Elimination of truncated recombinant protein expressed in Escherichia coli by removing cryptic translation initiation site

• We observed a truncated version of a yeast protein expressed in Escherichia coli.
• The truncated protein resulted from cryptic initiation from a GTG codon.
• Mutating the cryptic Shine Dalgarno site eliminated the truncated protein.
• Introducing cryptic sites for other proteins did not produce truncated proteins.

Undesirable truncated recombinant protein products pose a special expression and purification challenge because such products often share similar chromatographic properties as the desired full length protein. We describe here our observation of both full length and a truncated form of a yeast protein (Gcn5) expressed in Escherichia coli, and the reduction or elimination of the truncated form by mutating a cryptic Shine-Dalgarno or START codon within the Gcn5 coding region. Unsuccessful attempts to engineer in a cryptic translation initiation site into other recombinant proteins suggest that cryptic Shine-Dalgarno or START codon sequences are necessary but not sufficient for cryptic translation in E. coli.