Efficient Antibody Assembly in E. coli Periplasm by Disulfide Bond Folding Factor Co-expression and Culture Optimization

Abstract

Molecular chaperones and protein folding factors of bacterial periplasmic space play important roles in assisting disulfide bond formation and proper protein folding. In this study, effects of disulfide bond protein (Dsb) families were investigated on assembly of 3F3 Fab, an antibody inhibitor targeting matrix metalloproteinase-14 (MMP-14). By optimizing DsbA/C co-expression, promoter for 3F3 Fab, host strains, and culture media and conditions, a high yield of 30-mg purified 3F3 Fab per liter culture was achieved. Produced 3F3 Fab exhibited binding affinity of 34 nM and inhibition potency of 970 nM. This established method of DsbA/C co-expression can be applied to produce other important disulfide bond-dependent recombinant proteins in E. coli periplasm.

Keywords: DsbA; DsbC; Fab; IgG; Over-expression; Periplasm; Protein folding factor.

Figure 1. Expressed 3F3 heavy and light chains failed assembling to Fab

(A) Construct of 3F3 Fab expression cassette with P_PhoA promotor and STII leaders. FLAG tag and His-tag are at the C-termini of light and heavy chains. (B) Purification results of four Fabs 2B5, 3A2, 3F3, and 3E9 [13] from their BL21 periplasmic fractions using anti-FLAG resin, resulting in capture of Fabs and light chain (LC) fragments (VL-CL). 3F3 LC MW = 27 kDa. (C) Purification results of four Fabs from their BL21 periplasmic fractions using Ni-NTA resin, resulting in capture of Fabs and heavy chain (HC) fragments (VH-CH1). 3F3 HC MW = 30 kDa. Further quantification showed assembled 3F3 Fab (MW = 57 kDa) had a yield <10 μg per litter of culture.

Figure 2. Folding factor DsbA/C construct designs and expression results

(A) Four constructs were applied in this paper: expression of either DsbA or DsbC under an arabinose promoter P_BAD; bicistronic co-expression of DsbA and DsbC under one P_BAD promoter (AC-1P); monocistronic co-expression of DsbA and DsbC under two separate P_BAD promoters (AC-2P). (B) SDS-PAGE analysis of periplasmic fractions. Cultures were induced with 0.2% arabinose. DsbA MW = 23 kDa; DsbC MW = 25 kDa; n.c. = negative control; M = protein marker.

Figure 3. Effects of disulfide bond enzymes DsbA/C on 3F3 Fab assembly

Western blotting results of overnight cultures at 30°C in 2×YT. Jude-I was the host. 3F3 Fab was expressed under a P_PhoA promotor. No DsbA/C co-expression served as the negative control (n.c.). Bands were detected with anti-His-HRP.

Figure 4. Promoter, host and culture temperature optimizations

(A) 3F3 Fab expression construct with P_Lac and pelB leaders. 0.2% arabinose was used for DsbA/C expression. 0.1 mM IPTG was used for Fab induction. Western blotting results using host cells Jude-I or BL21 at (B) 30 °C or (C) 37°C overnight in 2×YT. Bands were detected with anti-Fab-HRP. The red arrows in (B) and (C) indicate the assembled 3F3 Fab and 3F3 Fab with leader peptide respectively.

Figure 5. High yields of 3F3 Fab using (A) rich media TB and (B) fed-batch media EnBase

Co-expression was induced with 0.2% arabinose and 0.1 mM IPTG. Western blotting bands were detected with anti-Fab-HRP. The red arrow in (B) indicates truncated Fab.

Figure 6. Binding affinity and inhibition potency of 3F3 Fab

Binding EC₅₀ and inhibition IC₅₀ were determined using ELISA and FRET assays respectively. Purified 3F3 Fab sample after concentration (inset) was used for measurements.