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Optimization of DsRed production in Escherichia coli : Effect of ribosome binding site sequestration on translation efficiency
Author(s) -
Pfleger Brian F.,
Fawzi Nicolas J.,
Keasling Jay D.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20630
Subject(s) - escherichia coli , ribosomal binding site , green fluorescent protein , ribosome , translation (biology) , mutagenesis , protein engineering , saturated mutagenesis , fluorescent protein , binding site , recombinant dna , biology , chemistry , gene , microbiology and biotechnology , computational biology , biochemistry , messenger rna , enzyme , mutation , rna , mutant
DsRed‐Express, a popular reporter protein, cannot be expressed in Escherichia coli using a consensus ribosome binding site (RBS) potentially due to basepairing in the RBS that inhibits translation initiation. Saturation mutagenesis was used to probe for a gene sequence that minimized basepairing in the RBS while maintaining the same spectral properties and maturation characteristics as DsRed‐Express. The new DsRed, designated here as RFP EC for E. coli optimized red fluorescent protein, fluoresces 2.5 times greater than DsRed‐Express when expressed from the same vector. © 2005 Wiley Periodicals, inc.