Cell-free protein expression (in vitro transcription/translation) is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment, which lends itself to specific labeling with fluorescence, biotin, radioactivity or heavy atoms, via modified charged tRNA’s or amino acids. Cell-free protein expression systems provide quick access to proteins of interest and remain a staple in the collection of tools available for the elucidation of cellular pathways and mechanisms(1) as well as for high-throughput screening for drug discovery(2)(3). The advent of cell-free systems with higher expression levels has broadened the applications to include NMR-based structural proteomics and membrane protein purification. The open environment of the cell-free system grants flexibility, allowing addition of components during protein synthesis such as liposomes/detergents or microsomal membranes for membrane proteins, and it is impervious to synthesis of toxic proteins. Both prokaryotic (E. coli) and eukaryotic (rabbit reticulocyte [RRL], wheat germ and insect) protein synthesis systems are commercially available. With these systems, the input templates can be either plasmid DNA, PCR DNA or mRNA.
The eukaryotic cell-free protein synthesis systems (RRL and wheat germ) are either translation systems that are primed with mRNA or coupled transcription/translation (TnT®) systems supplemented with the optimal phage RNA polymerases (T7, SP6 or T3) and primed with plasmid DNA or PCR DNA containing the T7, SP6 or T3 promoter. Prokaryotic S30 transcription/translation systems rely on endogenous transcription machinery or may be supplemented with T7 RNA polymerases. Cell-free transcription and /or translation systems continue to offer considerable utility, especially in functional proteomics, and the recent development of the higher yield expression systems has expanded their application.
Numerous approaches can be used to study protein interactions, including immunoprecipitations, co-immunoprecipitations, pull-downs and protein microarrays, all of which can be used in conjunction with cell-free protein synthesis. Domain mapping, a method for identifying regions of proteins that interact, can be achieved by integrating pull-downs and in vitro protein synthesis. The SMC-1 (cohesion subunit) domain that binds to the microtubule-bound RNA export factor 1, Rae-1, was mapped using Flag pull-downs and protein expression in a RRL TnT® system(4). The Rae-1 binding site was pinpointed to a 21-amino acid region of SMC-1 947–967 and subsequently verified in HeLa cells. In a second example of domain mapping, the human requiem protein, REQ, binding domain mapping was determined for several partners, Brm, BRG 1, BAF60a , Ini 1 and p52, using GST pull-downs and RRL translation systems(5).
Functional protein microarrays have become not only a tool for protein interactions but a potential for diagnostics in ascertaining autoantibodies binding to their cognate antigens in plasma. Nucleic Acid Programmable Protein Array (NAPPA)(6),(7) consists of biotinylated DNA encoding open reading frames (ORFs) as GST fusions. The slides are coated with avidin and anti-GST antibody, so that the DNA is captured by the avidin and the cell-free expressed proteins are captured by the anti-GST antibody. Moreover, NAPPA has been demonstrated to be a powerful tool in examining autoimmune diseases(8). Plasma from patients with Ankylosing Spondylitis (AS), a common inflammatory rheumatic disease, was screened with two high-density NAPPA slides consisting of 3498 RRL-expressed proteins. The autoantigens were identified and analyzed to determine signaling cascades and tissue origin. Patients with AS had autoantibody responses towards connective, skeletal and muscular tissue, unlike those patients with Rheumatoid Arthritis and healthy controls. In a slight modification of the NAPPA experiment, researchers printed linear DNA as GFP C-terminal fusions with the anti-GFP antibody for capture and immobilization of fluorescent Arabidopsis proteins(9). A hybrid cell-free expression system (E. coli S30/wheat germ) was employed so that expression levels would be appropriate for mass spectrometry analysis. A third, unique protein array that also uses cell-free protein synthesis to generate the array is the HaloLink™ Protein Array. This array is distinguished by the covalent and oriented capture of the HaloTag® proteins generated by cell-free expression onto the slide via a HaloTag® ligand(10),(11).
