The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144,111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 pg * ml-' * day-'. The recombinant product was purified by a combination of ion-exchange and metalchelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Aminoterminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichiu coli when provided with its physiological substrates, i. e. hydrogen peroxide and chloride ions.

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A hybrid between human tissue-type plasminogen activator (t-PA) and human single chain urokinase-type plasminogen activator (scu-PA) was obtained by ligation of cDNA fragments encoding the NH2-terminal amino acids 1 to 67 of t-PA and the COOH-ter-minal amino acids 136 to 411, of scu-PA. Both this chimaeric cDNA and cDNA encoding scu-PA were expressed in a mammalian system (HAK-cells) using bovine papilloma virus (BPV) derived vectors. Two stable cell lines were obtained which secreted the recombinant hybrid and the scu-PA at 1 μg/ml and 2 μg/ml u-PA related antigen respectively into the culture medium. Following purification by Zinc chelate Sepharose, immunoadsorption chromatography, benzamidine-Sepharose and Ultrogel AcA44 gel filtration, highly purified proteins were obtained with a yield of about 200 μg/1. SDS gel electrophoresis under reducing conditions showed single bands with M 43,000 and M 50,000 respectively. Following conversion to urokinase with plasmin, both proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with km1.4 and 0.5 μM and k2 0.0034 s and 0.0027 s . Neither protein bound specifically to fibrin.Thus the fusion of the finger-like domain of t-PA to the COOH-terminal part of scu-PA does not confer fibrin affinity of t-PA to this chimaeric protein. However, peptide material can be fused to the COOH-terminal part of scu-PA without perturbing its enzymatic properties.

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.

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