Beyond Refolding: The Case for Mammalian-Expressed Single-Chain Trimer MHCs

For years, recombinant major histocompatibility complexes (MHCs) have been expressed in bacterial systems with in vitro refolding. While this preserves the native MHC sequence, bacterial expression lacks post-translational modifications (PTMs) and risks misfolding. But what if we could bypass these challenges and produce fully functional MHCs directly in the form our cells naturally use?

In humans, these proteins are encoded within the MHC region of the genome (the human major histocompatibility complex), where highly polymorphic MHC genes and MHC alleles generate diverse human leukocyte antigen profiles across HLA alleles in the population.

What are MHC complexes and why are they important?

MHC complexes are composed of three chains: a peptide, an invariable light chain called β2-microglobulin (B2M), and a highly polymorphic heavy chain (HLA in humans) (Figure 1). These complexes are built from MHC molecules that sit on the cell surface of most nucleated cells. MHC complexes present sets of 8 to 14 residue antigenic peptides on their surface. These peptides are presented to T cells and hence are critical in adaptive immunity. In practice, peptide antigens (often short peptide fragments of abnormal proteins) are stabilized in the peptide binding groove (also described as the peptide binding cleft) through peptide binding interactions defined by amino acid side chains that help bind peptides with allele-specific preferences. The interaction between MHCs and TCRs is the basis for immune surveillance to detect invading pathogens and also enlighten researchers of cancer immunotherapy development, such as adaptive T cell therapies and TCR-mimic antibody discovery. To facilitate development of these therapies, it is crucial to have biologically active and physiologically relevant MHC complexes as the study tools. This is especially true when researchers track antigen presentation and antigen processing steps that shape antigen specificity and downstream immune response readouts.

MHC class I vs MHC class II in antigen presentation

The major histocompatibility complex class includes both MHC class I and class II systems. MHC class I molecules primarily present intracellular peptides to CD8 T cell populations, including cytotoxic T cells, supporting detection of infected or transformed cells. In contrast, MHC class II molecules typically present peptides derived from extracellular proteins and help coordinate broader adaptive responses. Together, these MHC class pathways shape the quality and breadth of cell responses observed in functional assays.

Why antigen presenting cells matter

Professional antigen presenting cells such as dendritic cells and b cells use mhc molecules to display peptide antigens to t cells, driving t cell activation and expanding antigen specific t cells and other specific t cells from a polyclonal t cell pool. This antigen presentation process depends on the correct pairing of peptide and hla molecule (a given human leukocyte antigen variant), which can be influenced by hla amino acid modifications in engineered contexts.

Figure 1. Structure of MHC-peptide complex.

Traditional Approach to MHC Production: E. coli expression and in vitro refolding

Traditionally, MHCs are expressed in E. coli and refolded in vitro, which is a complicated and labor-intensive process. This process involves expressing the heavy and light chains separately and refolding them in the presence of an excess of peptide. This process is laborious and carries the risk of misfolding MHCs. However, the biggest advantage of bacterial expression is that it maintains the original MHC sequence, something valued by researchers who want to ensure they’re accurately identifying epitope-specific antibodies or antigen-specific T cells. For some workflows, researchers may also use refolded proteins to validate peptide binding algorithms or cross-check peptide mhc binding predictions and peptide binding algorithms by directly measuring binding or stability with a defined antigenic peptide or the same peptide across multiple alleles.

Figure 2. Process of MHC expression in E. coli and in vitro refolding. 

Modern Approach to MHC Production: Single-Chain Trimer MHC Expression from Mammalian Systems

A more novel approach to MHC production is the development of single-chain trimer (SCT) MHC complexes. The concept of SCT MHCs was first detailed in “Translational and basic applications of peptide-MHC single chain trimers” published in the Journal of Immunology (2), which laid the groundwork for their enhanced stability and functional advantages.  These SCT MHCs combine the heavy chain, β2-microglobulin, and peptide into a single polypeptide chain, expressed in mammalian cells, effectively addressing several limitations of bacteria-refolded MHCs (Figure 3). In this design, a peptide linker connects components within SCT constructs, helping ensure the displayed cell epitope peptide remains stably positioned for TCR recognition.

Figure 3. Process of single-chain trimer MHC expression in mammalian systems. 

SCT expression simplifies the expression process and the number of steps to production. Additionally, it stabilizes MHC complexes and reduces the risk of peptide displacement by other peptides. This results in more accurate and reliable detection of antigen-specific T cells. In certain cases, when expression systems require fine-tuning to improve folding efficiency or to accommodate specialized structural designs, custom protein expression services can support these specific production needs. Moreover, refolded proteins always pose a risk of structural uncertainty. 

