Expression and Purification of Soluble, Glycosylated T‐cell Receptors in Chinese Hamster Ovary Cells

Abstract

T‐cell receptors (TCRs) are glycoproteins that are expressed on the surface of T cells, which are a major component of the adaptive immune response. The glycans on the constant domain of this protein are involved in interactions with other proteins, such as galectin‐3, which mediates the immune response1. Additionally, altering the glycosylation of this protein has been associated with diseases such as Multiple Sclerosis1. Herein, we aim to express a soluble, glycosylated TCR in Chinese hamster ovary (CHO) suspension cells and purify it with the goal of characterizing the effects of glycosylation on TCR structure and function. To date, a soluble, unglycosylated TCR was expressed once, in 1990,2 but has not been replicated since. This project aims to express a glycosylated, soluble TCR with more current technology, which should result in more efficient expression.This project began with the design of a solubilized glycoprotein. We selected a starting sequence that has known binding affinity to ovalbumin and proceeded with modifications for optimized expression. The transmembrane sequence was removed after the last cysteine and replaced with a glycosylphosphatidylinositol (GPI) ‐ anchored sequence. This targets the protein to the endoplasmic reticulum (ER) and Golgi apparatus to ensure glycosylation. Following expression and glycosylation, the TCR should be localized to the surface of the cell, where the GPI sequence can be enzymatically cleaved using phosphatidylinositol‐phospholipase C (PI‐PLC), resulting in a soluble released protein. The α and β chains of the heterodimer were combined into a single plasmid using a 2A peptide. The use of 2A peptides instead of a more traditional technique, such as internal ribosome entry site (IRES), results in more efficient folding due to expression of stoichiometric ratios of both chains of the protein, as compared to a higher concentration of a single chain.The protein is transfected in CHO‐S cells and the expressed protein is harvested after 24 hours. The protein of interest is purified by affinity chromatography using a nickel column, followed by analysis with SDS‐PAGE. Imaging of the gels after surface cleavage with PI‐PLC and cell lysis confirmed that the protein was not localized to the plasma membrane as it was not recovered after enzymatic cleavage. However, the protein is purified from the inside of the cell after cell lysis. To determine the cellular localization of the protein we are using immunofluorescence using fluorescently labeled antibodies to target the His tag on the protein, as well as the ER, Golgi, and plasma membrane. Currently we are addressing the incorrect localization and analyzing methods to redirect the protein to the cell surface.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.