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 cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). 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 --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 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.

Highlights

  • 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)

  • We have developed a new cell-free co-expression method for rapid self-assembly of discoidal nanolipoprotein particles containing membrane protein (MP1⁄7NLPs)

  • Atomic force microscopy of samples containing bR separated by SEC revealed that 58% of the particles had an increased height (Fig. 4 and Table I) confirming bR incorporation and in agreement with previous Atomic Force Microscopy (AFM) characterization of bR structure in native membrane sheets [38]

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Summary

Introduction

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). As we have reported previously, NLPs have an average height of 5.0 Ϯ 0.5 nm with diameters ranging from 10 to 60 nm (Ϯ3%), depending upon the apolipoprotein and lipid used to make the NLP [10] These methods of in situ generation of NLPs have not been demonstrated previously in cell-free reactions, which may allow for a greater range of cofactors and additives for obtaining improved solubility and function. The first involves lengthy refolding after cell-free expression, whereas the second involves cell-free protein expression in the presence of lipids [19, 20] Both methods produced functional bacteriorhodopsin (bR) liposomes but with limited solubility and stability [19, 20]. This method may provide a general cellfree approach for membrane protein expression, making a wider range of membrane proteins available for study compared with current techniques

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