HIGH-LEVEL SECRETION IN PICHIA-PASTORIS AND BIOCHEMICAL-CHARACTERIZATION OF THE RECOMBINANT KRINGLE-2 DOMAIN OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR

The kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) h as been expressed in Pichia postoris cell lines GS115 and KM71, This c onstruct contained a hexahistidine sequence at the C-terminus of the k ringle to aid in purification by immobilized metal-ion-affinity chroma tography, The exact amino acid sequence of the isolated kringle was EA EAYV-[K2(tPA)]SR(H)(6), where [K2(tPA)] represents amino acid sequence residues C-1-C-82 Of the kringle domain (residues 180-261 of tPA), Th e clones of the yeast transformants provided large amounts of the reco mbinant (r)-[K2(tPA)]-containing polypeptide at levels that allowed re ady purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter, Purification of the kringle dom ain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni+-affinity chromatography or lysine-Sepha rose affinity chromatography. N-linked glycans were present on approx, 30% of this yeast-expressed material, at N-5 Of the kringle (correspo nds to N-11 of the particular construct, N-184 of full-length tPA). Th e expressed recombinant kringle recognized a conformation-specific mon oclonal antibody generated against tPA that is directed to the K2 doma in of the protein, interacted properly with various omega-amino acid l igands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution H-1-NMR, The re sults demonstrate that the P. pastoris system can be employed to obtai n large amounts of secreted and properly folded kringle domains.