A Leucine Zipper Dimerization Strategy to Generate Soluble T Cell Receptors Using the Escherichia coli Expression System

T cell-mediated adaptive immunity plays a key role in immunological surveillance and host control of infectious diseases. A better understanding of T cell receptor (TCR) recognition of pathogen-derived epitopes or cancer-associated neoantigens is the basis for developing T cell-based vaccines and immunotherapies. Studies on the interaction between soluble TCR α:β heterodimers and peptide-bound major histocompatibility complexes (pMHCs) inform underlying mechanisms driving TCR recognition, but not every isolated TCR can be prepared in soluble form for structural and functional studies using conventional methods. Here, taking a challenging HIV-specific TCR as a model, we designed a general leucine zipper (LZ) dimerization strategy for soluble TCR preparation using the Escherichia coli expression system. We report details of TCR construction, inclusion body expression and purification, and protein refolding and purification. Measurements of binding affinity between the TCR and its specific pMHC using surface plasmon resonance (SPR) verify its activity. We conclude that this is a feasible approach to produce challenging TCRs in soluble form, needed for studies related to T cell recognition.


Introduction
T lymphocytes are vital mediators of adaptive immunity. Each αβ T cell contains a unique αβ T cell receptor (TCR) that engages with antigens and initiates signaling transduction [1][2][3]. T cells develop in the thymus, where they undergo TCR β-selection, positive selection, and negative selection before migrating to peripheral lymphoid tissues [4][5][6][7]. T cells remain naïve until they have encountered their specific antigens in the form of a peptide-bound major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells such as dendritic cells [8] and macrophages [9]. TCR recognition of pMHC leads to proliferation and differentiation into either CD4 + or CD8 + effector T cells [8,10]. V(D)J recombination during T cell development generates a highly diverse repertoire of TCRα and TCRβ chains, needed for sampling polymorphic pMHCs [11][12][13][14]. TCR diversity is essential for providing the immune system with the ability to combat novel diseases [15]. For example, recent studies demonstrate the critical role of HIV-1-specific cellular immunity in maintaining spontaneous control of HIV without medications [16,17]. A deeper structural understanding of T cell recognition of pathogen-derived pMHC complexes is fundamental to both vaccine development and immunotherapeutic approaches for viruses and cancers, but this has been technically challenging [18,19].
Although some TCR and TCR-pMHC complex structures have been determined, many are difficult to obtain, and thus, answers to many biological questions remain elusive [1,20].

Inclusion Body Expression and Extraction
TCRα-pET22b(+) and TCRβ-pET22b(+) constructs were transformed into E. coli BL 21(DE3) competent cells (purchased from Invitrogen, Inc., Waltham, MA, USA) for protein expression. Transformed cells were cultured in Luria-Bertani (LB) medium (purchased from United States Biological, Inc., Salem, MA, USA). When cell density reached an OD 600 value of~0.8, the expression was induced with 1 mM IPTG at 37 • C for 4 h. Inclusion body pellets were then suspended and lysed via sonification in extraction buffer (50 mM Tris-HCl, 1 M NaCl, 2% Triton-X100, pH 9.0). Inclusion bodies were separated at 10,000 rpm for 10 min. To sufficiently lyse the cells and improve recombinant protein purity, inclusion bodies were suspended and lysed twice or more. Inclusion bodies were washed three times using wash buffer (50 mM Tris, 20 mM EDTA, pH 8.5). Purified inclusion bodies were solubilized in 6 M guanidine-HCl and stored at −20 or −80 • C.

Protein Purification and Examination
Dialyzed protein solution was filtered using a PES 0.22 µm membrane (purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), purified by nickel-NTA (Ni-NTA) (purchased from Qiagen, Inc., Hilden, Germany), and purified by gravity-flow chromatography, size exclusion chromatography (SEC) (products of GE Healthcare, Inc., Chicago, IL, USA). Refolded proteins at each step were evaluated by SDS-page gel. Protein concentration was measured by NanoDrop™ 2000 (product of ThermoFisher Scientific, Inc., Waltham, MA, USA).

