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

Published on Dec 1, 2006in Biotechnology Progress2.41
· DOI :10.1021/bp060110v
Toru Ezure9
Estimated H-index: 9
(Shimadzu Corp.),
Takashi Suzuki12
Estimated H-index: 12
(Shimadzu Corp.)
+ 7 AuthorsOsamu Nishimura24
Estimated H-index: 24
(Shimadzu Corp.)
We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and /?-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.
  • References (0)
  • Citations (59)
Cited By59
#1Aidan Tinafar (U of T: University of Toronto)
#2Katariina Jaenes (U of T: University of Toronto)
Last.Keith Pardee (U of T: University of Toronto)H-Index: 14
view all 3 authors...
#1Giada Lo Gullo (Sapienza University of Rome)H-Index: 1
#2Rosanna Mattossovich (National Research Council)H-Index: 2
Last.Dario Benelli (Sapienza University of Rome)H-Index: 12
view all 6 authors...
#1Wan-Qiu Liu (ShanghaiTech University)H-Index: 2
#2Lingkai Zhang (ShanghaiTech University)H-Index: 2
Last.Jian Li (ShanghaiTech University)H-Index: 2
view all 4 authors...
#1Sung Sun Yim (Columbia University)H-Index: 2
#2Nathan I. Johns (Columbia University)
Last.Harris H. Wang (Columbia University)H-Index: 25
view all 11 authors...
#1Upasana Rai (CCMB: Centre for Cellular and Molecular Biology)H-Index: 1
#2Rakhi Sharma (CCMB: Centre for Cellular and Molecular Biology)
Last.Mandar V. Deshmukh (CCMB: Centre for Cellular and Molecular Biology)H-Index: 9
view all 3 authors...
#1Jessica G. Perez (NU: Northwestern University)H-Index: 3
#2Jessica C. Stark (NU: Northwestern University)H-Index: 6
view all 3 authors...
View next paperCell-free translation reconstituted with purified components.