Part:BBa_K4963000
Cannabinoid Receptor 1 (Modified)
The modified part is an optimized receptor protein with an open side for fusion, retrieved from Cannabinoid receptor 1. The active binding sites for the purpose of this project (binding sites for honokiol and AM - 6538) were found restricted to a certain domain of the protein. To optimize the receptor, the excessive part was shed to obtain a modified binding domain still expressible on its own. The resultant is a coherent cylindrical structure functioning as the receptor. The part may be used as a receptor/carrier for usual cannabinoid receptor targets as a shorter, more effective version.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 345
Illegal PstI site found at 1044 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 345
Illegal PstI site found at 1044 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 345
Illegal PstI site found at 1044 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 345
Illegal PstI site found at 1044 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 843
This new part presents three distinct advantages/features:
1. Removal of redundant domains, allowing for more directed binding.
2. Reduction of alternative binding sites for other biological agents, which may
interfere with the system.
3. Increasing the probability of competitive inhibition/release of honokiol (our drug) by AM - 6538 (our antagonist), by restricting them to a smaller space.
Figure 1 depicts the modifications made.
Fig. 1 Cannabinoid Receptor whole with removed part (red) and retained part (purple) highlighted. Image created on AutoDock Tools.
Extraction and Usage
The modified CB1 construct was obtained from GenScript in the cloning plasmid pUC57 with the restriction site specified against the type II restriction enzyme EcoRV. The plasmid was digested with EcoRV. For the plasmid, 1µl of sample was digested against 1µl EcoRV in a 30µl reaction set for 4 hours. However, pUC57 was undigested.
The procedure was then repeated, with the reaction set for 8 hours. Negative results were obtained i.e. pUC57 was still undigested. The reaction was again repeated, now set for 12 hours, but to no avail.
In consultation with IDT, the restriction targets were modified to EcoR1 and HindIII. 1 µl each of both enzymes was used to digest 1µl pUC57. The construct was now successfully digested.
Fig. 2 Restriction Digestion digestion with EcoR1 and HindIII; gel electrophoresis results shown. pUC57 (digested) shown in lane1. Lane 5 shows positive control. Upper band in lane1 (red) is empty pUC57, at 2710bp. Lower band in lane1 is CB1, at 1320 bp.
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