Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment.
A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter-Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the posttranslational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microg of glycosylated gp120/ml of reaction mixture.
Tarui
H
H
Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, Japan.
Murata
M
M
Tani
I
I
Imanishi
S
S
Nishikawa
S
S
Hara
T
T
D000818:Animals; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis; D011312:Pressure; D014176:Protein Biosynthesis; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*
D015699:HIV Envelope Protein gp120