can be extended to other MHC molecules.