Citation

  • Authors: Shen Y. et al.
  • Year: 2018
  • Journal: Protein Expr Purif 141 44-51
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK-293F

Method

900 mL of HEK293 cell suspension at 1 x 10^6 cells/mL were prepared on the day before transfection. The cells were cultured overnight at 37°C, 125 rpm and 5% CO2 levels. Next day, 500 μg HE4 plasmid, diluted into 100 mL of serum-free medium, was mixed with 750 μL of pure FectoPRO ® DNA transfection reagent, and incubated at 25 C for 10 min after homogenization. The 100 mL FectoPRO ® /DNA transfection mix were added to the cells, then cultured until harvest and purification.

Abstract

Human epididymis protein-4 (HE4) may serve as a putative biomarker for the early diagnosis, therapy and especially prognosis of ovarian, lung and breast cancer. Detection and targeting of HE4 using the anti-HE4 antibody could be one of the effective strategies for the cancer diagnosis and treatment in clinical practice. In this study, a high-efficiency expression system was established to purify recombinant HE4. We obtained high purity HE4 in 400 mg quantity from 1 L culture supernatant of HEK293F cells. CCK-8 and cell cycle assays indicated that the purified recombinant HE4 protein could promote SKOV3 cell cycle and proliferation at the concentration of 0.1 mg/L. Furthermore, an anti-HE4 high-affinity monoclonal antibody 9C3 (ka = 8.1 × 106 1/MS, kd = 4.4 × 10-5 1/S, KD = 5.5 × 10-12 M) was prepared using hybridoma technique and analyzed by surface plasmon resonance technology using this HE4 protein. Differential Scanning calorimeter (DSC) analysis showed that 9C3 had a commendable thermal stability with the Tm value of 73 °C. Analyses of western blot, immunohistochemistry and immunofluorescence showed that the 9C3 was highly specific to HE4 in human cancer cells and tissues. In conclusion, our study designed a method to prepare human recombinant HE4 with high yield and generated a high-affinity anti-HE4 monoclonal antibody that might have potential for basic research and clinical application.

Pubmed