Part:BBa_K3195004
Trx-His-EK tagged subtilisin-like protease
Subtilisin-like protease is a kind of alcalase found in amphioxus (Branchiostoma) similar to subtilisin protease. In our project, it has been used to degrade microcystin with another enzyme called carboxypeptidase Z/N.
Usage and Biology
Microcystin Degradation
Similar to subtilisin, our subtilisin-like protease found in amphioxus (Branchiostoma)[1] can catalyze the hydrolysis of protein to amino acid. Subtilisin has been widely used in all walks of life even in laundry.
Interestingly, we found the subtilisin-like protease can degrade not only proteins but also microcystins.BBa_K3195004 was created for mass expression of this enzyme in E.coli(BL21). We remove the signal peptide and optimize its condons. Moreover, we added Trx-His-EK tag at N-terminals for the convenience of purification.We test the activity of this enzyme in degrading microcystin and measured by HPLC-MS-MS.In the wet lab experiments, we found that carboxypeptidase Z/N and subtilisin-like protein could not decompose microcystins independently, but when the two enzymes were put into the experiment, they could decompose one of the microcystins, microcystin RR.
In our project, we cloned this part into pET32a vector and transformed it into E.coli(DH5α) for mass production of subtilisin-like protease .
Characterization
Experession
Optimal Conditions The biobrick was cloned in pET32a expression vector and transformed into E.coli. We decided to evaluate the results by changing two variables in order to determine the optimal protein expression conditions:1. Induction strategy 2. Temperature and time for induction. Bacteria starter obtained by incubation at 37°C and harvested by centrifugation. The results are in Figure 2.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 447
Illegal XhoI site found at 1645 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 889
Illegal AgeI site found at 1120 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 967
//chassis/prokaryote/ecoli
//function/degradation
origin | Branchiostoma |
protein | subtilisin-like protease |
tag | Trx-His-EK |