Abstract
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.
Highlights
Monoclonal antibodies represent an important platform for the development of biotherapeutic products
When the NISTmAb was expressed in SHuffle cells [12] it was observed that in addition to the fulllength heavy and light chains forming the desired product, termed “eNISTmAb,” a copurifying truncated product derived from the heavy chain was formed
When the NISTmAb was expressed in SHuffle cells, a truncated product from the heavy chain was observed (Fig. 1, lane 1, marked by an asterisk) [12]
Summary
When the NISTmAb was expressed in SHuffle cells (eNISTmAb), a truncated product from the heavy chain was observed (Fig. 1, lane 1, marked by an asterisk) [12]. To identify the common mRNA sequence required for the internal initiation, the region upstream (85 nucleotides) and downstream (92 nucleotides) of the GTG codon in eNISTmAb was constructed in-frame with a green fluorescent protein (GFP) reporter under the control of a T7 promoter and cloned into the pET-21a expression vector (Fig. S3). The reporter construct with GTG as the initiation codon resulted in appreciable fluorescence, whereas substituting GTG for any of the other three codons encoding Val (GTA, GTC, and GTT) abolished fluorescence (Fig. 3A) This observation was expected as the latter three codons are not known to serve as translation start sites. GFP median fluorescence intensity (MFI) of the ATG population [19,737] was more than an order of magnitude greater than that of the GTG population [1,147], and other NNN constructs showed per-cell GFP MFIs comparable with both the ATG proϪ construct and untransformed E. coli BL21(DE3)
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