Molecular Cloning, Sequencing, and Expression inEscherichia coliof Human Preprourokinase cDNA

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Abstract
A complementary DNA 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 complementary DNA 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 complementary DNA sequence fits the published amino acid sequence data with 3 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 the clone. A large Bgl I fragment derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains were 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 MW and being specifically recognized by antibodies raised against natural human urokinase.