Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNA.
A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008970:Molecular Weight; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D004792:Enzyme Precursors; D004247:DNA; D014568:Urokinase-Type Plasminogen Activator