Masaaki Ito

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.

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.

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.

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.