Improving protein solubility: the use of the Escherichia coli dihydrofolate reductase gene as a fusion reporter.

Liu, J-W; Boucher, Y; Stokes, HW; Ollis, DL

Improving protein solubility: the use of the Escherichia coli dihydrofolate reductase gene as a fusion reporter.

Liu, J-W Boucher, Y Stokes, HW Ollis, DL

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Publication Type:: Journal Article
Citation:: Protein Expr Purif, 2006, 47 (1), pp. 258 - 263
Issue Date:: 2006-05

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Field	Value	Language
dc.contributor.author	Liu, J-W	en_US
dc.contributor.author	Boucher, Y	en_US
dc.contributor.author	Stokes, HW	en_US
dc.contributor.author	Ollis, DL	en_US
dc.date.available	2005-11-18	en_US
dc.date.issued	2006-05	en_US
dc.identifier.citation	Protein Expr Purif, 2006, 47 (1), pp. 258 - 263	en_US
dc.identifier.issn	1046-5928	en_US
dc.identifier.uri	http://hdl.handle.net/10453/8602
dc.description.abstract	We have devised a strategy for screening mutant libraries for enzyme variants with enhanced solubility. The method is based on the observation that Escherichia coli can become insensitive to the antibiotic trimethoprim (TMP) if dihydrofolate reductase (DHFR) is expressed at an appropriate level. DHFR is a very soluble protein and can be expressed at levels that exceed normally lethal concentrations of TMP. In our approach, the gene encoding an insoluble target protein is placed in a vector so that the translated protein will be fused to DHFR. The resulting fusion protein will form inclusion bodies and inactivate DHFR-the cells will be susceptible to TMP. Mutations to the target protein that make it more soluble will also make the fusion protein more soluble so that DHFR will be at least partially active-the cells will be resistant to TMP. As the solubility of the target protein increases, the cells will become more resistant to TMP. The system was tested with a putative acetyltransferase (ACE) from a strain of the marine bacterium Vibrio fischerii. The gene encoding this protein was of interest since it is part of a mobile gene cassette within an integron array of the strain in question. After multiple rounds of shuffling and selection, ACE mutants were produced that had significantly improved solubility.	en_US
dc.language	eng	en_US
dc.relation.ispartof	Protein Expr Purif	en_US
dc.relation.isbasedon	10.1016/j.pep.2005.11.019	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Escherichia coli	en_US
dc.subject.mesh	Tetrahydrofolate Dehydrogenase	en_US
dc.subject.mesh	Acetyltransferases	en_US
dc.subject.mesh	Recombinant Fusion Proteins	en_US
dc.subject.mesh	Directed Molecular Evolution	en_US
dc.subject.mesh	Genes, Reporter	en_US
dc.subject.mesh	Solubility	en_US
dc.title	Improving protein solubility: the use of the Escherichia coli dihydrofolate reductase gene as a fusion reporter.	en_US
dc.type	Journal Article
utslib.citation.volume	1	en_US
utslib.citation.volume	47	en_US
utslib.location.activity	United States	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0699 Other Biological Sciences	en_US
dc.location.activity	ISI:000237604200032	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
utslib.copyright.status	closed_access
pubs.issue	1	en_US
pubs.publication-status	Published	en_US
pubs.volume	47	en_US

Abstract:

We have devised a strategy for screening mutant libraries for enzyme variants with enhanced solubility. The method is based on the observation that Escherichia coli can become insensitive to the antibiotic trimethoprim (TMP) if dihydrofolate reductase (DHFR) is expressed at an appropriate level. DHFR is a very soluble protein and can be expressed at levels that exceed normally lethal concentrations of TMP. In our approach, the gene encoding an insoluble target protein is placed in a vector so that the translated protein will be fused to DHFR. The resulting fusion protein will form inclusion bodies and inactivate DHFR-the cells will be susceptible to TMP. Mutations to the target protein that make it more soluble will also make the fusion protein more soluble so that DHFR will be at least partially active-the cells will be resistant to TMP. As the solubility of the target protein increases, the cells will become more resistant to TMP. The system was tested with a putative acetyltransferase (ACE) from a strain of the marine bacterium Vibrio fischerii. The gene encoding this protein was of interest since it is part of a mobile gene cassette within an integron array of the strain in question. After multiple rounds of shuffling and selection, ACE mutants were produced that had significantly improved solubility.

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http://hdl.handle.net/10453/8602