A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody.
A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK). The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA. An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl. SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, 376-8515, Gunma, Japan.
W
W
Dansithong
S
S
Paul
Y
Y
Kojima
K
K
Kamiya
T
T
Shinozawa
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D016207:Cytokines; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D000078792:Midkine; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D016207:Cytokines; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D000078792:Midkine