title: Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli.
abstract: We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence. A large portion of the secreted protein is associated with an insoluble cellular fraction. This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione. Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography. The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed. Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen [Castellino et al. (1986) Arch. Biochem. Biophys. 247, 312-320]. Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM. Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids [Winn et al. (1980) Eur. J. Biochem. 104, 579-586], we have shown the lysine binding site of t-PA kringle-2 to have a preference for a ligand with 8.8-A separation between amine and carboxylate functions. Charge interactions with the epsilon-amino group of L-lysine are important in binding since the affinities for N epsilon-acetyl-L-lysine, L-arginine, and gamma-guanidinobutyric acid are decreased greater than 2000-fold, 200-fold, and 12-fold, respectively, relative to the affinity for L-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Biomolecular Chemistry Department, Genentech, Inc., South San Francisco, California 94080.
authors.forename: S
authors.initials: S
authors.lastname: Cleary
authors.forename: M G
authors.initials: MG
authors.lastname: Mulkerrin
authors.forename: R F
authors.initials: RF
authors.lastname: Kelley
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator