Expression Optimization of Anti-CD22 scFv-Apoptin Fusion Protein Using Experimental Design Methodology

Abstract

Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein.

Methods: Four variables (cell optical density at induction, IPTG concentration, induction temperature, and induction time) were tested using experimental design.

Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield.

Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein.

Keywords: Recombinant protein; Single-chain antibodies; Fusion proteins; E. coli.

Fig. 1

SDS-PAGE analysis of the recombinant scFv-apoptin. Total protein (20 µg) was loaded in each lane in each sample of cell lysate. Lane 1, protein size marker (kDa); lane 2, scFv-apoptin expressed at abs_ind = 1.5, IPTG = 0.1 mM, temperature = 25 °C, and time = 24 h; lane 3, scFv expressed under the same condition except the time of induction that was 4 h. The recombinant protein (44 kDa) is shown by arrow.