Protein Digestion
To enzymatically cleave the LZ, the purified TCRαβ was mixed with homemade TEV protease [29] in 10 mL cleavage buffer (20 mM Tris-HCl, 200 mM NaCl, pH 8.0) in a TCR:TEV mass-ratio of 50:1 at 4 • C for over 24 h. The protein-enzyme mixture was gently resuspended every 8-10 h. Following TEV digestion of the LZ, 1 mL Ni-NTA resin (equilibrated with 20 mM Tris-HCl, 200 mM NaCl, pH 8.0) was added to the proteinenzyme mixture and resuspended at 4 • C to allow the Ni-NTA resin to bind His-tagged nontarget proteins. Reverse Ni-NTA gravity-flow chromatography was used to remove the 6xHis tagged LZ and TEV protease. Digested TCR, without a His tag, was collected in the flow-through and further purified by SEC.

Surface Plasmon Resonance
To examine soluble TCR activity, TCR and corresponding pHLA binding affinity was measured via Biacore S200 (product of GE Healthcare, Inc., Chicago, IL, USA). The biotinylated pHLA was coated on a Cytiva Series S sensor SA chip (purchased from Cytiva, Inc., Marlborough, MA, USA), and the refolded TCR served as the analyte flowed over the chip surface. Biotinylated pHLAs were obtained from Dr. Søren Buus (University of Copenhagen, København, Denmark).

Software for Data Analysis
Structural analysis was performed in Pymol (Version 2.4.1, Schrödinger, Inc., New York, NY, USA). Protein gel graphic figures were generated by ChemiDocTMTouch Imaging System (Bio-rad Laboratories, Inc., Hercules, CA, USA). Protein sequences were analyzed using Protein Identification and Analysis Tools on the ExPASy Server (https://web.expasy. org/protparam/). TCRs and pHLAs binding affinities were analyzed by Biacore Insight Evaluation Software(Biacore S200, Cytiva, Inc., Marlborough, MA, USA). Figure 1A is an example of a TCRαβ engaging with a pMHC in the highest resolution TCR-pMHC complex structure available (PDB, 1OGA), reported by the Yvonne Jones Lab at the University of Oxford (Oxford, United Kingdom) [27]. The yellow and blue ribbons represent the TCRα and TCRβ chains, respectively. The class I MHC heavy chain is shown in green and the beta-2 microglobulin (β2m) light chain in magenta. The antigenic peptide is represented by a red line lying in the antigen-binding domain of the heavy chain.

TCRα and TCRβ Chain Construct Design and Protein Production Workflow
(https://web.expasy.org/protparam/). TCRs and pHLAs binding affinities were analyzed by Biacore Insight Evaluation Software(Biacore S200, Cytiva, Inc., Marlborough, MA, USA). Figure 1A is an example of a TCRαβ engaging with a pMHC in the highest resolution TCR-pMHC complex structure available (PDB, 1OGA), reported by the Yvonne Jones Lab at the University of Oxford (Oxford, United Kingdom) [27]. The yellow and blue ribbons represent the TCRα and TCRβ chains, respectively. The class I MHC heavy chain is shown in green and the beta-2 microglobulin (β2m) light chain in magenta. The antigenic peptide is represented by a red line lying in the antigen-binding domain of the heavy chain.

