title: Synthesis and secretion of a fibrinolytically active tissue-type plasminogen activator variant in Escherichia coli.
abstract: A gene encoding a variant (lacking amino acids 6-173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.
authors.affiliation: KabiGen AB, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Waldenström
authors.forename: E
authors.initials: E
authors.lastname: Holmgren
authors.forename: A
authors.initials: A
authors.lastname: Attersand
authors.forename: C
authors.initials: C
authors.lastname: Kalderén
authors.forename: B
authors.initials: B
authors.lastname: Löwenadler
authors.forename: B
authors.initials: B
authors.lastname: Rådén
authors.forename: L
authors.initials: L
authors.lastname: Hansson
authors.forename: G
authors.initials: G
authors.lastname: Pohl
mesh_terms: D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator