Enhancing full-length antibody production by signal peptide engineering

Abstract

Background: Protein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal peptide is fused to the N-terminus of the heterologous protein. The signal peptide mediates translocation of the heterologous protein from the cytoplasm to the periplasm and is cleaved during the translocation process. It was previously shown that optimization of the translation initiation region (TIR) which overlaps with the nucleotide sequence of the signal sequence improves the production of heterologous proteins. Despite the progress, there is still room to improve yields using secretion as a means to produce protein complexes such as full-length monoclonal antibodies (mAbs).

Results: In this study we identified the inefficient secretion of heavy chain as the limitation for full-length mAb accumulation in the periplasm. To improve heavy chain secretion we investigated the effects of various signal peptides at controlled TIR strengths. The signal peptide of disulfide oxidoreductase (DsbA) mediated more efficient secretion of heavy chain than the other signal peptides tested. Mutagenesis studies demonstrated that at controlled translational levels, hydrophobicity of the hydrophobic core (H-region) of the signal peptide is a critical factor for heavy chain secretion and full-length mAb accumulation in the periplasm. Increasing the hydrophobicity of a signal peptide enhanced heavy chain secretion and periplasmic levels of assembled full-length mAbs, while decreasing the hydrophobicity had the opposite effect.

Conclusions: This study demonstrates that under similar translational strengths, the hydrophobicity of the signal peptide plays an important role in heavy chain secretion. Increasing the hydrophobicity of the H-region and controlling TIR strengths can serve as an approach to improve heavy chain secretion and full-length mAb production in E. coli.

Fig. 1

Electron microscopy of E. coli expressing full-length hu5D5. a–b Transmission electron microscopy (TEM) images of 64B4 host strain expressing the empty vector pBR322 (a) or pBR-ssSTII0.3-ssSTII1-hu5D5 (b). White arrowheads point to inclusion bodies and black arrows point to the periplasm. c–d Immunostaining EM probing for light chain using anti-LC antibody (c) or heavy chain using anti-Fc antibody (d). Black arrows point to the periplasm. Black arrowheads point to gold particles indicating the cellular localization of light chain or heavy chain. Scale bars equal to 200 nm. The complete plasmid sequences were confirmed by DNA sequencing

Fig. 2

The amino acid sequences of signal peptides and the hydrophobicity of the H-region. The N-terminus, H-region, and the C-terminus were assigned following previous literature [35, 42, 53, 60]. The average and sum hydrophobicity of the H-region were calculated using the Eisenberg scale [61]

Fig. 3

The effects of signal peptide hydrophobicity on full-length hu5D5 production. For all the constructs, light chain was fused to ssSTII0.3 and heavy chain was fused to ssSTII1, ssDsbA1, ssPhoA1, ssMalE1, ssSTII1 S-13L, ssSTII1 S-13I, ssDsbA1 L-9S, or ssDsbA L-9I. Top panel the non-reduced soluble protein samples from the cell lysates were separated by electrophoresis followed by Western blot analysis. Heavy chain-containing species are marked based on the apparent molecular weight. Middle panel Western blot of soluble heavy chain in the periplasm. The periplasmic protein samples were reduced with 0.2 M DTT. Bottom panel Western blot of reduced total heavy chain in the whole cell lysates. All the blots were probed with the HRP-conjugated anti-Fc antibody

Fig. 4

The effects of signal peptide hydrophobicity on the cellular localization of inclusion bodies. TEM of E. coli expressing pBR-ssSTII0.3-ssSTII1-hu5D5, pBR-ssSTII0.3-ssDsbA1-hu5D5, pBR-ssSTII0.3-ssMalE1-hu5D5, pBR-ssSTII0.3-ssPhoA1-hu5D5, pBR-ssSTII0.3-ssSTII1 S-13L-hu5D5, or pBR-ssSTII0.3-ssDsbA1 L-9S-hu5D5. Black arrows point to the periplasmic space, and the white arrowheads point to the inclusion bodies

Fig. 5

The effects of signal peptide hydrophobicity on full-length (a) mAb1 and (b) mAb2 production. For all the constructs, light chain was fused to ssSTII0.3 and heavy chain was fused to either ssSTII1, ssSTII1 S-13L, ssDsbA1, or ssDsbA1 L-9S. 64B4 harboring the empty vector (ev) pBR322 was loaded as the negative control. Top panel the non-reduced soluble protein samples from the cell lysates were separated by electrophoresis followed by Western blot probed with HRP-conjugated anti-Fc antibody. Heavy chain-containing species are marked based on the apparent molecular weight. Middle panel Western blot of soluble heavy chain in the periplasm. The periplasmic protein samples were reduced by 0.2 M DTT. Bottom panel Western blot of reduced total heavy chain in the whole cell lysate