Engineered Chimera Protein Constructs to Facilitate the Production of Heterologous Transmembrane Proteins in E. coli

Abstract

To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, E. coli has the lowest cost and fastest turnover. However, many of the TMPs expressed in E. coli are misfolded. Several strategies have been developed to either direct the foreign TMPs to E. coli's membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of Mycobacterium tuberculosis, HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs' expression in E. coli.

Keywords: E. coli expression host of heterologous transmembrane proteins; protein engineering; soluble transmembrane proteins; transmembrane protein fusion strategies.

Figure 1

Schematic representation of the pMBP signal peptide (SP)-GPCR and pMBP-GPCR chimera constructs used to produce functional GPCRs in E. coli plasma membranes. In some cases, a protease site between pMBP and GPCR was introduced to remove the tag after purifying the protein.

Figure 2

Mistic fusion strategy to produce heterologous TMPs in E. coli. (A) NMR structure of mistic, PDB code 1YGM. The N- and C-termini are indicated. The protein folds into a 4-helix bundle. (B) Fusion strategy used to produce the aKv1.1 channel and aKv1.1 6TM. The figure was adopted from Ref. [20] with permission from Elsevier (License number 5665041200130). Only one mistic copy was fused to the N-terminal of aKv1.1. (C) Fusion strategy to produce eukaryotic rhodopsin variants. The figure adopted from Ref. [22] with permission from Elsevier (license number 5665050773775). Two copies of mistic were fused to the N- and C-termini of the TMPs.

Figure 3

MBP-Vpu chimera construct forms soluble oligomers. (A) Size exclusion chromatography (SEC) of purified 50 µM MBP-VPu (blue) shows that the protein forms oligomers with molecular weight greater than 250 kDa. The SEC of a mixture of protein molecular weight standards is in white brown, and the peaks corresponding to proteins with different molecular weights are indicated. The figure was adopted from Ref. [24] with permission from Elsevier (license number 5665051248242; accessed on 9 November 2023). (B) cryoEM analysis of soluble MBP-Vpu oligomers revealed predominantly hexamers and hexamer-to-heptamer equilibrium. The MBP moieties of each MBP-Vpu monomer are colored in pink, gray, shades of purple, and red. The electron density, which most likely represents the Vpu oligomerization core, is colored in blue. The figure was adopted from Ref. [25] under the conditions of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/; accessed on 9 November 2023).

Figure 4

Soluble apoAI–EmrE studies by SAXS. (a–c) Multiple views of the reconstructed particle envelope calculated ab initio from the dimer SAXS data (red circles in (d)) using DAMMIF [70]. The asterisk (*) in (a) denotes ApoAI lacking its 43-residue globular N-terminal domain. (d) Comparison between the experimental scattering profile of the dimer (red circles) and the theoretical profile calculated for the proposed model using CRYSOL software version 3.0 (solid line). The figure was reproduced from Ref. [26] under the conditions of Creative Commons Attribution 4.0 International License. Note, in addition to the C-terminal apoAI tag, the EmrE protein had an N-terminally fused mMBP tag as well. The dimeric state of EmrE was preserved in the soluble form.