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, 99 (23), 14652-7

A Cell-Free Protein Synthesis System for High-Throughput Proteomics

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A Cell-Free Protein Synthesis System for High-Throughput Proteomics

Tatsuya Sawasaki et al. Proc Natl Acad Sci U S A.

Abstract

We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.

Figures

Fig 1.
Fig 1.
Template activities of mRNAs having different UTRs. For each construct, the 5′ component (cap or Ω) is indicated first, followed by the 3′ component [length of UTR and presence of poly(A)]. Protein synthesis activity was measured as hot-trichloroacetic acid-insoluble radioactivity by using luciferase mRNA. (a) mRNA concentration was 0.1 μM for cap/549⋅pA and cap/549 and 0.2 μM for the other mRNAs. (b) Incubations were done for 4 h.
Fig 2.
Fig 2.
A cell-free expression vector and its performance. (a) Schematic illustration of pEU. (b) SDS/PAGE analysis of GFP produced during 14 days of reaction. mRNA produced by transcription of circular pEU was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1-μl aliquot of the mixture was run on the gel, and protein bands were stained with CBB. The arrow shows GFP; “st” designates an authentic GFP band (0.5 μg).
Fig 3.
Fig 3.
Parallel expression of cDNA into proteins by using the split-primers PCR technique. (a) Design of the split-type primers for the introduction of the required UTRs into cDNA sequences. (b and c) Expected PCR-generated DNAs and mRNA, respectively. (d) Split-primer PCR-generated products. (e) Transcription was carried out with 10-μl aliquots from the PCR samples in a 100-μl reaction. Transcripts were visualized by electrophoresis. (f) All of the transcript was used for batch-mode translation (50 μl, 4 h), and the protein products were analyzed by SDS/PAGE autoradiography.
Fig 4.
Fig 4.
A high-throughput production for screening proteins from cDNA libraries. Authentic (A) and GST-fused (G) proteins in the reaction mixtures after a semiautomated PCR/transcription and translation from 54 different cDNAs separated by SDS/PAGE and stained with CBB. T and S mark total translation product and the supernatant fraction, respectively, after centrifugation at 30,000 × g for 15 min.
Fig 5.
Fig 5.
Activity and folding of the cell-free-produced polypeptides. (a and b) Autophosphorylation activity of five Arabidopsis protein kinases. SDS/PAGE and CBB-stained gel of the partially purified products (a) and the autoradiogram (b). Lanes 1–6 in a and b represent At1g07150, At5g49760, At2g02800, At5g62710, and At4g35500, respectively. NC denotes samples from the reaction mixture incubated in the absence of mRNA. M denotes protein size marker. (c) HSQC spectrum of the hypothetical protein of the flowering locus T protein produced in the cell-free.

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