Part:BBa_K3195004

Trx-His-EK tagged subtilisin-like protease

Subtilisin-like protease is a kind of alcalase 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.

Figure 1.The abscissa represent 28 enzymes in our project and the unit of ordinate value is mg/L. The ordinate value represents the remained microcystin RR(MC-RR) in the degraded sample. The right picture shows the results of degradation by each enzyme, and the left one shows the results of mixing each enzyme with enzyme 8(carboxypeptidase Z/N).The sample 12(in red) was degraded by subtilisin-like protease.

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.

Figure 2. Expression tests of the target protein.
Annotations:
MW. Molecular weight marker.
Ø. Non-induced bacteria culture (negative control).
NO.1 NO.2 NO.3. Three parallel E.coli strains.
NPE.native protein extract. DPE.denatured protein extract.

After analyzing the electrophoretic pattern, we found the optimal conditions for mass expression of subtilisin-like protease : E.coli should be inducted at 37℃ for 4 hours.

Small Scale Purification

In order to determine the feasibility of our mass protein purification, we conducted 200mL purification tests in DPE conditions for target protein(Lysis buffer: PBS, pH7.5, 10% Glycerol; Washing buffer: PBS, pH7.5, 1% Triton X 100, 5 mM EDTA; Denaturing buffer: PBS, pH7.5, 8M urea(or 2%NLS); Dialysis buffer: PBS, pH7.5, 8M urea(or 0.2%NLS) ). Starting materials were prepared after production in optimal native expression conditions described earlier. Final sample was qualitative by SDS-PAGE, quantitative by Bradford method.The result is in Figure 2. The total mass is 2 mg.

Figure 3. Coomassie blue staining. Reducing-PAGE analysis. 2µg of sample loaded.

Homology Modeling

For better understanding its mechanism we predicted its three-dimensional structure. By comparing its sequence with cathepsin B, we found several action sites of cathepsin B could find similar short peptide structures in these two amino acid sequences. CYX-184 was found in subtilisin-like protease.

Figure 4.The three dimension prediction of subtilisin-like protease.

We speculate that subtilisin-like protease are involved in the decomposite of microcystin RR, howvever due to some reasons in its degradation microenvironment, it cannot limit the movement(rotation,vibration,ect) of microcystin RR. Only together with carboxypeptidase Z/N, they can decompose microcystin RR.

Reference

[1]He Chunpeng, Han Tingyu, Liao Xin, Zhou Yuxin, Wang Xiuqiang, Guan Rui, Tian Tian, Li Yixin, Bi Changwei, Lu Na, He Ziyi, Hu Bing, Zhou Qiang, Hu Yue, Lu Zuhong and Chen J.-Y. Phagocytic intracellular digestion in amphioxus (Branchiostoma)285Proc. R. Soc. B http://doi.org/10.1098/rspb.2018.0438

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 447
  Illegal XhoI site found at 1645
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 889
  Illegal AgeI site found at 1120
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 967