Figure 2.

Construction, expression, and selective purification of polyhistidine-tagged pro-urokinase. (Upper part) In the BamHI– XhoI excised pcDNAneoI, a double strand oligonucleotide encoding the twelve COOH-terminal amino acids of pro-uPA followed by a “spacing-arm,” a stretch of six histidines and a stop codon, was inserted. A large BamHI fragment containing the remainder of pro-uPA gene was subsequently inserted in the unique BamHI site (see Materials and Methods). (Lower part) The plasmids either encoding pro-uPA (pcDNAuPA) or Hispro-uPA^wt (pcDNA6xHis-tagged uPA) were transiently transfected in HeLa cells: 48 h later, aliquots of the serum-free conditioned media from the transfectants were incubated with 5B4 anti-uPA (5B4) or with Ni-NTA (Ni²⁺) or with an irrelevant antibody (−), and the resulting samples were analyzed by SDSPAGE under reducing conditions.