Understanding Cellular Events
Exploiting the components or lack of specific components within a cell-free protein system to examine mechanisms of cellular events can be advantageous. The ubiquitin-proteasome activity in RRL allows examination of molecular mechanisms involving degradation/turnover of proteins. A recent study using RRL identified novel substrates (isoforms 1 and 2 of human glucokinase) for the ubiquitin-conjugating enzymes(12). By exploiting endogenous O-GlcNAcylation activity of RRL(13), researchers were able to translate deletion mutants of LXRα and LXRβ (liver X receptor) to determine which domain(s) contained the O-GlcNAc site(s)(14). RRL also was employed to examine the specific protease activity from human rhinovirus (HRV) and poliovirus (PV). Site-directed mutagenesis coalesced with in vitro protein synthesis permitted the identification of amino acids that could be crucial to interactions or enzymatic activities present during viral infection. Specifically, this study examined the mechanism for cleavage of Nup62 (nucleoporins) during infection with HRV and PV by site-directed mutagenesis of Nup62. The activity of HRV2 2A protease on Nup62 mutants expressed in vitro revealed six different positions that were cleaved(15).
Cell-free systems also may be used to gain understanding of virus and cellular stress mechanisms. Cap-dependent translation involves more than 12 translation initiation factors that recruit the 40S subunit to the 5´ end of the mRNA, whereas cap-independent translation, exhibited by cellular stress or viral mRNAs, can initiate translation using only an internal ribosome entry site (IRES). Thus IRES are RNA elements that recruit ribosomes without the initiation factors, so that only IRES containing mRNAs are translated during times of cellular stress or viral infection. The primer-extension inhibition or toeprinting assay has become an increasingly popular method to investigate the mechanisms of IRES in viral mRNAs. Not only can the toeprinting assay be done with purified ribosomal complexes(16), it also can be done directly in RRL(17)(18)(19). The mRNA is translated and elongation is arrested with inhibitors such as ediene or cycloheximide, locking the position of the ribosome on the transcript. The exposed region of the mRNA is copied into cDNA using a specific labeled primer and reverse transcriptase, generating toeprinting fragments dispensing information on the IRES mechanisms for recruitment.
Historically, most drug screens using eukaryotic translation systems have been secondary screens to assess the selectivity of small molecules for antibacterial properties. Hsp90 has emerged as a promising target for the development of anti-tumor agents(2). A novel primary screen based on the abundant supply of Hsp90 in the RRL to refold heat-denatured firefly luciferase has been developed(3). This screen is able to identify small-molecule inhibitors of either the N-or C-terminus of Hsp90. In a screen of 20,000 compounds (Z-factor = 0.62), 120 compounds were identified as potent Hsp90 inhibitors(3).
Membrane Protein and/or NMR Applications
Only about 250 structures of unique integral membrane proteins have been determined, which represents less than 1 % of the known protein structures despite recent advances in X-ray crystallography and NMR spectroscopy(20). Obtaining sufficient quantity of functional purified membrane protein for analysis is tremendously difficult. With the emergence of the high-yield expression systems (S30 and wheat germ), the synthesis of functional membrane proteins has been met with some success(21)(22). One example is cytochrome c oxidase (CcO) from Paracoccus denitrificans, a membrane protein complex composed of three distinct subunits that contain two redox -active copper centers and two heme A molecules. CcO has been synthesized successfully and assembled in an S-30 cell free system with the addition of E. coli cell membrane fractions(23). Spectral analysis also revealed that the complex was enzymatically active.
Recently, two exciting papers have emerged that combine cell-free expression of membrane proteins with NMR(24)(25). In the first study, the mechano-sensitive channel of large conductance from E. coli (MscL) a 75Kda homopentameric membrane protein was synthesized in the presence of detergents with 13C, 15N-label on the 16 isoleucine and on the 3 threonine residues in S30 cell-free system. Solid-state NMR was used for structure determination. In the second study, three backbone structures are revealed for the transmembrane (TM) domains of the three classes of E. coli histidine kinase receptors (HKRs). The TM domains were subjected to a cell-free combinatorial dual-labeling strategy and NMR providing insight to the structural motif of the TM domains. Two classes of the HRKs (ArcB and QseC) are both two-helical motifs, while the third class (KdpD) comprises a four-helical bundle with shorter second and third helices. This unique strategy gives rise to fast determination of vital backbone structures of membrane proteins.
Long a staple of the molecular biology laboratory, cell-free expression systems continue to be a vital tool for life science researchers. Such systems are proving invaluable tools in high-throughput studies and drug screens, and improvement to the systems, including the development of high-yield systems, are making it possible for researchers to study difficult proteins including transmembrane proteins. As life scientists begin to ask questions of cellular systems, of how proteins and other molecules in cells interact, cell-free expression systems will continue to be an integral part of these investigations.