Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins--evaluation of two anti-His-tag monoclonal antibodies.
A tag comprising four to six histidines genetically fused to the protein of interest (His-tag) has been widely used to purify proteins by immobilized metal-ion affinity chromatography (IMAC). Here we report the utilization of the same tag twice in series, first for IMAC and subsequently for immunoaffinity purification. Both steps are based on completely different physical principles and can therefore remove different contaminants. Two anti-His-tag antibodies (3D5 and PentaHis) were characterized for their binding and elution properties using the BIAcore surface plasmon resonance biosensor. The dissociation constant of the PentaHis antibody was determined to be 1 x 10(-8) M and for the 3D5 antibody 3.4 x 10(-7) M at pH 7.4. Imidazole in the sample did interfere with binding, whereas chelating agents such as EDTA and high salt did not. The antibody 3D5 was coupled to a column matrix and used for a coupled two-step purification, in which the IMAC column is eluted with EDTA and the eluent is loaded directly on the immunoaffinity column. This method may constitute a very general procedure to purify proteins to near homogeneity without the need to tailor conditions individually, and it may thus be very attractive for high-throughput screening programs and for developing general protocols for clinical grade material.
Müller
K M
KM
Biochemisches Institut, Universität Zürich, Switzerland.
Arndt
K M
KM
Bauer
K
K
Plückthun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004492:Edetic Acid; D006639:Histidine; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007132:Immunoglobulin Isotypes / Q000302:isolation & purification; D007202:Indicators and Reagents; D007700:Kinetics; D008670:Metals; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012685:Sepharose; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007202:Indicators and Reagents; D008670:Metals; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D006639:Histidine; D009532:Nickel; D012685:Sepharose; D004492:Edetic Acid