Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter.
The human tissue plasminogen activator (h-tPA) cDNA was fused either with the leader sequence of the killer toxin of Kluyveromyces lactis or with the Saccharomyces diastaticus glucoamylase leader peptide and cloned in the yeast expression vector under the control of the inducible USAgal/CYC1 promoter. The recombinant tPA is produced in yeast as a single-chain glycosylated polypeptide of 66-72 kDa, which accumulates intracellularly associated with a membrane fraction. Using two-step fed-batch fermentation, a productivity up to 100 mg/l of active intracellular tPA was obtained.
Martegani
E
E
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Forlani
N
N
Mauri
I
I
Porro
D
D
Schleuning
W D
WD
Alberghina
L
L
D005285:Fermentation; D005690:Galactose; D005822:Genetic Vectors; D006801:Humans; D007716:Kluyveromyces / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012440:Saccharomyces / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis*
D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator; D005690:Galactose