Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system.
The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.
Kobatake
E
E
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Ebisawa
A
A
Asaka
O
O
Yanagida
Y
Y
Ikariyama
Y
Y
Aizawa
M
M
D017123:Bacteriophage T7 / Q000235:genetics; D019149:Bioreactors; D002474:Cell-Free System; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D013205:Staphylococcal Protein A; D012259:Ribonuclease, Pancreatic