title: 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.
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. zyz1977627@gmail.com.
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Protein Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. liu.peter@gene.com.
authors.forename: Peter
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of Protein Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. gan.yutian@gene.com.
authors.forename: Yutian
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Department of Protein Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. Sandoval.wendy@gene.com.
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
authors.affiliation: Department of Pathology, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. katakam.anand-kumar@gene.com.
authors.forename: Anand Kumar
authors.initials: AK
authors.lastname: Katakam
authors.affiliation: Department of Pathology, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. reichelt.mike@gene.com.
authors.forename: Mike
authors.initials: M
authors.lastname: Reichelt
authors.affiliation: Department of Pathology, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. rangell.linda@gene.com.
authors.forename: Linda
authors.initials: L
authors.lastname: Rangell
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA way, South San Francisco, CA, 94080, USA. reilly.dorothea@gene.com.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Reilly
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D057927:Hydrophobic and Hydrophilic Interactions; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D019897:Periplasm / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D021382:Protein Sorting Signals