TCRα and TCRβ Chain Construct Design and Protein Production Workflow
While soluble MHCs are relatively straightforward to prepare, producing soluble TCRs remains a major challenge. To overcome this bottleneck, a pair of charge complementary LZ sequences were added to the C-terminus of each TCRα and TCRβ chain (Figure 1A,B). Additionally, to facilitate protein purification, a 6xHis tag was added to the TCRα chain C terminal end, and a TEV protease cleavage sequence was inserted between the LZ and TCRα and β chains. Engineered TCRα and TCRβ chains from the HIV-specific CTL clone C8 (a QW9-HLA-B57-tetramer sorted CTL clone) were expressed as inclusion bodies in E. coli and refolded into soluble protein. To increase TCRαβ stability, we introduced two free cysteine amino acids in each TCR chain, one within the LZ and the other within the constant domain of each TCRα and β chain ( Figure 1B). Νotably, we also attempted to engineer an LZ construct without free cysteines but failed to produce our target protein. During proper refolding, four free cysteines form two inter-disulfide bonds to lock the dimer ( While soluble MHCs are relatively straightforward to prepare, producing soluble TCRs remains a major challenge. To overcome this bottleneck, a pair of charge complementary LZ sequences were added to the C-terminus of each TCRα and TCRβ chain ( Figure 1A,B). Additionally, to facilitate protein purification, a 6xHis tag was added to the TCRα chain C terminal end, and a TEV protease cleavage sequence was inserted between the LZ and TCRα and β chains.
Engineered TCRα and TCRβ chains from the HIV-specific CTL clone C8 (a QW9-HLA-B57-tetramer sorted CTL clone) were expressed as inclusion bodies in E. coli and refolded into soluble protein. To increase TCRαβ stability, we introduced two free cysteine amino acids in each TCR chain, one within the LZ and the other within the constant domain of each TCRα and β chain ( Figure 1B). Notably, we also attempted to engineer an LZ construct without free cysteines but failed to produce our target protein. During proper refolding, four free cysteines form two inter-disulfide bonds to lock the dimer ( Figure 1C). LZ charge complementarity on the TCRα and TCRβ facilitate TCRα-TCRβ heterodimer but prevent TCRα-TCRα and TCRβ-TCRβ dimerization. Homemade TEV protease was then used for digestion to remove the LZ ( Figure 1C).

Inclusion Bodies Expression, Purification and Solubilization
A high yield of engineered C8 TCRα (termed C8A-LZ(+)) and TCRβ (termed C8B-LZ(−)) inclusion bodies were expressed in E. coli. The C8B-LZ(−) inclusion bodies fused with an acidic LZ sequence were purified and denatured easily by following general protocols. However, after applying the same extraction protocol to C8A-LZ(+) inclusion bodies, inclusion body pellets were difficult to solubilize using urea. Guanidine-HCl allowed for better solubility but consistently resulted in high A260/280 values, measured by NanoDrop TM 2000 ( Figure 2A). A pure protein sample A260/280 ratio is~0.55, and a higher ratio may indicate contamination of isolated proteins with DNA [30]. This could result from E. coli extracted inclusion bodies or soluble proteins having a high isoelectric point (pI) due to numerous positively charged residues. Our alkaline LZ fused C8A-LZ(+) has a globally high pI of 9.17, and the LZ(+) itself possesses a pI of 10.56 ( Figure 2B). These data suggested an optimized protocol was needed for C8A-LZ(+) inclusion body purification.

