Many metabolic and biosynthetic pathways can influence protein product attributes during translation and post-translational modification (i.e. sequence variation, glycosylation, oxidation, proteolysis, etc.). For both recombinant and native protein biosynthesis, the resulting protein product attributes are analogous to a historical record of cellular events. Of particular interest are sequence variants caused by amino acid misincorporation. Believed to be universal, amino acid misincorporation is the inherent errant replacement of an amino acid by another. In biological systems, high-level amino acid misincorporation can cause loss of proteostasis and disease through the compounded accumulation of errors. And in biopharmaceutical applications, since protein product function is derived from the product’s attributes, elevated sequence variant frequency has been shown to result in adverse effects such as product instability, structure change, aggregation, and the potential for immunogenicity. Amino acid starvation, fast cell growth, overproduction, and oxidative stress, which are physiological conditions that can be observed in disease states and during biopharmaceuticals production, can all significantly exacerbate mistranslation by orders of magnitude. In an effort to elucidate the major sequence variant mechanisms, to our best knowledge, our work is the first investigation of amino acid misincorporation through systematic amino acid starvation. Our results reveal that one-base mismatches during codon recognition and tRNA misacylation are both common and major mechanisms. Also, different cell lines share common misincorporation patterns suggesting that a conserved set of mechanisms and error propensities exists.

Implications of protein sequence variants derived from systematic starvation

255th National Meeting (2018)

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