Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment

• Published in: Applied microbiology and biotechnology 2001-05, Vol.55 (4), p.446-453
• Main Authors: Tarui, H, Murata, M, Tani, I, Imanishi, S, Nishikawa, S, Hara, T
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Animals; Biological and medical sciences; Biotechnology; Cell-Free System; creatine; creatine kinase; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycosylation; guanosine triphosphate; High pressure; high pressure treatment; HIV Envelope Protein gp120 - biosynthesis; Insects; Kinases; magnesium; Methods. Procedures. Technologies; Nitrogen; Noctuidae; Others; phosphates; potassium; Pressure; Protein Biosynthesis; spermidine; Spodoptera - cytology; Spodoptera - metabolism; Spodoptera frugiperda; Various methods and equipments
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530000534

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 post-translational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microgram of glycosylated gp 120/ml of reaction mixture.