Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli

J Biol Chem. 2019 Nov 29;294(48):18046-18056. doi: 10.1074/jbc.RA119.011008. Epub 2019 Oct 11.


Monoclonal antibodies (mAbs) represent an important platform for the development of biotherapeutic products. Most mAbs are produced in mammalian cells, but several mAbs are made in Escherichia coli, including therapeutic fragments. The NISTmAb is a well-characterized reference material made widely available to facilitate the development of both originator biologics and biosimilars. Here, when expressing NISTmAb from codon-optimized constructs in E. coli (eNISTmAb), a truncated variant of its heavy chain was observed. N-terminal protein sequencing and mutagenesis analyses indicated that the truncation resulted from an internal translation initiation from a GTG codon (encoding Val) within eNISTmAb. Using computational and biochemical approaches, we demonstrate that this translation initiates from a weak Shine-Dalgarno sequence and is facilitated by a putative ribosomal protein S1-binding site. We also observed similar internal initiation in the mAb adalimumab (the amino acid sequence of the drug Humira) when expressed in E. coli Of note, these internal initiation regions were likely an unintended result of the codon optimization for E. coli expression, and the amino acid pattern from which it is derived was identified as a Pro-Ser-X-X-X-Val motif. We discuss the implications of our findings for E. coli protein expression and codon optimization and outline possible strategies for reducing the likelihood of internal translation initiation and truncated product formation.

Keywords: Escherichia coli (E. coli); GTG codon; NISTmAb; Shine-Dalgarno sequence; codon optimization; mAb; monoclonal antibody; protein expression; recombinant protein expression; translation initiation; translation regulation.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adalimumab* / biosynthesis
  • Adalimumab* / genetics
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Immunoglobulin Heavy Chains* / biosynthesis
  • Immunoglobulin Heavy Chains* / genetics
  • Peptide Chain Initiation, Translational*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics


  • Immunoglobulin Heavy Chains
  • Recombinant Proteins
  • Adalimumab