Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Abstract

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

Figure 1. In vitro folding of the KcsA protein obtained by CFPS

A) The tetrameric KcsA channel is depicted in ribbon representation. B) SDS-PAGE gel showing the native KcsA channel (lane 1) and the KcsA polypeptide obtained by CFPS (Lane 2). C) SDS−PAGE gel showing the KcsA protein before (lane 1) and after incorporation into lipid vesicles (lane 2). In B and C, the monomer (M) and the tetramer (T) bands are indicated. D) Size exclusion chromatography of the in vitro folded KcsA (iv-KcsA) channel. Inset: SDS-PAGE gel with lane 1) cellular expressed KcsA and 2) iv-KcsA. E) A representative single-channel trace for the iv-KcsA channel recorded at +150 mV in 10 mM succinate/150 mM KCl (pH 4.0) inside and 10 mM HEPES/150 mM KCl (pH 7.0) outside. F) Single channel currents as a function of voltage for iv-KcsA (○) and the cellular expressed KcsA (□) channels in the above solutions. The error bars represent the SD for 3 or more measurements.

Figure 2. Structure of the selectivity filter of the in vitro folded KcsA channel

A) Structure of the selectivity filter of iv-KcsA channel. The 2 F_o-F_c electron density of the selectivity filter of iv-KcsA channel contoured at 3.0 σ is shown with residues 71–80 as sticks, and the K⁺ ions in the selectivity filter shown as purple spheres. B) Superimposition of residues 71–80 of the iv- KcsA (red) and the cellular expressed KcsA channel (blue).

Figure 3. In vitro folding of the MVP protein obtained by CFPS

A) Schematic diagram showing the gating of the MVP channel. The MVP channel opens on membrane hyperpolarization and closes on depolarization. B) The native MVP channel migrates as a tetramer on SDS-PAGE (lane 1) while the unfolded MVP channel migrates as a monomer (lane 2). The MVP polypeptide obtained by CFPS was folded using lipid vesicles. C) SDS−PAGE gel showing the MVP protein before (lane 1) and after incorporation into lipid vesicles (lane 2). The monomer (M) and the tetramer (T) bands are indicated. D) Size exclusion chromatography of the in vitro folded MVP channel (iv-MVP). Inset: SDS-PAGE gel showing the purified iv-MVP channel. E) Representative single-channel traces for the iv-MVP channel recorded at −200 mV in a POPE: POPG (3:1) bilayer with 10 mM HEPES/150 mM KCl (pH 7.5) on both sides of the membrane. F) Single channel currents as a function of voltage for iv-MVP (○) and the cellular expressed MVP (□) channels in the above solutions. G) The voltage dependence of the open probability of the iv-MVP channels. The open probability is normalized to the value observed at −200 mV. The data are plotted as mean ± SD for 3 or more measurements.

Figure 4. CFPS of the LeuT transporter

A) Structure of the LeuT transporter shown in ribbon representation. B) SEC of the cellular expressed (blue), the unfolded (black) and the in vitro folded (red) LeuT transporter (iv-LeuT). The elution profile is observed for iv-LeuT is similar to the cellular expressed LeuT and is very distinct from the elution profile observed for the unfolded LeuT. C) Alanine uptake assay by the cellular expressed and iv-LeuT transporter. Alanine uptake assays were used to examine the functionality of LeuT. Time course of ¹⁴C-Ala uptake by iv-LeuT (red circles) and the cellular expressed LeuT (blue squares) in the presence of a Na⁺ gradient is shown. ¹⁴C-Ala uptake is not observed in the absence of a Na⁺ gradient for the in vitro folded (red open circles) or the cellular expressed transporter (blue open squares). Error bars indicate the SD for 3 measurements.

Scheme 1. Strategy for membrane protein expression by combining cell-free synthesis with in vitro folding

The various advantages of cell-free synthesis of membrane proteins is described.