Part:BBa_K314012
CapD_CP
A circularly permuted [http://www.parts.igem.org/Part:BBa_K314011 CapD] with a Foldit-designed linker.
Usage and Biology
To test BBa _K314012, it was inserted into a pET29b+ vector using Kunkel's Mutagenesis. CapD_CP was then produced and purified as described in the [http://2010.igem.org/Team:Washington/Protocols/50mLPurificationCapD UW 2010 iGEM Team's Small Scale Protein Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki]. A protein gel was also run to determine physical qualities. The resulting data is shown below.
We also ran the purified protein samples thought a mass spectrometer to make sure the firs methionine was being removed by the natural E. Coli methionine aminopeptidase and that the protein was the expected size. Our results indicate that a massive majority of the protein in our purified sample was within .02% error of the predicted CapD_CP size.
The substrate vs. reaction rate curve above plots the PDG cleaved per enzyme per hour (y-axis) as a function of substrate concentration (x-axis). As observed in the curve above, high substrate concentrations suffered from substrate inhibition under the conditions our enzyme was assayed. Kinetic constants, kcat and Km, taken from our plotted curves are shown in the table above.
The protein gel above shows the single band seen CapD_CP. This makes quantifying active CapD_CP extremely easy when correcting for enzyme concentration.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1489
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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