Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system

Abstract

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.

Figure 1. Development of a bacterial secretion system for the production of a soluble and secreted single-chain antibody.

scFv was fused with the bacterial secretory carrier protein osmY to produce a good yield of soluble scFv secreted into the LB medium and to circumvent scFv inclusion body formation in the cytoplasm.

Figure 2. Construction and expression of secreted αEGFR.

(a) αEGFR was fused with the N- or C-terminus of the osmY gene to form αEGFR-osmY and osmY-αEGFR fusion proteins, respectively. αEGFR expressed in the periplasmic space (pelB-αEGFR) was used as the control. H, Histidine tag. (b) αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR plasmids were transformed into BL-21 to obtain αEGFR-osmY/BL21, osmY-αEGFR/BL21, and pelB-αEGFR/BL21 cells, respectively. The expression of αEGFR was confirmed by western blot analysis using an anti-histidine tag antibody. Lane 1, BL21 as negative control; Lane 2, αEGFR-osmY/BL21; Lane 3, osmY-αEGFR/BL21; and Lane 4, pelB-αEGFR/BL21.

Figure 3. Solubility and secretion of αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR in different components.

The transformed cells were induced with IPTG and the presence of αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR were detected by western blot analysis using an anti-histidine tag antibody in (a) concentrated LB medium, (b) soluble lysate, and (c) insoluble protein (pellet), as described in the Materials and Methods section. Lane 1, BL21 as negative control; Lane 2, αEGFR-osmY/BL21; Lane 3, osmY-αEGFR/BL21; and Lane 4, pelB-αEGFR/BL21.

Figure 4. Function of secreted αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR.

The growth medium of αEGFR-osmY/BL21, osmY-αEGFR/BL21, and pelB-αEGFR/BL21 was added to the EGFR-positive MDA-MB-468 cells, and the binding activity of the αEGFR fusion protein was detected by ELISA using an anti-histidine tag antibody.

Figure 5. Antigen-binding activity of αEGFR-osmY, osmY-αEGFR, and refolded pelB-αEGFR.

The fusion protein αEGFR-osmY and osmY-αEGFR were purified by using a Ni-column, and the control pelB-αEGFR was purified by using the Ni-column under denaturing/refolding conditions. The binding activities of different concentrations of αEGFR against EGFR-positive cells (MDA-MB-468) were determined by ELISA using an anti-histidine tag antibody.