Protein evolution via amino acid and codon elimination.
BACKGROUND
Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.


RESULTS
The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.


CONCLUSIONS
The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.
Goltermann
Lise
L
Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.
Larsen
Marie Sofie Yoo
MS
Banerjee
Rajat
R
Joerger
Andreas C
AC
Ibba
Michael
M
Bentin
Thomas
T
D000595:Amino Acid Sequence / Q000235:genetics; D003062:Codon / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D016296:Mutagenesis; D010649:Phenylalanine; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics*; D017384:Sequence Deletion
D003062:Codon; D011506:Proteins; D049452:Green Fluorescent Proteins; D010649:Phenylalanine