Cell-free protein synthesis or coupled in vitro transcription-translation (IVTT) is a means for small-scale synthesis of proteins, especially those that are toxic to cells. The ability to easily manipulate the reaction components and conditions makes this method particularly amenable to automation and miniaturization, enabling a myriad of applications covering both functional and structural proteomics (1,2,3,4,5,6,7). The most popular cell-free translation systems are extracts derived from Escherichia coli, wheat germ, rabbit reticulocytes, and insect cells (1,2,3,4,5,6,7). Recent efforts to improve IVTT-based protein synthesis include both the development of different cell extract formulations and the generation of new bacterial strains for making cell extracts by mutating the genes involved in the catabolism of certain amino acids (8). Modifications to enhance the yield of protein and to reduce the cost of using IVTT technology have also been published (9). These include the development of substrate- and energy-source replenishment protocols (10,11) and a continuous-flow cell-free translation system (12). An IVTT system based on reconstitution of purified proteins (13) is also available commercially. In addition, cell-free protein synthesis has also been achieved using microfluidic array devices (14) and immobilized DNA templates (15,16). These advancements have led to the commercialization of IVTT technology that is fully scalable from lab bench to production scale and designed to work with conventional bacterial cell culture reactors. Such large-scale production would enable next-generation protein therapeutics for the treatment of human disease (7) (www.sutrobio.com).

PCR-generated DNA preparations are often used as templates for IVTT synthesis of proteins. For example, split-primer PCR technology bypasses the requirement for the construction of expression plasmids because the regulatory sequences needed for expression can be incorporated into the template during PCR amplification (2,5,17) (www.promega.com). This strategy allows the construction of PCR-generated DNA for production of many proteins in parallel and eliminates many time-consuming cloning steps, and also lends itself to robotic automation for high-throughput protein expression. However, PCR is not convenient for template preparation on a very large scale. In addition, in some cases laboratories may already have suitable plasmid constructs available for cell-free protein synthesis, or may be using one of a recently described set of ligation-independent IVTT vectors (18). For these reasons, there are situations in which expression plasmids may be the preferred template choice.

For cases in which IVTT is performed with plasmids, it would be desirable to reduce the time and labor requirements for template preparation. Convenient, isothermal amplification of an expression plasmid can be done by multiply-primed rolling circle amplification (RCA) using the high-fidelity DNA polymerase of bacteriophage φ29 and exonu-clease-resistant random hexamer primers (19). Although DNA amplified this way has been shown to be suitable for genetic analysis such as DNA sequencing and genotyping, the suitability of RCA-generated DNA, which is known to have interruptions in both strands and a highly branched structure (19), has not yet been demonstrated for coupled IVTT applications. If such DNA templates are suitable for IVTT-based protein synthesis, it would be possible to carry out single-tube and high-throughput cell-free synthesis of proteins, starting from minute quantities of crude expression plasmids down to the amount present in single bacterial colonies. In this report, we provide evidence that multiply-primed RCA-DNA is suitable for IVTT-based protein synthesis, and that it is feasible to do single-tube RCA-coupled IVTT reactions starting with <100 pg input DNA.

We evaluated the relative efficiency of IVTT-based protein synthesis from RCA-amplified DNA compared with purified plasmid DNA by using a commercially available translation kit. The expression plasmid, which contains an optimized ribosomal binding site sequences for E. coli translation, carries the GFP gene with a C-terminal His₆ tag under the control of a T7 promoter. To generate RCA-DNA, 1 ng of the GFP expression plasmid was amplified for 90 min at 30°C using the GenomiPhi V2 DNA amplification kit (GE Healthcare, Piscataway, NJ, USA), and the amplification product was quantified using the PicoGreen assay (Invitrogen, Carlsbad, CA, USA). As expected, the 20-µL amplification reaction produced ~5 µg RCA-DNA, which was used for the IVTT reaction without any further purification. A 50-µL IVTT reaction was carried out for 4–6 h at 30°C as per the RTS 100 E. coli HY Kit protocol (Roche, Indianapolis, IN, USA) using 500 ng unpurified amplification product (RCA-DNA), 500 ng purified plasmid DNA, or 100 pg plasmid DNA to mimic the content of non-amplified plasmid in 500 ng RCA-DNA. The 100 pg plasmid is negligible compared with the 500 ng template required for IVTT reactions and was considered the no-template control. After IVTT, the reaction mixture was stored overnight at 4°C to allow folding of the GFP protein to occur to completion. The relative amount of GFP protein made was evaluated by measuring the fluorescence of the expressed protein using a fluorescence plate reader (Tecan US, Inc., Durham, NC, USA). Results shown in Figure 1 demonstrate that RCA-DNA is nearly as efficient as plasmid DNA for protein synthesis by IVTT. It should be noted that whereas the plasmid DNA was purified, supercoiled DNA, the RCA-DNA was an amplification reaction product used directly, without purification. Our study did not include sufficient data to determine the cause of the small difference in protein synthesis between purified and amplified DNA, but we suggest that salt or some other component of the amplification reaction process may have affected yields. The presence of the His tag in the newly synthesized protein was demonstrated by purification of the GFP protein using His-trap spin columns (GE Healthcare). Analysis of the purified proteins using SDS-PAGE (Figure 2) shows that IVTT-generated His-tagged GFP could be readily purified in this manner, and that its apparent size is as expected. In a similar manner, using a rabbit reticulocyte extract, we have also demonstrated the production of luciferase from an amplified expression plasmid (data not shown).