Releasing profiles of gene products from recombinant Escherichia coli in a high-voltage pulsed electric field.
Release of recombinant proteins from gene-engineered Escherichia coli by applying a pulsed electric field (PEF) to a cell suspension was studied. When E. coli/pNC1, which produces beta-glucosidase and accumulates it in cytoplasm, was exposed to PEF, the most effective release of this enzyme was achieved in the cell suspension of 5% glycine and 15% PEG solution under 10kV/cm and 280J/ml of a PEF in a needle-plate electrode chamber. However, the amount of released beta-glucosidase by PEF treatment was only 26% of that by ultrasonic treatment. On the other hand, alpha-amylase produced by E. coli/pHI301A and accumulated in the periplasmic space could be easily released by PEF treatment. When this recombinant E. coli was suspended in 0.9% NaCl and 10% PEG solution and exposed to 10kV/cm and 200J/ml of a PEF in a plate-plate electrode chamber, 89% of intracellular alpha-amylase with nine-times higher specific activity compared with that by ultrasonic treatment was released. The release tendency of cellobiohydrolase, produced by E. coli/pNB6 and accumulated in both the cytoplasm and periplasmic space, was intermediate between those of beta-glucosidase and alpha-amylase. In this case, 70% of cellobiohydrolase with 1.9-times higher specific activity compared with that by ultrasonic treatment could be released when E. coli/pNB6 was suspended in 15% PEG and 10kV/cm and 200J/ml of a PEF was applied in a needle-plate electrode chamber. These results indicated that PEF treatment could easily disrupt the outer membrane, but it was difficult to disrupt the cytoplasmic membrane simultaneously. Therefore, PEF treatment is useful for easy release of periplasmic protein with selectivity.
Ohshima
T
Department of Biological and Chemical Engineering, Gunma University, Tenjin-cho 1-5-1, Kiryu, Gunma-ken, Japan
Hama
Y
Sato
M