Part:BBa_K3629013
Modified PfCBHI expression construct with gibson homology sequences and 6X His tag
Usage and Biology
CHIMERIC PROTEIN CREATION To develop this modified PfCBHI we utilized a hybridized catalytic domain combining the sequence from P. funiculosum and T. reesei. These modifications allow the protein to operate at more moderate conditions for Y. lipolytica than the wild type protein. The linker sequence and cellulose-binding module were then added from the P. funiculosum wild type. These modifications were shown to increase the productivity of the protein and widen the operating parameters for the protein.
Structural models were generated to get a starting point for the protein. The predicted structure was then protonated in silico at numerous pHs. After protonation, the structures were solvated in water and underwent molecular dynamic simulation. Metrics around the simulations were taken and our modelling showed protein stability within the pH 3 to 7 range.
Design
The native signal peptide from P. funiculosum was removed so it would not interfere with the fused Lip2 secretion tag native to Y. lipolytica.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2725
Illegal EcoRI site found at 2546 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2546
Illegal NheI site found at 75
Illegal SpeI site found at 2726
Illegal PstI site found at 2740
Illegal NotI site found at 7
Illegal NotI site found at 2733 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2546
Illegal BglII site found at 1322
Illegal BamHI site found at 2631
Illegal XhoI site found at 87 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2726
Illegal EcoRI site found at 2546 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal EcoRI site found at 2546
Illegal XbaI site found at 16
Illegal SpeI site found at 2726
Illegal PstI site found at 2740
Illegal NgoMIV site found at 804 - 1000COMPATIBLE WITH RFC[1000]
None |