Expanded chemical diversity sampling through whole protein evolution.
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.
Baldwin
Amy J
AJ
Cardiff University, School of Biosciences, Main Building, Park Place, Cardiff, UK CF10 3AT.
Arpino
James A J
JA
Edwards
Wayne R
WR
Tippmann
Eric M
EM
Jones
D Dafydd
DD
D019943:Amino Acid Substitution; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D015202:Protein Engineering; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D018388:Codon, Terminator; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010649:Phenylalanine; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D000604:Amino Acyl-tRNA Synthetases