Selection, purification, and characterization of a HER2-targeting soluble designed ankyrin repeat protein by E. coli surface display using HER2-positive melanoma cells

Abstract

ABSTRACTHuman epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells. DARPin selected from the library could be expressed in soluble form with a yield of 70 mg/L. After purified by two continuous and easy steps, the purity of DARPin was 90% as established by SDS-PAGE and RP-HPLC. Selected DARPin showed significant HER2-targeting ability with an affinity of 1.05 ± 0.47 µM. MTT assay demonstrated that at the concentration of 640 nM, the selected DARPin dimer could inhibit the SK-BR-3 growth at a rate of 36.63 and 46.34% in 48 and 72 hr incubation separately, which was similar to trastuzumab (43.12 and 49.14% separately). These findings suggested that it was an effective method to select antibody mimetic DARPin by bacteria surface display combined with live cells sorting and provided a drug candidate for cancer therapy.