Optimization of DsRed production in Escherichia coli: Effect of ribosome binding site sequestration on translation efficiency

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 RFPEC 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.

[1]  M. Zimmer,et al.  Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. , 2002, Chemical reviews.

[2]  J. Keasling,et al.  Controlling Messenger RNA Stability in Bacteria: Strategies for Engineering Gene Expression , 1997, Biotechnology progress.

[3]  Christina D. Smolke,et al.  Coordinated, Differential Expression of Two Genes through Directed mRNA Cleavage and Stabilization by Secondary Structures , 2000, Applied and Environmental Microbiology.

[4]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[5]  L. Gold,et al.  Posttranscriptional regulatory mechanisms in Escherichia coli. , 1988, Annual review of biochemistry.

[6]  M. Dreyfus,et al.  In the absence of translation, RNase E can bypass 5' mRNA stabilizers in Escherichia coli. , 1998, Journal of molecular biology.

[7]  Jan van Duin,et al.  Control of prokaryotic translational initiation by mRNA secondary structure , 1990 .

[8]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[9]  W. Bentley,et al.  Biotechnological applications of green fluorescent protein , 2003, Applied Microbiology and Biotechnology.

[10]  D. Bumann,et al.  Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria , 2003, FEBS letters.

[11]  Jan van Duin,et al.  Translational initiation on structured messengers : another role for the Shine-Dalgarno interaction , 1994 .

[12]  B. Glick,et al.  Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed) , 2002, Nature Biotechnology.

[13]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[14]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[15]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  L. Lindahl,et al.  Correlation of translation efficiency with the decay of lacZ mRNA in Escherichia coli. , 1994, Journal of molecular biology.