title: 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.
authors.affiliation: Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Ezure
authors.forename: Takashi
authors.initials: T
authors.lastname: Suzuki
authors.forename: Shoken
authors.initials: S
authors.lastname: Higashide
authors.forename: Eiichi
authors.initials: E
authors.lastname: Shintani
authors.forename: Kohki
authors.initials: K
authors.lastname: Endo
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Shikata
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ito
authors.forename: Koji
authors.initials: K
authors.lastname: Tanimizu
authors.forename: Osamu
authors.initials: O
authors.lastname: Nishimura
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002457:Cell Extracts; D002460:Cell Line; D002474:Cell-Free System; D005615:Freezing; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007915:Lepidoptera / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D002457:Cell Extracts; D019476:Insect Proteins; D011994:Recombinant Proteins