Cell-free synthesis of functional aquaporin Z in synthetic liposomes.

The challenges involved in producing sufficient quantities of aquaporins for precise biophysical characterization have limited our knowledge of this important class of molecules. This article describes a cell-free protein synthesis method for producing high concentrations of the E. coli water transporter, aquaporin Z (AqpZ), in synthetic liposomes. To our knowledge, this is the first report of in vitro synthesis of a membrane protein directly into synthetic liposomes with verified function, (i.e., transport activity and selectivity). Titration of DOPC lipid vesicles added to the cell-free reaction show that production yields of active AqpZ are dependent on the concentration of DOPC lipid vesicles added to the cell-free reaction, with 224 +/- 24 lipids required per aquaporin monomer. Supplementation of the signal recognition particle receptor (FtsY) to the cell-free reaction increases production of vesicle-associated AqpZ but not active AqpZ. Cell-free reactions using 7 mg/mL lipids that were not supplemented with FtsY produced 507 +/- 11 microg/mL of vesicle-associated AqpZ that exhibited a specific water transport activity of (2.2 +/- 0.3) x 10(-14) cm(3) s(-1) monomer(-1). Proteinase K protection, activation energy determination, and selectivity against glycerol and urea transport also confirmed the production of correctly folded AqpZ. This technique is capable of producing milligram quantities of aquaporin that can be readily assayed for function, facilitating biophysical characterization and high-throughput analysis.

An integrated cell-free metabolic platform for protein production and synthetic biology.

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

High yield cell-free production of integral membrane proteins without refolding or detergents.

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Affinity purification of lipid vesicles.

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.