High-yield production of recombinant human myelin oligodendrocyte glycoprotein in SHuffle bacteria without a refolding step

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a model for central nervous system (CNS) autoimmune demyelinating diseases such as multiple sclerosis (MS) and MOG antibody-associated disease (MOGAD). Immunization with the extracellular domain of recombinant human MOG (rhMOG), which contains pathogenic antibody and T cell epitopes, induces B cell-dependent EAE for studies in mice. However, these studies have been hampered by rhMOG availability due to its insolubility when overexpressed in bacterial cells, and the requirement for inefficient denaturation and refolding. Here, we describe a new protocol for the high-yield production of soluble rhMOG in SHuffle cells, a commercially available E. coli strain engineered to facilitate disulfide bond formation in the cytoplasm. SHuffle cells can produce a soluble fraction of rhMOG yielding >100 mg/L. Analytical size exclusion chromatography multi-angle light scattering (SEC-MALS) and differential scanning fluorimetry of purified rhMOG reveals a homogeneous monomer with a high melting temperature, indicative of a well-folded protein. An in vitro proliferation assay establishes that purified rhMOG can be processed and recognized by T cells expressing a T cell receptor (TCR) specific for the immunodominant MOG_35-55 peptide epitope. Lastly, immunization of wild-type, but not B cell deficient, mice with rhMOG resulted in robust induction of EAE, indicating a B cell-dependent induction. Our SHuffle cell method greatly simplifies rhMOG production by combining the high yield and speed of bacterial cell expression with enhanced disulfide bond formation and folding, which will enable further investigation of B cell-dependent EAE and expand human research of MOG in CNS demyelinating diseases.

Keywords: Autoantigen; B cells; Experimental autoimmune encephalomyelitis (EAE); Myelin oligodendrocyte glycoprotein (MOG); Purification; SHuffle cells.

Figure 1.. Sequence and structure of MOG.

A) Amino acid sequence of human MOG. rhMOG is highlighted in purple with residues numbered in the absence and presence (in parentheses) of signal peptide. The disulfide bond of rhMOG is denoted with a dashed line. All cysteine residues of MOG are denoted in bold. B) Comparison of mouse MOG and rhMOG structures. Disulfide bonds are shown in ball-and-stick representation (sulfur atom in yellow). The rhMOG structure was predicted using Alphafold2. Structure renderings were created using ChimeraX.

Figure 2.. Effects of host strain and induction temperature on the expression of soluble fraction rhMOG.

A) Comparison of insoluble (“I”) and soluble (“S”) lysate fractions from A) BL21 (DE3) E.coli rhMOG and SHuffle E.coli rhMOG induced at 37 °C for 4 hr and B) SHuffle E.coli rhMOG induced at lower temperatures for 24 hr. All samples were analyzed by reducing SDS-PAGE and stained with Coomassie blue. Asterisk denotes the expected migration position of rhMOG.

Figure 3.. rhMOG is a pure, homogeneous and thermostable protein.

A) Reducing SDS-PAGE of rhMOG stained with Coomassie Blue. B) SEC-MALS analysis of rhMOG. Sample was separated on a Superdex 200 Increase 3.2/300 column at 0.15 mL/min in PBS. Normalized light scattering (blue, left axis) and calculated molecular weight (red, right axis) are shown. C) Effect of reducing agent on the thermostability of rhMOG. Differential scanning intrinsic tryptophan fluorescence of rhMOG in the absence (green) and presence (orange) of 10 mM DTT. Melting temperature (T_m) is denoted with a dashed line. Insert shows a close-up view of the structure of human MOG (Alphafold2 model) in the same orientation as that shown in Figure 1B). The sole tryptophan and disulfide bond of rhMOG are shown in ball-and-stick representation (grey carbon, blue nitrogen, yellow sulfur).

Figure 4.. Functional activity of rhMOG in vitro and in vivo.

A) Western blot of purified rhMOG using MOG 35-55-specific primary antibody NYRMOG. B) In vitro proliferation of CD4+ T cells isolated from 2D2 transgenic mice and stimulated with MOG 35-55 peptide (filled square) or rhMOG protein (filled circle). Mean proliferation, expressed as stimulation index (SI), and standard error of the mean (SEM) are shown. C) In vivo induction of B cell-dependent EAE in mice. Clinical score was determined daily for wild type (“WT”, filled triangle) and B cell deficient (“μMT”, open triangle) mice that were immunized with rhMOG. Mean clinical score and SEM are shown.