Lekteplase--a secreted tissue plasminogen activator derivative from Escherichia coli.
Fermentation studies of batch-mode cultivation in 4-L fermenters were carried out to obtain an active recombinant DNA-derived tissue plasminogen activator (t-PA) deletion mutant, lekteplase, secreted and correctly folded from Escherichia coli. The OmpA signal sequence was used to deliver the heterologous product composed of kringle 2 plus serine protease domain (K2S) to the medium. Supplementing the complex medium with 10% glycerol and 20 mmol/l magnesium chloride led to an increase in cell numbers with final cell density reaching an OD600 of 24. The expression level of lekteplase in the medium detected by sandwich ELISA was 100 mg/L. Enzymatic activity of lysine-sepharose purified product was demonstrated by amidolytic assay, in vitro fibrin clot lysis, and copolymerization PAGE.
Manosroi
Jiradey
J
Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand.
Tayapiwatana
Chatchai
C
Manosroi
Aranya
A
Beer
Jürgen
J
Bergemann
Klaus
K
Werner
Rolf Günter
RG
D001483:Base Sequence; D019149:Bioreactors; D015153:Blotting, Western; D003018:Clot Retraction; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005990:Glycerol / Q000494:pharmacology; D007118:Immunoassay; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology*
D015636:Magnesium Chloride; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator; C449559:lekteplase; D005990:Glycerol