The History of Refolding MHCs: Building MHC Molecules from the Ground Up

Bacteria-refolded major histocompatibility complex (MHC) molecules, a key innovation in immunology, are a type of recombinant protein produced in bacteria that mimic natural MHC molecules. The development of this technology established a cost-effective and scalable method to produce large quantities of soluble, functional MHC molecules, revolutionized the study of T-cell immunology, drug discovery, and vaccine development.

The Early Days: Challenges with Native MHC

Before the development of bacteria-refolded MHC, researchers relied on obtaining MHC molecules from natural sources, primarily from mammalian cells, which was extremely challenging, inefficient, and expensive. Native MHC molecules are complex, membrane-bound glycoproteins that are difficult to purify in soluble form and require substantial effort to produce in large quantities. These challenges significantly limited the pace of research on T-cell recognition and the development of MHC-based diagnostics and therapeutics.

The quest for a more efficient production method led scientists to explore recombinant protein expression systems. While various systems like yeast and insect cells were considered, the bacterial expression system became a highly attractive option due to its low cost, rapid growth, and high yield. However, a major hurdle remained: the proper folding and assembly of the MHC-peptide complex.

The Breakthrough: Breaking it Down to Build it Up

The breakthrough came from a fundamental shift in thinking: instead of trying to mimic the cell's natural assembly process, scientists decided to build the molecule from the ground up, in vitro (in a test tube).

The foundational concept, pioneered in the early 1990s by labs like those of David Garboczi, Mark M. Davis, and John Altman was surprisingly straightforward:

1. Produce the Parts Separately in Bacteria: The genes for the MHC heavy chain and β2-microglobulin light chain were inserted into E. coli bacteria. The bacteria, unable to perform mammalian-style glycosylation, produced large quantities of simple, "naked" protein chains. However, these chains were produced as insoluble, misfolded clumps known as inclusion bodies which, ironically, was the key to the method's success.

2. Dissolve and Refold: The inclusion bodies were purified and then dissolved in a harsh denaturing chemical (like urea or guanidine hydrochloride) that unraveled the misfolded proteins into random coils.

3. The Magic of Refolding: This denatured mixture of heavy chain and β2-microglobulin was then diluted into a refolding buffer. This buffer contained the final, crucial ingredient: the specific peptide antigen of interest. As the proteins slowly renatured, the peptide acted similarly to a molecular scaffold, guiding the heavy chain and light chain to fold around it and form a stable, well assembled ternary peptide-MHC complex.

4. Purification: Correctly refolded MHC-peptide complexes were then purified from misfolded aggregates and other contaminants using chromatography techniques.

This process, now famously known as the "refolding" or "dilution" method, was a revelation.

E.coli-refolded peptide-MHC complex

Key Milestones and Refinements

The development was not a single event but a series of critical advancements:

• Early 1990s: Proof-of-concept studies demonstrated that functional MHC-I could be generated from E. coli inclusion bodies. The initial yields were low, but it proved the principle worked.

• Mid-to-Late 1990s: The protocol was systematically optimized. Many scientists tweaked the refolding buffer conditions such as redox agents (to help form disulfide bonds), pH, temperature, and additive concentrations to dramatically improve yields and reproducibility.

• The Tetramer Revolution: The most impactful application emerged in 1996 from the lab of Dr. John Altman. By biotinylating the refolded MHC complex and attaching it to fluorescent streptavidin, they created MHC Tetramers. These four-armed tools could bind specifically to T-cells with the correct receptor, allowing researchers to identify, count, sort, and study antigen-specific T-cells directly from blood or tissue samples with unprecedented precision. This transformed immunology from an inferential science to a direct observational one.

• 2000s - Present: The methodology expanded to include more unstable MHC alleles and was adapted for MHC class II molecules, which present antigens to helper T-cells and are even more complex to produce. Automation and high-throughput refolding platforms were developed, enabling the screening of thousands of peptides for T-cell responses, a critical need for modern vaccine and neoantigen discovery.

Bacteria-refolded MHC Development Timeline

Impact and Legacy

The development of bacteria-refolded MHC technology is a classic example of a methodological breakthrough catalyzing an entire field. Its impact is profound:

• Basic Research: It provided a limitless supply of pure, defined MHC-peptide complexes for structural studies (like X-ray crystallography) to understand immune recognition at an atomic level.

• T-cell Monitoring: MHC Tetramers became the gold standard for tracking T-cell responses in infections (like HIV, COVID-19), cancer, and autoimmune disorders.

• Vaccine and Drug Discovery: It enables the high-throughput identification of immunogenic epitopes, accelerating the development of new vaccines and T-cell-based therapies.

• Adoptive Cell Therapy: The ability to identify and isolate cancer-specific T-cells using tetramers is directly applicable to designing personalized cell therapies for cancer patients.

While newer methods exist such as mammalian-expressed single chain trimer peptide-MHCs, the principles of bacterial refolding remain a cornerstone of immunological research, paving the way for modern vaccine, TCR-based therapy research and development.

Exploring KACTUS E.coli-refolded MHC production capability

KACTUS excels in the production of high-quality, bacteria-refolded MHC-peptide complexes. Leveraging our robust refolding technology, we offer a comprehensive catalog of MHC products covering major alleles and hot peptide antigens. In addition, we also provide custom production services tailored to your specific research needs. Our strong capabilities ensure a reliable supply of these essential tools for immunology research, accelerating your work in vaccine development, T-cell therapy, and neoantigen discovery

Product Validation Data

Biotinylated Human HLA-A*02:01&B2M&P53 R175H (HMTEVVRHC) Monomer Protein (MHC-HE011B)

PE-Labeled Human HLA-A*11:01&B2M&KRAS WT (VVVGAGGVGK) Tetramer Protein (MHC-HM429TP)

Product list

Click here to browse KACTUS 70+ E.coli-refolded MHC monomers & tetramers catalog products and inquire about custom MHC production services!

Reference：

Garboczi, D. N., Hung, D. T., & Wiley, D. C. (1992). HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proceedings of the National Academy of Sciences, 89(8), 3429–3433.

Altman, J. D., Reay, P. A., & Davis, M. M. (1993). Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli. Proceedings of the National Academy of Sciences, 90(22), 10330–10334.

Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., & Davis, M. M. (1996). Phenotypic analysis of antigen-specific T lymphocytes. Science, 274(5284), 94–96.

Garboczi, D. N., & Utz, U. (2003). The MHC-Peptide Complex and T Cell Receptor. In Protein Structures: A Practical Approach (2nd ed., Vol. 2). Oxford University Press.

Davis, M. M., Altman, J. D., & Newell, E. W. (2011). Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis. Nature Reviews Immunology, 11(8), 551–558.

Bakker, A. H., & Schumacher, T. N. (2005). MHC multimer technology: current status and future prospects. Current Opinion in Immunology, 17(4), 428–433.