Inclusion Bodies Expression, Purification and Solubilization
A high yield of engineered C8 TCRα (termed C8A-LZ(+)) and TCRβ (termed C8B-LZ(−)) inclusion bodies were expressed in E. coli. The C8B-LZ(−) inclusion bodies fused with an acidic LZ sequence were purified and denatured easily by following general protocols. However, after applying the same extraction protocol to C8A-LZ(+) inclusion bodies, inclusion body pellets were difficult to solubilize using urea. Guanidine-HCl allowed for better solubility but consistently resulted in high A260/280 values, measured by NanoDrop TM 2000 ( Figure 2A). A pure protein sample A260/280 ratio is ~0.55, and a higher ratio may indicate contamination of isolated proteins with DNA [30]. This could result from E. coli extracted inclusion bodies or soluble proteins having a high isoelectric point (pI) due to numerous positively charged residues. Our alkaline LZ fused C8A-LZ(+) has a globally high pI of 9.17, and the LZ(+) itself possesses a pI of 10.56 ( Figure 2B). These data suggested an optimized protocol was needed for C8A-LZ(+) inclusion body purification. To address the concern of C8A-LZ(+) inclusion body DNA contamination, we combined multiple strategies (summarized in Figure 2C) to reduce DNA and inclusion body binding: (1) Lysis buffer and wash buffer pH was increased to lower protein surface charge; (2) Lysis buffer and wash buffer salt concentration was increased to improve ion strength and weaken DNA-protein interaction; (3) Arginine was added to lysis buffer as To address the concern of C8A-LZ(+) inclusion body DNA contamination, we combined multiple strategies (summarized in Figure 2C) to reduce DNA and inclusion body binding: (1) Lysis buffer and wash buffer pH was increased to lower protein surface charge; (2) Lysis buffer and wash buffer salt concentration was increased to improve ion strength and weaken DNA-protein interaction; (3) Arginine was added to lysis buffer as a DNA binding competitor; (4) Additional sonications were performed during lysis for increased separation of protein and DNA; (5) 6 M Guanidine hydrochloride was used to solubilize inclusion bodies. Utilizing these strategies, we observed much lower A260/280 for C8A-LZ(+) (Figure 2A).
Interestingly, we also found that a lower temperature can improve DNA contamination removal. Inclusion bodies solubilized in guanidine-HCl precipitate into white pellets after overnight storage at −20 • C. Proteins were then spun down at 12,000 rpm (4 • C) to remove pellets, and A260/280 of the supernatant reached~0.7 (Figure 2A).

Refolding of Soluble C8 TCR
The refolding system for this challenging C8 TCR required optimization of several buffer components. We considered three important factors: pH, GSH:GSSG ratio, and C8A-  Figure 2D). As reported by Coutard B. et al., acidic pI proteins prefer alkaline refolding buffers, while alkaline pI proteins prefer acidic refolding buffers [31]. The C8A-LZ(+) has a global pI of 9.17. On one hand, C8 TCRα and TCRβ without LZs have highly acidic pIs ( Figure 2B) that optimally refold at pH 8.0; on the other hand, the two LZ sequences have complementary charges that facilitate C8 TCRαβ dimerization and require a pH closer to 7. Moreover, a relatively alkaline pH provides an oxidized environment for disulfide bond formation. Meanwhile, the ratio of GSH:GSSG was also optimized to promote proper disulfide bond formation. Lastly, based on the stability and solubility of each C8A-LZ(+) and C8B-LZ(−) chain, varying ratios of C8A-LZ(+):C8B-LZ(−) in the refolding system were tested as well.
Following experimentation, several conditions were optimized and summarized in Figure 2D,E. Refolded C8A-LZ(+):C8B-LZ(−) inclusion bodies were dialyzed in two manners: (i) A series of buffers with varying denaturant concentrations (ii) The same buffers without any denaturants. The yield of successfully refolded protein under each condition was identified by SDS-page gel. Our results indicated (1) a GSH:GSSG ratio of 5 mM:0.5 mM and a C8A-LZ(+):C8B-LZ(−) ratio of 1:1.5 were optimal (2) two dialysis exchanges with double distilled water (ddH 2 O) only followed by two exchanges with 10 mM Tris buffer (pH 8.0) were optimal for dialysis.

Soluble C8 TCR Purification
We scaled up our refolding volume to 1 L once our refolding system was optimized. To separate the soluble protein from the refolding system, we combined nickel affinity purification and SEC. We first concentrated our refolded protein from 1 L to 100 mL before incubating the protein for over 2 h with Ni-NTA Resin (purchased from Qiagen, Inc., Hilden, Germany). After washing and eluting the Ni-NTA resin with 10 mM to 200 mM imidazole at stepwise concentrations, we used SDS-page gels to identify the components of each sample fraction ( Figure 3A,B).

