A cell-free protein synthesis system for high-throughput proteomics

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 PCRgenerated 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. With the sequencing of the genomes of various species, attention has turned to the structure, properties, and functional activities of proteins. However, rapid progress in the study of proteins encoded by the newly discovered genes is based on the availability of sufficient amounts of a large number of proteins. Currently, three strategies are being used for protein production: chemical synthesis, in vivo expression, and cell-free protein synthesis. The first two methods have severe limitations: chemical synthesis is not practical for the synthesis of long peptides (1), and in vivo expression can produce only those proteins that do not affect the physiology of the host cell (2). Cell-free translation systems, in contrast, can synthesize large proteins with a speed and accuracy that approach those of in vivo translation (3, 4), and they can express proteins that would otherwise interfere with host cell physiology. However, these systems are reputed to be unstable and thus inefficient (5). One of the most convenient and promising eukaryotic cell-free translation systems is based on wheat embryos that store all of the components of translation in a concentrated dried state, ready for protein synthesis as soon as germination starts. Recently, we found that conventional wheat germ extracts contain the RNA N-glycosidase tritin and other inhibitors of translation such as thionin, ribonucleases, deoxyribonucleases, and proteases. These inhibitors originate from the endosperm (6, 7). Extensive washing of wheat embryos to eliminate endosperm contaminants has resulted in extracts with a high degree of stability and activity. By using mRNA having 5Ј-m 7 GpppG (cap) and a poly(A)-tail (pA) with this extract, the translation reaction proceeds for Ͼ60 h, when it is performed in a dialysis bag with a continuous supply of substrate...

Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay

“…ORFs of human E3 ubiquitin ligases were amplified from a set of National Institutes of Health mammalian gene correction clones by PCR using target protein-specific sense primers beginning with the S1 sequence and the antisense primers AODA2306 (for pBluescriptR-based clones (27)) or pDONR221_ 1 st A4080 (5Ј-ATCTTTTCTACGGGGTCTGA-3Ј for pCMV-SPORT6, pOTB7, pCR4-TOPO, and pCR-BluntII-TOPObased clones). A second split-primer PCR was carried out using an SPu sense primer containing an SP6 promoter sequence, a deSP6E01 sense primer containing a GST tag sequence (28), and the respective AODA2303 (for AODA2306 (27)) or pDO-NR221_2 nd A4035 (5Ј-ACGTTAAGGGATTTTGGTCA-3Ј for pDONR221_1 st A4080) antisense primer. Expression of GSTtagged recombinant proteins was performed on 96-well plates using the GenDecoder 1000 protein synthesizer (CellFree Sciences, Japan) according to the protocol of the manufacturer.…”

“…This eukaryotic cellbased in vitro translation method offers several advantages over the E. coli system, particularly allowing the high-throughput expression of recombinant proteins from cloned ORF resources in vitro (18,19). In this study, to generate the protein library, template DNAs containing an ORF were amplified from a collection of National Institutes of Health mammalian gene correction clones encoding 96 RING finger-type E3 ubiquitin ligases and fused with a 5Ј terminal SP6 promoter and GST tag sequences by split primer PCR (28,39). Agarose gel electrophoresis and ethidium bromide staining of the split PCR product confirmed that 92 DNA templates were synthesized successfully (supplemental Fig.…”

Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease inNicotiana benthamiana

“…30 The recombinant NbAC was synthesized by a robotic protein synthesizer, Protemist ® DT (CellFree Sciences) according to the manufacturer's instructions.…”

RNA recognition mechanism of eukaryote tRNA (m7G46) methyltransferase (Trm8–Trm82 complex)

“…Yeast Trm8-Trm82 heterodimer was synthesized by a wheat germ in vitro translation system [25][26][27][28]. Because there was a 6 · His tag sequence expressed at the N-terminus of Trm82, the synthesized enzyme could be purified by Ni-NTA superflow column chromatography and DE52 column chromatography [28].…”