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Ubiquitin Carboxy Hydrolase‐L3 (UCH‐L3): A Molecular Sensor of Protein Stability?
Author(s) -
RomoFewell Octavio,
Navarro Mario,
Love John J.
Publication year - 2010
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.24.1_supplement.463.14
Subject(s) - hydrolase , ubiquitin , biochemistry , enzyme , hydrolysis , chemistry , deubiquitinating enzyme , protease , in vivo , microbiology and biotechnology , biology , gene , genetics
The accurate assessment of protein stability, as well as exploring new means to enhance protein stability are of keen interest. To achieve this we are exploring the properties of ubiquitin carboxy hydrolase‐L3 (UCH‐L3), a protease that cleaves proteins N‐terminally fused to ubiquitin. Based on the hydrolysis rate we are exploring whether UCH‐L3 is capable of differentiating between proteins with different thermal stabilities. First, we developed a panel of mutants derived from streptococcal protein G that span a thermal stability range from 28–100 °C. The genes for these variants were fused N‐terminal to the gene for ubiquitin. To explore the activity of UCH‐L3 in vivo, E. coli (BL21) cells were cotransformed with plasmids coding for both the enzyme and the fusions in compatible plasmids. After protein induction the hydrolysis rates were estimated with SDS‐PAGE. For further characterization, ex vivo and in vitro assays were run with cell extracts and the purified enzyme and substrates. In addition, size exclusion chromatography and CD (Circular dicrhoism) studies were performed in order to determine the oligomerization state and the melting temperature (Tm) of the substrates. In the three formats tested in vivo , ex vivo and in vitro , the hydrolysis was similar and consistent. Hydrolysis of the different constructs by UCH‐L3 is correlated to the thermal stability of each particular test protein. Interestingly, the enzyme is capable of differentiating between substrates with only one amino acid change, even for a position far from the cleavage site. Founding has been provided by National Science Foundation, Consejo Nacional De Ciencia y Tecnologia, Mexico, and by the NIH/NIGMS SDSU MARC Program 5T34GM08303.