Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines.
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 micrograms.ml-1.day-1. The recombinant product was purified by a combination of ion-exchange and metal-chelate 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. Amino-terminal 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 Escherichia coli when provided with its physiological substrates, i.e. hydrogen peroxide and chloride ions.
Moguilevsky
N
N
Department of Applied Genetics, Université Libre de Bruxelles, Nivelles, Belgium.
Garcia-Quintana
L
L
Jacquet
A
A
Tournay
C
C
Fabry
L
L
Piérard
L
L
Bollen
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000032:analysis / Q000096:biosynthesis* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000494:pharmacology
D002241:Carbohydrates; D011994:Recombinant Proteins; D009195:Peroxidase