Journal
CHEMBIOCHEM
Volume 17, Issue 13, Pages 1250-1256Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201600117
Keywords
amber codon; blue-white screening; codon context; genetic code expansion; high-throughput screening; protein engineering
Funding
- National Natural Science Foundation of China [81530090, 21572015, 813611680027]
- National Basic Research Program of China (973 Program) [2010CB12300]
- Special Project for Significant New Drugs Development grant [2013ZX09103003020]
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The effect of codon context on amber codon-guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re-explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ- gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X-gal. Large-scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual-luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria.
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