Enhancing full-length antibody production by signal peptide engineering

Yizhou Zhou,Linda Rangell,Peter Liu,Anand Kumar Katakam,Wendy Sandoval,Yutian Gan,Mike Reichelt,Dorothea Reilly

doi:10.1186/s12934-016-0445-3

Abstract

BackgroundProtein 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).ResultsIn 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0445-3) contains supplementary material, which is available to authorized users.

Highlights

Protein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies
Production of full‐length hu5D5 in the periplasm of E. coli and challenges in antibody chain secretion We started with a humanized anti-MET hu5D5.v2 antibody [32] as a model IgG1 to study the production of full-length monoclonal antibodies (mAbs) in the periplasm of E. coli
Simmons et al previously constructed signal sequence of Heat-Stable Enterotoxin II (ssSTII) variants with various translation initiation region (TIR) [31] and showed that lower TIR strengths resulted in increased full-length mAb production [33]

Summary

Introduction

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. Protein secretion to the periplasm of Escherichia coli offers an attractive route to produce heterologous proteins that contain disulfide bonds [1,2,3,4]. In this approach, the N-terminus of the heterologous protein is fused to a signal peptide that mediates translocation of the protein from the cytoplasm to the periplasm. The yields of heterologous proteins in the periplasm are often reported to be low [1, 3]

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Results

Discussion

Conclusion