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.