Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins.

Kim Wals,Huib Ovaa

doi:10.3389/fchem.2014.00015

Abstract

Unnatural amino acid (UAA) incorporation by amber codon suppression offers scientists a powerful tool to modify the properties of proteins at will. UAA incorporation has been used for a plethora of fundamental research applications and, more recently, also for the selective modification of therapeutic proteins. In this review most recent developments in Escherichia coli codon expansion and, unnatural amino acid incorporation are discussed together with some remarkable recent developments in improved efficient UAA incorporation. We focus on the generation of proteins that hold promise for future therapeutic applications that would be impossible to obtain without unnatural amino acid incorporation, including the generation of bi-specific antibodies and antibody drug conjugates.

Highlights

Unnatural amino acid (UAA) incorporation by amber codon suppression offers scientists a powerful tool to modify the properties of proteins at will
MUTANT SELECTION WITH SINGLE GENETIC CONSTRUCTS The classical method to generate UAA-accepting mutants of orthogonal aminoacyl-transfer RNA (tRNA) synthetase (aaRS) and iso-tRNA pairs for E. coli (Xie and Schultz, 2005b), as described by P.G
Whereas exploiting the ε-amino group of lysine to covalently modify it with polyethylene glycol chains (PEG) generally yields heterogeneous products, for homogenous proteins site-specific reactive groups can be inserted into proteins by amber codon suppression

Summary

Introduction

Unnatural amino acid (UAA) incorporation by amber codon suppression offers scientists a powerful tool to modify the properties of proteins at will. The majority of therapeutic proteins are produced in eukaryotic and mammalian cell systems, E. coli protein expression is in general cheaper, more susceptible to genetic modifications, and versatile with regard to mutant library development.

Results

Conclusion