Cell-free co-expression of functional membrane proteins and apolipoprotein forming soluble nanolipoprotein particles.

@article{citeulike:3175478, 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, which leads 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 a delta 1-49 apolipoprotein A-1 fragment (delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoyl-phosphatidylcholine (DMPC), results in production of functional bacteriorhodopsin (bR), as demonstrated by a 5 nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FTIR difference spectra for the bR -> M transition. Importantly, the functional bR is solubilized in discoidal bR-NLPs as determined by atomic force microscopy (AFM). A survey study of other membrane proteins co-expressed with delta49A1 scaffold protein also significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general approach for expressing membrane proteins enabling further studies.}, address = {Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA 94551-9900.}, author = {Cappuccio, Jenny A A. and Blanchette, Craig D D. and Sulchek, Todd A A. and Arroyo, Erin S S. and Kralj, Joel M M. and Hinz, Angela K K. and Kuhn, Edward A A. and Chromy, Brett A A. and Segelke, Brent W W. and Rothschild, Kenneth J J. and Fletcher, Julia and Katzen, Federico and Peterson, Todd C C. and Kudlicki, Wieslaw A A. and Bench, Graham and Hoeprich, Paul D D. and Coleman, Matthew A A. }, citeulike-article-id = {3175478}, doi = {http://dx.doi.org/10.1074/mcp.M800191-MCP200}, issn = {1535-9484}, journal = {Molecular \& cellular proteomics : MCP}, keywords = {cloning, membrane-protein}, month = {July}, posted-at = {2008-08-31 01:33:35}, priority = {2}, title = {Cell-free co-expression of functional membrane proteins and apolipoprotein forming soluble nanolipoprotein particles.}, url = {http://dx.doi.org/10.1074/mcp.M800191-MCP200}, year = {2008} }

TY - JOUR ID - citeulike:3175478 L3 - citeulike-article-id:3175478 N2 - 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, which leads 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 a delta 1-49 apolipoprotein A-1 fragment (delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoyl-phosphatidylcholine (DMPC), results in production of functional bacteriorhodopsin (bR), as demonstrated by a 5 nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FTIR difference spectra for the bR -> M transition. Importantly, the functional bR is solubilized in discoidal bR-NLPs as determined by atomic force microscopy (AFM). A survey study of other membrane proteins co-expressed with delta49A1 scaffold protein also significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general approach for expressing membrane proteins enabling further studies. JF - Molecular & cellular proteomics : MCP SN - 1535-9484 AD - Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA 94551-9900. TI - Cell-free co-expression of functional membrane proteins and apolipoprotein forming soluble nanolipoprotein particles. KW - cloning KW - membrane-protein AU - Cappuccio, Jenny A AU - Blanchette, Craig D AU - Sulchek, Todd A AU - Arroyo, Erin S AU - Kralj, Joel M AU - Hinz, Angela K AU - Kuhn, Edward A AU - Chromy, Brett A AU - Segelke, Brent W AU - Rothschild, Kenneth J AU - Fletcher, Julia AU - Katzen, Federico AU - Peterson, Todd C AU - Kudlicki, Wieslaw A AU - Bench, Graham AU - Hoeprich, Paul D AU - Coleman, Matthew A PY - 2008/07/04/ UR - http://dx.doi.org/10.1074/mcp.M800191-MCP200 ER -