Poly[G] improved protein productivity of cell-free translation by inhibiting mRNase in wheat germ extract

Abstract

Degradation of template mRNA lowers the efficiency of cell-free translation. Although cell-free translation system using wheat germ extract (WGE) is usually supplemented with placental RNase inhibitor (PRI) to stabilize mRNA, the decay rate of mRNA, however, was still fairly rapid. For elucidating cause of mRNA degradation in wheat germ cell-free translation system, the authors applied activity staining in SDS-PAGE gel to investigate ribonuclease activities in WGE. RNases or nucleases of molecular mass of about 65, 42, and 37 kDa were detected in WGE, and named WGa, WGb and WGc, respectively. WGb and WGc were nucleases that degraded both RNA and DNA, and WGa was an RNase. Polyguanylic acid (5′), poly[G], which was known to have affinity with RNases, strongly inhibited RNase activity in WGE. Addition of poly[G] to wheat germ cell-free translation system resulted in 7-fold increase in protein productivity. Luciferase mRNA that was added in the cell-free translation system as a template was not degraded in 90 min in the presence of poly[G] while it was degraded by 80% in 30 min in the absence of poly[G]. The authors concluded that poly[G] improved the protein productivity of wheat germ cell-free translation system by inhibiting RNase activity that degrades template RNA.