RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

ABSTRACT: Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic

SUBMITTER: Yin G 

PROVIDER: S-EPMC5465077 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature