Expression and characterization of a DNase I-Fc fusion enzyme.
Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits.
Dwyer
M A
MA
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Huang
A J
AJ
Pan
C Q
CQ
Lazarus
R A
RA
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004278:DNA, Superhelical / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D018411:Spodoptera
D000199:Actins; D004278:DNA, Superhelical; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D003850:Deoxyribonuclease I; D010206:Papain