Practical SCT workflow notes (where SCTs can streamline production)
Teams building panels may use SCT expression to standardize outputs across alleles and peptides, tracking SCT protein expression and SCT expression yield (including expressed SCT protein yield) as part of process optimization. For library-style projects, SCT peptide library production may start from peptide encoded dna fragments and purified SCT plasmids, with verification steps such as restriction enzyme digest methods before expression and purification. Downstream, tetramer assembly may be standardized by controlling the final SCT tetramer concentration for consistent staining of antigen specific t cells in flow cytometry.

Building on this innovation, KACTUS recognized the inherent physiologic challenges and labor-intensive process of E. coli expression. Since our founding in 2018, we have made it our mission to engineer superior MHC complexes by leveraging the mammalian-expressed SCT design to eliminate the need for in vitro refolding. Our validation data has shown equivalent performance of mammalian-expressed SCT MHCs to E. coli refolded MHCs (Figure 4). Additionally, our mammalian expression systems mean the MHC complexes have PTMs including glycosylation. Because these molecules form stable structures under physiologic conditions, mammalian expression can support more consistent comparisons across mhc class I and MHC class II assay formats.

Figure 4. Comparison of ELISA activity data for E. coli refolded MHCs versus mammalian expressed (Expi293) SCT MHCs. 

Addressing Linker Concerns in SCT MHCs

Creating a single polypeptide chain between the peptide, heavy chain, and light chain, requires the addition of a linker. A common concern with SCTs is that the linker means a change in the original protein sequence. Researchers fear this change might interfere with TCR binding, affecting the physiological accuracy of antibodies or antigen-specific T cells identified using SCT MHCs. To address this concern, we performed extensive analysis of over 200 TCR-pMHC complex crystal structures to show that the linker in SCT MHCs does not interfere with the TCR-peptide-MHC interface. The linker is positioned away from the critical binding regions, ensuring that MHC-TCR interactions remain intact (Figure 5).

Figure 5. Structural analysis of TCR and peptide-MHC I/II interactions demonstrates the C-terminal linker does not interfere with the MHC-TCR binding interface. 

Should you choose SCT MHCs for your research?

Selection of recombinant MHC complexes will always need to be considered in the context of experiment goals and applications. Researchers who are targeting biologically relevant MHCs that contain innate PTMs may be better off using MHCs derived from mammalian cells for immunization and antibody screening. In this scenario, E. coli-sourced MHCs could even be used to counterselect and remove antibodies that have an affinity towards MHCs without PTMs. Conversely, there might be situations where researchers want antibodies specifically targeting MHCs without PTMs or with low PTMs. Then, MHCs derived from E. coli would be suitable for immunization and screening. 

That being said, advanced precision research in antibody drugs and T cell therapy increasingly requires the use of biologically relevant target proteins. The PTMs of mammalian-expressed SCT MHCs provide a distinct advantage of being more physiologically relevant when selecting for antibodies or T cells with high specificity and efficacy. This advantage becomes even more significant when researchers require precise control over MHC configurations, such as specific peptide loading or structural variations tailored to unique experimental designs. For example, researchers may isolate autologous T cells from peripheral blood mononuclear cells and evaluate cell responses to defined peptide/HLA pairs, including changes in inhibitory receptors that can modulate activation signals. In functional validation, assay readouts may include cytokines or other cell secreted markers, and some workflows include a cell rapid expansion protocol to increase cell numbers before downstream testing. When deeper specificity mapping is needed, teams may use a t cell cloning workflow or cell cloning workflow to derive monoclonal populations, including experiments using lysed target cells to confirm recognition under defined conditions.

Our MHC Class I and Class II Products & Services

Owing to the development of TCR mimic antibodies and T cell therapy, we’ve developed a large catalog selection of mammalian-expressed MHC complexes. This includes MHC Class I and Class II monomers and tetramers as well as peptide-ready MHCs which are MHC monomers or tetramers that can be loaded with an antigenic peptide in-house. Browse our full suite of products here or request a custom MHC complex. Additionally, we offer soluble TCR expression services and SPR analysis services to address MHC/TCR binding interactions. We also support related research through our SLC protein expression services, enabling high-quality production of membrane transport proteins for immune interaction studies.

References

1. Altman, J. D., H. Moss, P. A., R. Goulder, P. J., 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. https://doi.org/10.1126/science.274.5284.94
2. Hansen, T. H., Connolly, J. M., Gould, K. G., & Fremont, D. H. (2010). Translational and basic applications of peptide-MHCI single chain trimers. Trends in Immunology, 31(10), 363. https://doi.org/10.1016/j.it.2010.07.003