Expanded chemical diversity sampling through whole protein evolution

Abstract

A directed evolution method has been developed that allows random substitution of a contiguous trinucleotide sequence for TAG throughout a target gene for use in conjunction with an expanded genetic code. Using TEM-1 beta-lactamase and enhanced green fluorescent protein as targets, protein variants were identified whose functional phenotype was rescued in vivo when co-expressed with orthogonal tRNA-aminoacyl-tRNA synthase pairs that insert p-iodophenylalanine in response to UAG. Sequencing of the selected clones that retained the target protein function revealed that >90% of the variants contained in-frame TAG codons distributed throughout the target gene. Such an approach will allow broader sampling of new chemical diversity by proteins, so opening new avenues for studying biological systems and for adapting proteins for biotechnological applications. A common set of reagents allows the method to be used on different protein systems and in combination with an array of different unnatural amino acids, so helping to reveal the true potential for engineering proteins through expanded chemical diversity sampling.

Random Single Amino Acid Deletion Sampling Unveils Structural Tolerance and the Benefits of Helical Registry Shift on GFP Folding and Structure

“…Insertion of the engineered transposon MuDel (Jones, 2005) into the egfp gene encoding EGFP residing within the pNOM-XP3 plasmid was performed using an in vitro transposition and selection procedure described elsewhere (Baldwin et al., 2009) to generate the library egfp Δ 2504 . This is described in more detail in the Supplemental Experimental Procedures.…”

Unnatural amino acid mutagenesis-based enzyme engineering

“…Although this strategy was demonstrated with an antibody, it can also be extended to enzymes where the incorporation of novel amino acid codons into the genetic code can be advantageous for the evolution of enzymes with novel or enhanced functions. In addition, directed evolution methods have been developed that allow random substitution of a contiguous trinucleotide stop codon sequence (TAG) throughout a target gene for use in conjunction with site-specific incorporation [94].…”

“…In the current decade, the directed evolution strategy has been enhanced by an expanded set of amino acids that enlarge the sequence space of proteins and in turn increase the chances of evolving a desired mutant [69,[93][94][95]. Recent progress in protein evolution using UAAs is outlined below.…”

The juggernauts of biology

“…Over the course of the past 10 years, at least 50 amino acids have been successfully integrated using this method. New developments in the field have seen the introduction of novel methods to incorporate TAG mutations24, 25 and also development of new orthogonal aaRS systems. It is now possible to incorporate Naa into several common expression systems including Chinese hamster ovary (CHO) cells,26 Saccharomyces cerevisiae ,27 and a particularly robust system in Pichia pastoris 28.…”