C8-LZ TCR and QW9-B*57 Binding Affinity Measurements
To verify the functionality of our refolded C8-LZ, we tested its binding affinity with HLA-B*57, which presents the HIV Gag protein derived antigenic peptide QW9 (QW9_1). With a QW9 variant (QW9_2) bound to the same HLA-B*57 as a control, we performed surface plasmon resonance (SPR) to measure C8-LZ binding with QW9_1-B57 and QW9_2-B57. Using C8-LZ as the analyte, biotinylated QW9_1-HLA-B*57 and QW9_2-HLA-B*57 were coated on the same streptavidin sensor chip (Series S sensor SA). Refolded C8-LZ TCR had a measurable binding affinity with its specific peptide QW9_1 bound Fractions containing target protein were merged and concentrated using 30 kD molecular weight cut-off centrifugal filters (purchased from MilliporeSigma, Inc., Burlington, MA, USA) to a small volume for s200 Superdex SEC ( Figure 3C). Protein fractions were then Cells 2022, 11, 312 7 of 12 concentrated and loaded for a second and third round of s200 Superdex SEC ( Figure 3D,E). Results show the protein achieved high purity following three rounds of purification ( Figure 3F).

C8-LZ TCR and QW9-B*57 Binding Affinity Measurements
To verify the functionality of our refolded C8-LZ, we tested its binding affinity with HLA-B*57, which presents the HIV Gag protein derived antigenic peptide QW9 (QW9_1). With a QW9 variant (QW9_2) bound to the same HLA-B*57 as a control, we performed surface plasmon resonance (SPR) to measure C8-LZ binding with QW9_1-B57 and QW9_2-B57. Using C8-LZ as the analyte, biotinylated QW9_1-HLA-B*57 and QW9_2-HLA-B*57 were coated on the same streptavidin sensor chip (Series S sensor SA). Refolded C8-LZ TCR had a measurable binding affinity with its specific peptide QW9_1 bound HLA-B*57 ( Figure 4) with a dissociation constant of 130 µM, within the expected 1-200 µM range of TCR-pMHC binding [32]. However, no binding was detected for the QW9_2 variant bound HLA-B*57 ( Figure 4). These results suggest that our refolding strategy can produce a competent TCR capable of specific binding to its cognate pMHC.

C8-LZ TCR and QW9-B*57 Binding Affinity Measurements
To verify the functionality of our refolded C8-LZ, we tested its binding affinity with HLA-B*57, which presents the HIV Gag protein derived antigenic peptide QW9 (QW9_1). With a QW9 variant (QW9_2) bound to the same HLA-B*57 as a control, we performed surface plasmon resonance (SPR) to measure C8-LZ binding with QW9_1-B57 and QW9_2-B57. Using C8-LZ as the analyte, biotinylated QW9_1-HLA-B*57 and QW9_2-HLA-B*57 were coated on the same streptavidin sensor chip (Series S sensor SA). Refolded C8-LZ TCR had a measurable binding affinity with its specific peptide QW9_1 bound HLA-B*57 ( Figure 4) with a dissociation constant of 130 μM, within the expected 1-200 μM range of ΤCR-pMHC binding [32]. However, no binding was detected for the QW9_2 variant bound HLA-B*57 (Figure 4). These results suggest that our refolding strategy can produce a competent TCR capable of specific binding to its cognate pMHC.

TEV Digestion for Leucine Zipper Removal
Samples for crystallization require high purity and homogeneity. However, an LZ at the TCRαβ C-terminus could affect molecule packing for growing TCR or TCR-pMHC complex crystals [33]. To address this issue, we inserted a TEV protease cleavage site between the LZ and TCRαβ. We proceeded by using homemade 6xHis tagged TEV protease. Digested TCRαβ was then separated using a reverse Ni-NTA column. The LZ-6xHis, undigested TCR, and TEV protease could then bind the Ni-NTA column, while digested TCRαβ flowed through the column at a low imidazole concentration ( Figure 5A). Figure 5B depicts our digestion workflow. Furthermore, additional purification can be done via SEC. Figure 5C shows our digested C8 TCRαβ purified via s200 Superdex SEC.

