Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies.
Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.
Orsini
G
G
Department of Biotechnology, Farmitalia C. Erba, Milano, Italy.
Brandazza
A
A
Sarmientos
P
P
Molinari
A
A
Lansen
J
J
Cauet
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002845:Chromatography; D002850:Chromatography, Gel; D002872:Chromosome Deletion; D017886:Durapatite; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006801:Humans; D006882:Hydroxyapatites; D002479:Inclusion Bodies / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D010960:Plasminogen Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D005343:Fibrinolytic Agents; D006882:Hydroxyapatites; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D017886:Durapatite; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator