Research Project
2024585
DESCRIPTION: (Adapted from the Applicant's Abstract): Noninvasive methodologies for assaying enzymatic activity within cells are currently unable to differentiate between increased levels of protein expression and improved catalytic activity. In protein engineering, the former category mutants are often considered false positives and are usually the result of 'promoter up' mutations, rather than the desired physicochemical effect of improving enzyme efficiency. It is proposed to use the fluorescence signal from the green fluorescent protein (GFP) in fusion with enzymes to normalize for expression levels in single cells assayed by noninvasive optical techniques. In model studies, E-coli strains expressing a GFP-glucosidase fusion protein will be screened by quantitative imaging techniques. The chromogenic signal resulting from enzyme-catalyzed turnover of the substrate X-glu will be normalized by the fluorescence signal from the co-translated GFP tag, thus giving a measure of the specific activity of the enzyme. This optical screening procedure will be validated by constructing a series of GFP-glucosidase fusions in which the glucosidase gene product is either wild-type or impaired due to mutations affecting the rate of substrate turnover. Optical parameters (absorbance combined with fluorescence) will be measured on a cell sorter for selecting repetitively mutagenized bacteria to isolate enzymes with novel catalytic activities from very large mutagenized populations (>106 clones).
