Successful construction and stable expression of an anti-CD45RA scFv–EGFP fusion protein in Chinese hamster ovary cells

• We establish a method to construct an anti-CD45RA scFv3A4–EGFP fusion protein.
• The CHO cell expression system is as a new host to express the scFv3A4–EGFP.
• The scFv3A4–EGFP is persistently secreted to the medium in soluble form.
• The scFv3A4–EGFP is bifunctional and can be used in FACS and IF experiments.
• The method can be used to express other scFv–EGFP fusion proteins.

CD45RA has been found highly expressed on leukemia cells and may be a potential target of the disease. In this study, an anti-CD45RA single-chain antibody fragment (scFv3A4) was genetically linked to the N terminus of the enhanced green fluorescent protein (EGFP) to generate a scFv3A4–EGFP fusion protein. The scFv3A4–EGFP with a molecular weight of 57 kDa was stably expressed and secreted from the transfected CHO cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was 1350 μg/L. Flow cytometry analysis showed that the scFv3A4–EGFP had the same binding site and a very similar reactivity pattern with its parental murine monoclonal antibody (mAb) 3A4. Furthermore, comparing to conventional labeled 3A4-FITC antibody, the scFv3A4–EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection. Therefore, the scFv3A4–EGFP fusion protein can be a powerful tool to investigate the targeting of CD45RA on leukemia cells, biological activity of the target and possibly for the genetic manipulation of the antibody.