Masamitsu Shikata

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.

The ecdysteroid UDP-glucosyltransferase gene of Autographa californica nucleopolyhedrovirus alters the moulting and metamorphosis of a non-target insect, the silkworm, Bombyx mori (Lepidoptera, Bombycidae)

The Autographa californica nucleopolyhedrovirus (AcMNPV) does not infect the silkworm and molecular studies on silkworm insusceptibility have not been performed. In cultured cells of the silkworm, the expression of viral genes has been reported. The expression of AcMNPV genes and their effect in vivo and in vitro was studied. In this study, the early gene, the ecdysteroid UDP-glucosyltransferase (egt) gene of AcMNPV, which inactivates the insect moulting hormone by sugar conjugation, was examined to determine whether it would alter the growth of the silkworm. Using wild-type (wt) AcMNPV, the egt gene deletion virus (vEGTDEL), and the virus carrying the egt promoter-lacZ cassette in vEGTDEL (vEGTZ), the egt promoter-driven expression in cultured cells and in nonproductive infection of the silkworm was characterized. Infection of cultured cells with vEGTZ at three different doses occurred in a single cell manner. When budded wt AcMNPV was injected into the fourth and fifth instar larvae, an increase in the amount of virus occurred and caused abnormal larval growth, which resulted in the prolongation or skipping of the larval instar, premature pupation, or death during the pupal stage. For infection of the fourth instar larvae, precocious metamorphosis was observed. When the same amount of vEGTDEL was injected, the alteration of growth did not occur. These results suggest that the egt gene was expressed in the primary infected cells of the silkworm, and that the EGT was secreted into the haemocoel, which significantly altered larval growth.

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.