Cell‐Free Protein Synthesis System Prepared from Insect Cells by Freeze‐Thawing

Abstract

We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-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 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees 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 microg/mL of luciferase was synthesized in an Sf21 cell-free system at 25 degrees 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 Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

“…Genomic in silico analysis indicates that the organisms in the Lepidopteran order, including the Spodoptera genus, do not possess selenoproteins and are missing Sec incorporation machinery (16). Cells from Spodoptera frugiperda (Sf21) have been extensively used for protein expression, and a commercially available in vitro translation lysate from Sf21 cells provides robust translation activity (8). Because the addition of purified SBP2, eEFSec, and rabbit aminoacyl tRNA to Sf21 lysates did not result in Sec incorporation activity, 3 we combined mammalian cell lysate (supplying Sec incorporation factors) and Sf21 lysate (supplying ample general translation factors) to identify conditions where eEFSec or SBP2 may be limiting.…”

“…Physical contacts of this complex were mapped to the apical loop of the bSECIS and the C terminus of Domain IV (6). Mutations in the apical loop of bSECIS or deletion of Domain IV in SelB abolished the interaction of these two factors and, as a consequence, depleted Sec incorporation activity (7,8). The simple model for eukaryotic Sec incorporation is that a SBP2/SECIS/eEFSec/GTP/Sec-tRNA Sec complex is required for delivery of Sec-tRNA Sec to the A-site of the ribosome.…”

Disulfide bond formation of heterodimer and heterotrimer of human laminin-332 coiled-coil domains

“…We succeeded in synthesizing heterotrimers of LCC domains of LM-332 in a cell-free translation system derived from Spodoptera frugiperda 21 (Sf21) insect cells (21)(22)(23). With the advantages of this system, we could analyze the disulfide bond formation of heterodimers and heterotrimers using many mutants.…”

“…Protein synthesis was carried out using a Transdirect insect cell kit (Shimadzu, Kyoto, Japan) in non-reducing conditions. To check the heterodimer and heterotrimer formation, 6 μg of mRNAs of each α3, β3, and γ2 LCC domains and their mutants were mixed with other reagents of the kit to reach 50 μl of reaction volume following instruction (21)(22)(23). The reaction mixtures were incubated for 5 h at 25 o C.…”

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

“…19,20) The human laminin 3, 3, and 2 chains were expressed using 6 mg of mRNAs of each chain in a 50-ml reaction volume. The reaction mixtures were incubated for 5 h at 25 C. In heterodimer and heterotrimer assembly experiments, the human laminin 3, 3, and 2 chains were co-expressed using 12 or 18 mg of mRNAs in a 50-ml reaction volume.…”