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Screening for additives that stabilize enzymes at higher temperatures
Author(s) -
Coleman Jack,
Dai Lijun,
Patton Wayne F
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.909.4
Subject(s) - trehalose , enzyme , chemistry , polymerase , cryoprotectant , biochemistry , enzyme assay , dna , rna polymerase , thermal stability , chromatography , biophysics , microbiology and biotechnology , cryopreservation , rna , biology , organic chemistry , gene , embryo
In Molecular Biology, enzymes are often required that function at elevated temperatures. Enzymes produced by mesophilic organisms usually denature at elevated temperatures, followed by aggregate formation. We developed a rapid fluorescence‐based assay for assessing a range of parameters impacting the thermal stability of an enzyme. Overall, protein stability is monitored by a fluorogenic dye that selectively detects aggregated protein. Stability can be measured in the presence of different buffers, cryoprotectants and excipients. By systematically raising the temperature of the protein in solution, the temperature at which the protein aggregates (T agg ) can be determined. Using this method with Klenow DNA polymerase, we determined that trehalose significantly increases T agg . The DNA polymerase activity of the enzyme is significantly enhanced at 50°C in the presence of trehalose under the same conditions. Low amounts of the detergents, Tween20 ® and Triton X‐100 ® (0.05%) decreased T agg and also compromised enzyme activity, especially at elevated temperatures. T7 RNA polymerase was tested in a similar manner. Trehalose less significantly increased T agg , and had a more modest effect on increasing enzyme activity at 42°C. The described assay facilitates screening for buffers and additives that structurally stabilize a protein of interest at elevated temperatures. This work was supported by ENZO Life Sciences.