Kodai Machida

N -terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

Reconstitution of the human chaperonin CCT by co-expression of the eight distinct subunits in mammalian cells

The eukaryotic cytosolic chaperonin CCT (chaperonin-containing TCP-1) assists folding of newly synthesized polypeptides. The fully functional CCT is built from two identical rings, each composed of single copies of eight distinct subunits. To study the structure and function of the CCT complex and the role of each subunit, a rapid and efficient method for preparing a recombinant CCT complex is needed. In this work, we established an efficient expression and purification method to obtain human recombinant CCT. BHK-21 cells were infected with a vaccinia virus expressing T7 RNA polymerase and transfected with eight plasmids, each encoding any one of the eight CCT subunits in the T7 RNA polymerase promoter/terminator unit. The CCT1 subunit was engineered to carry a hexa-histidine tag or FLAG tag in the internal loop region. Three days later, cells were harvested for purification of the CCT complex through tag-dependent affinity chromatography and gel filtration. The purified recombinant CCT complexes were indistinguishable from the endogenous CCT purified from HeLa cells in terms of morphology and function. In conclusion, the co-expression system established in this study should be a simple and powerful tool for reconstitution of a large multi-subunit complex.

Production methods for viral particles

Viral particles and virus-like particles (VLPs) or capsids are becoming important vehicles and templates in bio-imaging, drug delivery and materials sciences. Viral particles are prepared by infecting the host organism but VLPs are obtained from cells that express a capsid protein. Some VLPs are disassembled and then re-assembled to incorporate a material of interest. Cell-free systems, which are amenable to manipulating the viral assembly process, are also available for producing viral particles. Regardless of the production system employed, the particles are functionalized by genetic and/or chemical engineering. Here, we review various methods for producing and functionalizing viral particles and VLPs, and we discuss the merits of each system.

Reconstitution of eukaryotic translation initiation factor 3 by co-expression of the subunits in a human cell-derived in vitro protein synthesis system

Many biologically important factors are composed of multiple subunits. To study the structure and function of the protein complexes and the role of each subunit, a rapid and efficient method to prepare recombinant protein complexes is needed. In this work, we established an in vitro reconstitution system of eukaryotic translation initiation factor (eIF) 3, a protein complex consisting of 11 distinct subunits. A HeLa cell-derived in vitro coupled transcription/translation system was programmed with multiple plasmids encoding the 11 eIF3 subunits in total. After incubation for several hours, the eIF3 complex was purified through tag-dependent affinity chromatography. When eIF3l, one of the nonessential subunits of eIF3, was not expressed, the eIF3 complex that was devoid of eIF3l was still obtained. Both the 11 subunits complex and the eIF3l-less complex were as active as native eIF3 as observed by a reconstituted translation initiation assay system. In conclusion, the cell-free co-expression system should be a feasible and rapid system to reconstitute protein complexes.

Cell-free RNA replication systems based on a human cell extracts-derived in vitro translation system with the encephalomyocarditisvirus RNA.

To study the relationship between translation and replication of encephalomyocarditisvirus (EMCV) RNA, we established a cell-free RNA replication system by employing a human cell extracts-based in vitro translation system. In this system, a cis-EMCV RNA replicon encoding the Renilla luciferase (R-luc) or GFP and the viral regulatory proteins efficiently replicated with simultaneous translation of the encoded protein. To examine how translation of the replicon RNA, but not the translated products, affected replication, a trans-EMCV RNA replicon encoding R-luc and the RNA replication elements was next constructed. The trans-replicon RNA replicated only in the presence of the regulatory proteins pre-expressed in trans. Incubation with cycloheximide, puromycin or a dominant-negative eukaryotic translation initiation factor 4A following expression of the regulatory proteins almost completely inhibited not only translation of the trans-replicon RNA but also replication of the RNA, suggesting that EMCV RNA translation promotes replication of the RNA. In conclusion, the cell-free RNA replication systems should become useful tools for the study of the viral RNA replication.

