Difference between revisions of "Part:BBa K3195003"
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[[Image:T--SEU-Nanjing-China--SDS-PAGE cathepsin B expression.png|360px|thumb|center|<b>Figure 3. Expression tests of the target protein. </b> | [[Image:T--SEU-Nanjing-China--SDS-PAGE cathepsin B expression.png|360px|thumb|center|<b>Figure 3. Expression tests of the target protein. </b> | ||
<br/>Annotations: | <br/>Annotations: | ||
− | <br/>MW. Molecular weight marker. | + | <br/><b>MW.</b> Molecular weight marker. |
− | <br/>Ø. Non-induced bacteria culture (negative control). | + | <br/><b>Ø</b>. Non-induced bacteria culture (negative control). |
− | <br/>16 and 37. Incubation temperature (°C) during induction with IPTG.Induction with IPTG 1mM during 16h at 16°C, or during 4h for other temperatures.<br/>NPE.native protein extract. DPE.denatured protein extract.]] | + | <br/><b>16 and 37</b>. Incubation temperature (°C) during induction with IPTG.Induction with IPTG 1mM during 16h at 16°C, or during 4h for other temperatures. |
+ | <br/><b>NPE.</b>native protein extract. <b>DPE.</b>denatured protein extract.]] | ||
[[Image:T--SEU-Nanjing-China--ExpressionGel1.png|360px|thumb|center|<b>Figure 4. Final sample QC of denatured condition</b> | [[Image:T--SEU-Nanjing-China--ExpressionGel1.png|360px|thumb|center|<b>Figure 4. Final sample QC of denatured condition</b> |
Revision as of 13:41, 16 October 2019
His-tagged Cathepsin B
This Cathepsin B from Branchiostoma belcheri tsingtauense is originally a kind of hydrolase in lysosome.In our project,it has been used to biodegrade microcystin,a kind of biotoxin synthesized by cyanobacteria.
Usage and Biology
Our BioBrick encodes a kind of cathepsin from Branchiostoma diverticulum epithelial cells. Cathepsin is a class of proteins found in the cells of various animal tissues, especially lysosome parts. Based on the results of bioinformatics analysis, the protein we found out belongs to cathepsin B. In Branchiostoma diverticulum epithelial cells, we speculate this kind of protein plays an important role in degrading cyanobacteria and microcystin. In fact, according to our experimental result, Cathepsin B protein has high efficiency in degradation of microcystin LR. So we construct a composite part so that other teams can easily use it. This BioBrick can be implemented in any host expression system by cloning it into an appropriate vector.
Characterization
Expression
The Biobrick was cloned in pET28b expression vector. After confirming the cloning by sequencing, the plasmid was transformed into E.coli DH5α. The transformation was confirmed by colony PCR. Cells (E. coli BL21 (DE3)) were cultured with the induction of IPTG under optimal expression condition. Ultrasound broke cells to separate proteins. A special sequence, His-Tag, was added to the end of the target protein. His-Tag can bind to metal Ni2+ ions, which is beneficial to the purification of target protein. The protein added with His-Tag can be purified by Ni2+ affinity chromatography column under non-denaturing conditions. SDS-PAGE was used to detect the expression of proteins after purification. The result was shown below.Activity assay
The BioBrick was characterized by measuring cathepsin B activity using Cathepsin B Activity Assay Kit. BioVision's Cathepsin B Activity Assay Kit is a fluorescence-based detection technique. Using AFC (7-amino-4-trifluoromethyl coumarin) labeled cathepsin-B priority substrate sequence RR. Cell solutes or other samples containing Cathepsin-B can digest RR-AFC and release free AFC. Free AFC can be easily quantified by fluorometer or fluorescence microtitrator plate.
We determined Km and Vmax for our cathepsin B by performing non-linear regression using Michaelis-Menten model as below. The two parameters were: Vmax=3749.56222 ± 87.82359 RFU/h Km=0.22768 ± 0.02604 mol/L
Molecular docking
In our project, we conducted homology modeling for better understanding its function. Then MOE Dock was used for molecular docking of cathepsin B with the small molecule(microcystin) and predicting the binding affinity. Small molecule was defined as ligand and cathepsin B as target. The binding site was identified by superpose cathepsin B with the original template structure, the position where the ligand in template structure was defined as binding site of cathepsin B. The docking workflow followed the “induced fit” protocol, in which the side chains of the receptor pocket were allowed to move according to ligand conformations, with a constraint on their positions. The weight used for tethering side chain atoms to their original positions was 10. The best ranked pose was selected as the final binding mode.
To investigate the binding affinity of CTS with microcystin, docking simulation studies were carried out. The docking scores are shown in table 1.
The binding model between cathepsin B and microcystin are shown in Figure 3. The nitrogen atoms of guanidine group of mcirocystin, regarded as hydrogen bond donor, forms hydrogen bonds with the backbone oxygen atom of Gly90 and Cys92, and with the side-chain chlorine atom of Cys184 in CTS respectively. The nitrogen atom of microcystin, regarded as hydrogen bond donor, forms a hydrogen bond with the backbone oxygen atom of Leu261 in cathepsin B.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 369
Illegal XhoI site found at 958 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 463
- 1000COMPATIBLE WITH RFC[1000]