Cell-free protein synthesis of CD1E and B2M protein and in vitro interaction

Abstract

CD1E, one of the most important glycolipid antigens on T cell membranes, is required for glycolipid antigen presentation on the cell surface. Cell-based recombinant expression systems have many limitations for synthesizing transmembrane proteins such as CD1E, including low protein yields and miss folding. To overcome these challenges, here we successfully synthesized high-quality soluble CD1E using an E.coli cell-free protein synthesis system (CFPS) with the aid of detergent. Following purification by Ni²⁺ affinity chromatography, we were able to obtain CD1E with ≥90% purity. Furthermore, we used the string website to predict the protein interaction network of CD1E and identified a potential binding partner━B2M. Similarly, we synthesized soluble B2M in the E.coli CFPS. Finally, we verified the interaction between CD1E and B2M by using Surface Plasmon Resonance (SPR). Taken together, the methods described here provide an alternative way to obtain active transmembrane protein and may facilitate future structural and functional studies on CD1E.

Keywords: B2M; CD1E; Cell-free protein synthesis; Membrane protein purification; Protein expression.