Purification and visualization of encephalomyocarditisvirus synthesized by an in vitro protein expression system derived from mammalian cell extract

Virus particles are promising vehicles and templates for vaccination, drug delivery and material sciences. Although infectious picornaviruses can be synthesized from genomic or synthetic RNA by cell-free protein expression systems derived from mammalian cell extract, there has been no direct evidence that authentic viral particles are indeed synthesized in the absence of living cells. We purified encephalomyocarditis virus (EMCV) synthesized by a HeLa cell extract-derived, cell-free protein expression system, and visualized the viral particles by transmission electron-microscopy. The in vitro-synthesized EMCV particles were indistinguishable from the in vivo-synthesized particles. Our results validate the use of the cell-free technique for the synthesis of EMCV particles.

Cell-free analysis of polyQ-dependent protein aggregation and its inhibition by chaperone proteins.

Protein misfolding and aggregation is one of the major causes of neurodegenerative disorders such as Alzheimers disease, Parkinsons disease and Huntingtons disease. So far protein aggregation related to these diseases has been studied using animals, cultured cells or purified proteins. In this study, we show that a newly synthesized polyglutamine protein implicated in Huntingtons disease forms large aggregates in HeLa cells, and successfully recapitulate the process of this aggregation using a translation-based system derived from HeLa cell extracts. When the cell-free translation system was pre-incubated with recombinant human cytosolic chaperonin CCT, or the Hsc70 chaperone system (Hsc70s: Hsc70, Hsp40, and Hsp110), aggregate formation was inhibited in a dose-dependent manner. In contrast, when these chaperone proteins were added in a post-translational manner, aggregation was not prevented. These data led us to suggest that chaperonin CCT and Hsc70s interact with nascent polyglutamine proteins co-translationally or immediately after their synthesis in a fashion that prevents intra- and intermolecular interactions of aggregation-prone polyglutamine proteins. We conclude that the in vitro approach described here can be usefully employed to analyze the mechanisms that provoke polyglutamine-driven protein aggregation and to screen for molecules to prevent it.

H + emission under room temperature and non-vacuum atmosphere from a sol–gel-derived nanoporous emitter

A high proton-conducting P2O5-SiO2 nanoporous glass rod was prepared via sol–gel technique, and its tip was sharpened by a meniscus-etching method. The glass rod shows proton conductivity of 1 × 10−3 at room temperature after absorption of water molecules. A palm-sized proton gun was prepared by utilizing the glass rod as a H+ emitter. A high voltage (~2.5 kV) was applied between the tip of glass rod and an extraction electrode, and a high ionic current was successfully observed even under non-vacuum atmosphere at room temperature. Protonation reaction for polyaniline was confirmed from the structural changes of C=N in quinone to protonated C–N+. New applications of proton implantation will be expected especially in bioscience and medical technology.Graphical Abstract

Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding

A subset of the proteome is prone to aggregate formation, which is prevented by chaperones in the cell. To investigate whether the basic principle underlying the aggregation process is common in prokaryotes and eukaryotes, we conducted a large-scale aggregation analysis of ~500 cytosolic budding yeast proteins using a chaperone-free reconstituted translation system, and compared the obtained data with that of ~3,000 Escherichia coli proteins reported previously. Although the physicochemical properties affecting the aggregation propensity were generally similar in yeast and E. coli proteins, the susceptibility of aggregation in yeast proteins were positively correlated with the presence of intrinsically disordered regions (IDRs). Notably, the aggregation propensity was not significantly changed by a removal of IDRs in model IDR-containing proteins, suggesting that the properties of ordered regions in these proteins are the dominant factors for aggregate formation. We also found that the proteins with longer IDRs were disfavored by E. coli chaperonin GroEL/ES, whereas both bacterial and yeast Hsp70/40 chaperones have a strong aggregation-prevention effect even for proteins possessing IDRs. These results imply that a key determinant to discriminate the eukaryotic proteomes from the prokaryotic proteomes in terms of protein folding would be the attachment of IDRs.

Protein Synthesis in vitro: Cell-Free Systems Derived from Human Cells

When researchers wish to obtain recombinant proteins, a primary choice of the method is in most cases the expression in E. coli. If this system does not work for the protein of interest, they may turn to insect or mammalian cells. Protein expression in vitro may be chosen if these in vivo expression systems do not give the protein satisfactorily. There are several reasons why expression of some recombinant proteins in living cells is poor. If the protein to be expressed is toxic to host cells or inhibitory for growth, it should be difficult to express the protein to a high level. The cell-free system is derived from the extract from broken cells, and therefore the above-mentioned problems that occur in the living cells, if not all, are avoidable.