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. (c) 2005 Elsevier Inc. All rights reserved.