Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines

Abstract

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.

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

“…54) relied on recombinant protein isolated from transfectants unable to synthesize the functional, normal subunits of MPO. The recombinant MPO produced in Chinese hamster ovary cells used for these studies has oligosaccharide side chains significantly different from native MPO, fails to undergo proteolytic processing into mature subunits, and is expressed only in the monomeric form (55). Such differences may have functional consequences.…”

Anti-native and recombinant myeloperoxidase monoclonals and human autoantibodies

“…Anti-recMPO ELISA (1 g/ml) was also performed with recombinant MPO purified from a CHO cell line kindly provided by N. Moguilevski (Université Libre de Bruxelles, Nivelles, Belgium) [12]. Positivity was defined as binding greater than the mean binding of 39 normal blood donors 2 s.d.…”

Stable Expression of Human H1‐histamine‐receptor cDNA in Chinese Hamster Ovary Cells

“…This plasmid encodes the complete human H, histamine receptor. Using plasmid 3604B digested with AutII, CHO-K1 cells (ATCC CCL61) were transfected by electroporation and selected as previously described (Moguilevsky et al, 1991). G418R-resistant clones were then isolated and transferred into 24-well plates for subsequent ['Hlmepyramine-binding assays.…”