Craig, Douglas B. and Schwab, Thomas and Sterner, Reinhard (2012) Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of beta-galactosidase. BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE, 90 (4). pp. 540-547. ISSN 0829-8211,
Full text not available from this repository. (Request a copy)Abstract
Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional beta-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized beta-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SINGLE-MOLECULE; ESCHERICHIA-COLI; ELECTROPHORETIC MOBILITY; CATALYTIC-ACTIVITY; ENZYME MICROHETEROGENEITY; ALKALINE-PHOSPHATASE; E. COLI; FREQUENCY; ENERGY; ASSAYS; beta-galactosidase; catalytic heterogeneity; translational error; E. coli; capillary electrophoresis |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 08 May 2020 06:11 |
| Last Modified: | 08 May 2020 06:11 |
| URI: | https://pred.uni-regensburg.de/id/eprint/18413 |
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