Analysis of plasmid-DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis.
Plasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis. We used this method to control the genetic stability during a fed-batch culture. The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000-22,000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non-cross-linked polyacrylamide. With this method the plasmid DNA from recombinant E. coli cultivation samples was analyzed within 30 min. Separation parameters such as gel concentration, capillary length and current were optimized for that purpose. The method was modified to allow the analysis of proteins. Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate-polyacrylamide filled capillaries. The method was also used to verify product purity after the down-stream process.
Hebenbrock
K
K
Institut für Technische Chemie, Universität Hannover, Germany.
Schügerl
K
K
Freitag
R
R
D002197:Capillary Action; D004247:DNA / Q000302:isolation & purification*; D015246:Deoxyribonuclease EcoRI; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification*
D011994:Recombinant Proteins; D004247:DNA; D015246:Deoxyribonuclease EcoRI