Application to TCRs beyond C8 TCR
To determine if our proposed strategy can be applied to TCRs beyond C8 TCR, we examined two additional TCR constructs, C8-Ig and C3-Ig, in which the TCRα chain was modified with an extra Ig fold domain ( Figure 6A). C8 is the same previously described TCR, and C3 is another HIV-specific TCR. This TCR-Ig construct design comes from one of our ongoing cross-link study projects, in which the Ig-fold domain is a target of another protein derived domain. Despite unsuccessful refolding of C8-Ig and C3-Ig TCRs using conventional approaches, the LZ strategy efficiently facilitated the refolding of these two TCRs. Following our optimized refolding and purification protocol described previously, we achieved high yields of these TCRs. Taking C8-Ig-LZ as an example, Ni-NTA column pu-rification is similar to purification for C8-LZ ( Figure 6B). Figure 6C displays s200 Superdex SEC for C8-Ig. Similarly, for C3-Ig-LZ, Figure 6D shows the result following the third-round of s200 Superdex SEC. C3-Ig-LZ protein fractions, alongside a conventionally refolded and purified C3 TCR for comparison, were examined by SDS-page gel ( Figure 6E). These results suggest that our engineered LZ for dimerization can serve as a general strategy for preparing soluble TCRs. Samples for crystallization require high purity and homogeneity. However, an LZ at the TCRαβ C-terminus could affect molecule packing for growing TCR or TCR-pMHC complex crystals [33]. To address this issue, we inserted a TEV protease cleavage site between the LZ and TCRαβ. We proceeded by using homemade 6xHis tagged TEV protease. Digested TCRαβ was then separated using a reverse Ni-NTA column. The LZ-6xHis, undigested TCR, and TEV protease could then bind the Ni-NTA column, while digested TCRαβ flowed through the column at a low imidazole concentration ( Figure 5A). Figure  5B depicts our digestion workflow. Furthermore, additional purification can be done via SEC. Figure 5C shows our digested C8 TCRαβ purified via s200 Superdex SEC.

Application to TCRs beyond C8 TCR
To determine if our proposed strategy can be applied to TCRs beyond C8 TCR, we examined two additional TCR constructs, C8-Ig and C3-Ig, in which the TCRα chain was modified with an extra Ig fold domain ( Figure 6A). C8 is the same previously described TCR, and C3 is another HIV-specific TCR. This TCR-Ig construct design comes from one of our ongoing cross-link study projects, in which the Ig-fold domain is a target of another protein derived domain. Despite unsuccessful refolding of C8-Ig and C3-Ig TCRs using conventional approaches, the LZ strategy efficiently facilitated the refolding of these two TCRs. Following our optimized refolding and purification protocol described previously, we achieved high yields of these TCRs. Taking C8-Ig-LZ as an example, Ni-NTA column purification is similar to purification for C8-LZ ( Figure 6B). Figure 6C displays s200 Superdex SEC for C8-Ig. Similarly, for C3-Ig-LZ, Figure 6D shows the result following the third-round of s200 Superdex SEC. C3-Ig-LZ protein fractions, alongside a conventionally refolded and purified C3 TCR for comparison, were examined by SDS-page gel ( Figure  6E). These results suggest that our engineered LZ for dimerization can serve as a general strategy for preparing soluble TCRs.

Discussion
To obtain soluble TCRs for structural, biophysical, and functional studies, we developed a general LZ dimerization strategy for challenging TCR production. Our biochemical data show that this strategy can generate soluble TCR α:β heterodimers using the E. coli expression system. Furthermore, our SPR data using a soluble C8 TCR show measurable binding with its specific pMHC while binding was abrogated by the same MHC pre-

Discussion
To obtain soluble TCRs for structural, biophysical, and functional studies, we developed a general LZ dimerization strategy for challenging TCR production. Our biochemical data show that this strategy can generate soluble TCR α:β heterodimers using the E. coli expression system. Furthermore, our SPR data using a soluble C8 TCR show measurable binding with its specific pMHC while binding was abrogated by the same MHC presenting an epitope variant. This is consistent with sophisticated TCR-pMHC specificity interactions [34]. Moreover, the LZ can be removed using TEV protease, making the digested TCR more suitable for structural studies, particularly for crystallography by reducing potential artifacts [33]. Of note, strategies for extracting TRA-LZ(+) inclusion bodies may be significant for the preparation of inclusion bodies or soluble forms of other alkaline proteins.
Given TCR recognition of an antigen presented by an MHC molecule is the critical first step for T cell function activation [8,10], detailed information regarding TCR-pMHC interactions is key to addressing basic immunological questions such as T cell cross-reactivity and viral immune escape [35] and can direct the development of immunotherapies such as TCR-like CARs targeting neoepitopes [36] and bispecific antibodies targeting both TCR and pMHC [37,38]. CAR-T cells are often designed by fusing T cell signaling transduction elements with an antibody's single-chain variable fragment (scFv), targeting a cancer biomarker [39]. However, neoantigens and viral epitopes presented by MHC molecules on cancer cells and infected cells pose as alternative targets. Therefore, a potent pMHC-specific TCR poses as a promising strategy for CAR-T or TCR-T cell therapy [40,41].
The antigen-binding fragment (Fab) region of an antibody has a highly similar 3dimensional architecture to TCRs, composed of Ig domains [42], as shown using representative examples in Figure 7A,B. However, unlike a TCR with an FG loop in the F and G β strands, Fab does not contain such a loop region ( Figure 7B). Interestingly, when superimposed, the Fab and TCR C termini share highly similar geometry ( Figure 7C). In order to produce soluble Fabs, gene fragments are typically integrated into an IgG framework and expressed in mammalian cells [43]. Fabs can then be digested from purified IgG; however, this process is costly. Based on the structural similarity of Fab and TCR, our LZ dimerization strategy for refolding may be explored as an approachable and economical way to produce Fabs for structural and therapeutic studies. and expressed in mammalian cells [43]. Fabs can then be digested from purified IgG; however, this process is costly. Based on the structural similarity of Fab and TCR, our LZ dimerization strategy for refolding may be explored as an approachable and economical way to produce Fabs for structural and therapeutic studies. Future directions of our ongoing work focus on structural and functional related experiments for C8 while also testing more TCRs to further assess the success rate of our approach. Our strategy provides an option for producing challenging soluble TCRs, such as our reported C8, C8-Ig and C3-Ig, that cannot be produced using conventional methods. Based on our long-term work on various TCRs, we propose that for a newly isolated Future directions of our ongoing work focus on structural and functional related experiments for C8 while also testing more TCRs to further assess the success rate of our approach. Our strategy provides an option for producing challenging soluble TCRs, such as our reported C8, C8-Ig and C3-Ig, that cannot be produced using conventional methods. Based on our long-term work on various TCRs, we propose that for a newly isolated TCR, the conventional method using cysteine engineered constant domains of TCRα and TCRβ for E. coli expression should be attempted first [23]. If unsuccessful, our present method should be attempted next, before the mammalian expression system as this final method introduces the potential of post-translational modifications and high-cost related concerns. The advantage to our LZ dimerization strategy using the E. Coli system is that it is an all-around feasible method that is low-cost, time-efficient, and suitable for structural studies. Our approach builds upon the conventional E. coli expression system strategy to allow for the soluble production of challenging TCRs, needed for studies related to T cell recognition.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.