Toru Ezure

Cell-free protein synthesis system prepared from insect cells by freeze-thawing.

We established a novel cell‐free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and β‐galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5′‐untranslated region (5′‐UTR) of mRNA. The extract was prepared by freeze‐thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5′‐UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5′‐UTRs tested. As a result, in a batch reaction approximately 71 μg of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell‐free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5′‐UTR of mRNA, approximately 45 μg/mL of luciferase was synthesized in an Sf21 cell‐free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell‐free systems may be a useful tool for simple synthesis in post‐genomic studies as a novel protein production method.

Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system.

To establish a strategy for the comprehensive identification of human N‐myristoylated proteins, the susceptibility of human cDNA clones to protein N‐myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell‐free protein synthesis system. One‐hundred‐and‐forty‐one cDNA clones with N‐terminal Met‐Gly motifs were selected as potential candidates from ∼2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N‐myristoylation was evaluated using fusion proteins, in which the N‐terminal ten amino acid residues were fused to an epitope‐tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N‐myristoylated. The metabolic labeling experiments both in an insect cell‐free protein synthesis system and in the transfected COS‐1 cells using full‐length cDNA revealed that 27 out of 29 proteins were in fact N‐myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N‐myristoylated proteins that have not been reported previously to be N‐myristoylated, indicating that this strategy is useful for the comprehensive identification of human N‐myristoylated proteins from human cDNA resources.

A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

Development of an Insect Cell-Free System

Cell-free protein synthesis systems offer production of native proteins with high speed, even for the proteins that are toxic to cells. Among cell-free systems, the system derived from insect cells has the potential to carry out post-translational modifications that are specific to eukaryotic organisms, as occurs in the rabbit reticulocyte system. In this review, we describe development of this insect cell-free system and its applications.

Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS.

Human Cu, Zn-superoxide dismutase (hSOD1) is a homodimer that coordinates one copper and one zinc ion per monomer. These metal ions contribute to its enzymatic activity and structural stability. In addition, hSOD1 maintains an intra-subunit disulfide bond formed in the reducing environment of the cytosol and is active under a variety of stringent denaturing conditions. We report the expression of hSOD1 in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) insect cells, and its structural analysis including the status of the sole intra-subunit disulfide bond by mass spectrometry. By using this system hSOD1 was obtained in a soluble active form after addition of Cu(2+) and Zn(2+) and was purified with a yield of approximately 33 microg from 1 ml of reaction volume. Both enzymatic and structural analyses of the recombinant hSOD1 indicate that it was completely identical to the protein isolated from human erythrocytes.

A simple fed-batch method for transcription and insect cell-free translation.

We tested a one batch reaction method for the transcription and the insect cell-free translation from undigested plasmids without any centrifugation steps. The efficiency of protein synthesis reached 74-112% of that achieved using the conventional procedure. This simplified method will help expedite the high throughput insect cell-free protein production.

Preparation of N-Acylated Proteins Modified with Fatty Acids Having a Specific Chain Length Using an Insect Cell-Free Protein Synthesis System

To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.

Expression and Chain Assembly of Human Laminin-332 in an Insect Cell-Free Translation System

Laminins are a family of large heterotrimeric glycoproteins comprising α, β, and γ chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the β3-γ2 heterodimer and the α3-β3-γ2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The α3-β3 and the α3-γ2 heterodimer were not detected, suggesting that the α3 chain can assemble with only β3-γ2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the β-γ heterodimer to the α-β-γ heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus

Single‐cell cloning is an essential technique for establishing genome‐edited cell clones mediated by programmable nucleases such as CRISPR‐Cas9. However, residual genome‐editing activity after single‐cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR‐Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single‐cell cloning of Cas9 RNP‐transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single‐cell cloning at 24, 48, 72 hr and 1 week post‐transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high‐resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long‐term incubation before single‐cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single‐cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures.