Purification of sufficiently gamma-carboxylated recombinant protein C and its derivatives. Calcium-dependent affinity shift in immunoaffinity and ion-exchange chromatography.
Protein C, which is an important anti-thrombotic factor in the blood coagulation cascade, undergoes several post-translational modifications. gamma-Carboxylation on nine glutamic acid residues at the N-terminal region of the light chain [gamma-carboxylated glutamic acid (Gla) domain] is considered to be critical for full anti-clotting activity. It is also known that when recombinant protein C is expressed in animal cells this particular modification is often lost. We were successful in preparing a monoclonal antibody (PC01) which distinguishes the sufficiently gamma-carboxylated protein from the rest by its specific affinity for the Ca(2+)-induced conformational change of the former, and thereby developed a simple process of purifying sufficiently gamma-carboxylated protein C. Culture supernatant of Chinese hamster ovary cell transformants was first applied to Q-Sepharose and recombinant protein C was partially purified. It was then loaded onto a PC01 affinity column in the presence of 5 mM calcium chloride. Sufficiently gamma-carboxylated protein C was retained while insufficient-carboxylated protein C quickly passed through. The former was eluted with 5 mM EDTA efficiently and with high purity, contained eight Gla units per molecule, and had similar anti-clotting activity. The flow-through was relatively impure protein C which contained five Gla units per molecule and showed limited anti-clotting activity. We extended the application of the Ca(2+)-induced conformational change to conventional ion-exchange chromatography. The sufficiently gamma-carboxylated protein C was found to elute earlier in the salt gradient from an anion-exchange column in the presence of 5 mM calcium chloride being fully separated from the insufficiently carboxylated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)
Ohsawa
K
K
Pharma Research Laboratories, Hoechst Japan Ltd., Saitama, Japan.
Kimura
M
M
Kurosawa-Ohsawa
K
K
Takahashi
M
M
Koyama
M
M
Abiko
Y
Y
Hirahara
K
K
Matsuishi
T
T
Tanaka
S
S
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007120:Immunochemistry; D011486:Protein C / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D011486:Protein C; D011994:Recombinant Proteins; D002118:Calcium