Releasing profiles of gene products from recombinant Escherichia coli in ahigh-voltage pulsed electric field

Citation
T. Ohshima et al., Releasing profiles of gene products from recombinant Escherichia coli in ahigh-voltage pulsed electric field, BIOCH ENG J, 5(2), 2000, pp. 149-155
Citations number
22
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology
Journal title
BIOCHEMICAL ENGINEERING JOURNAL
ISSN journal
1369-703X → ACNP
Volume
5
Issue
2
Year of publication
2000
Pages
149 - 155
Database
ISI
SICI code
1369-703X(200006)5:2<149:RPOGPF>2.0.ZU;2-A
Abstract
Release of recombinant proteins from gene-engineered Escherichia coli by ap plying a pulsed electric field (PEF) to a cell suspension was studied. When E. coli/pNC1, which produces beta-glucosidase and accumulates it in cytopl asm, was exposed to PEE the most effective release of this enzyme was achie ved in the cell suspension of 5% glycine and 15% PEG solution under 10 kV/c m and 280 J/ml of a PEF in a needle-plate electrode chamber. However, the a mount 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 10 kV/cm and 200 J/ml of a PEF in a pla te-plate electrode chamber, 89% of intracellular alpha-amylase with nine-ti mes higher specific activity compared with that by ultrasonic treatment was released. The release tendency of cellobiohydrolase, produced by E. coli/p NB6 and accumulated in both the cytoplasm and periplasmic space, was interm ediate between those of beta-glucosidase and alpha-amylase. In this case, 7 0% of cellobiohydrolase with 1.9-times higher specific activity compared wi th that by ultrasonic treatment could be released when E. coli/pNB6 was sus pended in 158 PEG and 10 kV/cm and 200 J/ml of a PEF was applied in a needl e-plate electrode chamber. These results indicated that PEF treatment could easily disrupt the outer membrane, but it was difficult to disrupt the cyt oplasmic membrane simultaneously. Therefore, PEF treatment is useful for ea sy release of periplasmic protein with selectivity. (C) 2000 Elsevier Scien ce S.A. All rights reserved.