Rapid purification of a new humanized single-chain Fv antibody/human interleukin-2 fusion protein reactive against HER2 receptor.
Human embryonic kidney 293 cells were transfected with plasmid pcDNA-H520C9scFv-rhIL-2 containing a chimeric cDNA encoding the humanized 520C9 scFv/recombinant human IL-2 fusion protein (H520C9scFv-rhIL-2). The transfected cells in plateau growing phase were cultured in serum-free medium for three days. The supernatant was collected, concentrated and purified using an affinity column packed with CNBr-activated Sepharose 4B coupled with anti-rhIL-2 mouse monoclonal antibody. The purified fusion protein was analyzed by ELISA, SDS-PAGE and Western blot. The fusion protein showed only one band in both silver stained electrophoresis gel and Western blot developed by ECL chemiluminescence system. Its molecular weight was confirmed to be about 45 kD. This fusion protein possessed binding specificity against p185 positive SKOV3 and B16/neu cells, and it might stimulate IL-2-dependent CTLL-2 cell proliferation as well.
Zhang
Wei-Yun
WY
Medical School, Nanjing University, Nanjing 210093, China. zhangwy@nju.edu.cn.
Yip
Tak-Chun
TC
Kwok
Cheuk-Sang
CS
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification*; D002460:Cell Line; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007668:Kidney / Q000276:immunology / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv