Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Abstract

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cellfree extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein ⌬1-49 apolipoprotein A-I fragment (⌬49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR 3 M transition. Importantly the functional bR was solubilized in discoidal bR⅐NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with ⌬49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

“…Transfer the dried protein-enriched filter papers to scintillation tubes (Zinsser Analytic), overlay with 3 mL of scintillation cocktail and let it shake on the orbital shaker for at least 1 h. Measure the incorporation of 14 C-leucine by liquid scintillation counting using the scintillation counter.…”

“…After incubating the lipid bilayer with the proteoliposomes, measure activity from the voltage-clamped lipid bilayers of the sec61 pore by a single channel amplifier single channel amplifier (EPC-10) and the data acquisition software Patchmaster connected to the multiplexer electronics port of the Orbit16 system [14][15][16]. Recordings are done at a sampling rate of 50 kHz with a 10 kHz Bessel filter (see Subheading 2.6, step 7).…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…This was accomplished by simultaneous cell-free expression of an apolipoprotein and BO in the presence of lipids and all-trans retinals, leading to the self-assembly of BR-containing nanolipoprotein particles (NLPs) [54]. In addition, high-yield cell-free synthesis of BR in the presence of small unilamellar liposomes has been demonstrated [122].…”

“…In a recent collaboration with M. Coleman at UC Davis, formerly a PhD student in my group, the rapid production of solubilized and functional BR in nanodiscs was demonstrated and characterized using FTIR difference spectroscopy [54]. This was accomplished by simultaneous cell-free expression of an apolipoprotein and BO in the presence of lipids and all-trans retinals, leading to the self-assembly of BR-containing nanolipoprotein particles (NLPs) [54].…”

One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

“…Consequently, detergents and membrane mimetics (3) are used to solubilize membrane proteins. To avoid cytotoxicity caused by in vivo expression and enable highthroughput production, cell-free systems are used to express membrane proteins in vitro (4)(5)(6)(7). Despite recent progresses, understanding the functions of membrane proteins is still a lengthy and difficult process.…”