http://www.abbs.info E-mail: [email protected]

ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(10): 707-712 CN 31-1940/Q

---

Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

Wei-Yun ZHANG*, Tak-Chun YIP¹, and Cheuk-Sang KWOK²

Medical School, Nanjing University, Nanjing 210093, China;

¹Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, China;

²Division of Radiation Oncology, City of Hope National Medical Center, Duarte, CA91010, USA

Abstract 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.

Key words single-chain Fv antibody; IL-2; fusion protein; human embryonic kidney 293 cells; affinity chromatography

-----------------

Received: June 1, 2004 Accepted: August 16, 2004

This study was supported by a grant from the Hong Kong Universities Research Council, and a Pilot Study grant from the Hong Kong Polytechnic University

*Corresponding author: Tel, 86-25-83686149; E-mail, [email protected]