CD74 is the invariant chain of MHC class II molecules and plays an essential role in proper folding, intracellular transport, and function of MHC II complexes in antigen presenting cells. Beyond its role in antigen presentation, CD74 also functions as a membrane receptor for the cytokine MIF, which is involved in the regulation of inflammatory, proliferative, and pro-survival signaling pathways. CD74 is a transmembrane protein with multiple isoforms (p33, p35, p41, and p43), which differ in their intracellular localization and function. The extracellular domain of CD74 can be proteolytically shed from the membrane, generating soluble forms that retain the ability to bind MIF and modulate its biological activity. Whether the additional thyroglobulin domain, characteristic of the p41 isoform, influences MIF binding to the soluble form of CD74 remains unclear. In this thesis, we therefore investigated the production of recombinant MIF and two soluble forms of the CD74 extracellular domain, s_p33 and s_p41, and assessed their ability to form complexes with MIF.
Recombinant proteins were expressed in Escherichia coli strains BL21[DE3]pLysS and Rosetta-gami B[DE3]pLysS. Expression of s_p33 and s_p41 was confirmed by Western blot using anti-CD74 antibodies. Proteins were expressed as fusions with a polyhistidine tag and therefore purified by Ni-NTA affinity chromatography. Since both s_p33 and s_p41 were predominantly found in the insoluble fractions of bacterial lysates, urea-based solubilization followed by refolding was performed. The efficiency of refolding, as well as the oligomeric state and the presence of aggregated species, were assessed by SDS-PAGE and gel filtration. We obtained elution profiles of MIF, consistent with its monomeric and trimeric form. We also used gel filtration to assess the s_p33–MIF and s_p41–MIF interactions. In the s_p33 and s_p41 samples, we detected predominantly aggregated forms, indicating limited refolding efficiency and a low amount of correctly folded soluble protein. Consequently, the concentrations of biologically active forms of s_p33 and s_p41 were too low for reliable assessment of binding to MIF, and a formation of a complex therefore could not be confirmed. It was also not possible to conclude whether the thyroglobulin domain in s_p41 affects MIF binding. Prior to further interaction analysis, additional optimization of expression, purification, and refolding of recombinant CD74 forms is required, primarily to obtain larger amounts of correctly folded and soluble protein.
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