Part:BBa_K3979005:Design
T. aestivum Wild Type Chitinase
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 712
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 7
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 73
Illegal NgoMIV site found at 621 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
While designing the sequence of the recombinant chitinase, there were several considerations that we needed to keep in mind. Firstly, since we planned to express the chitinase in E. coli, it became necessary that the final molecular weight was less than 100 kDa to enable robust protein folding post expression in the chassis. This imposed a constraint on the size and number of wild-type domains we could join. Secondly, since we aimed to use the chitinase as a therapeutic, the activity of our enzyme as a function of temperature and pH were essential parameters to keep in mind. Our interactions with distinguished crystallographers made us realize that these parameters would depend heavily on the temperature and pH activity curves of the wild-type chitinases we chose. Hence, we decided on wild-type enzymes that retained a significant fraction of their activities at physiological temperatures and pH.
Source
Chitinase II precursor from Triticum aestivum GenBank ID: AAD28730.1
References
- Singh A, Kirubakaran SI, Sakthivel N. Heterologous expression of new antifungal chitinase from wheat. Protein Expr Purif. 2007 Nov;56(1):100-9. doi: 10.1016/j.pep.2007.06.013. Epub 2007 Jul 12. PMID: 17697785.
- Bartholomew ES, Black K, Feng Z, et al. Comprehensive Analysis of the Chitinase Gene Family in Cucumber (Cucumis sativus L.): From Gene Identification and Evolution to Expression in Response to Fusarium oxysporum. Int J Mol Sci. 2019;20(21):5309. Published 2019 Oct 25. doi:10.3390/ijms20215309
- O. Trott, A. J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010) 455-461 DOI 10.1002/jcc.21334