Part:BBa_K3195002

GST tagged carboxypeptidase Z/N
Carboxypeptidase is a class of exopeptidases that can specifically degrade a peptide from its C-terminal of and release free amino acids. In our project, we found Carboxypeptidase Z/N can decompose microcystin RR with another enzyme called subtilisin-like protease.

Usage and Biology

Carboxypeptidase can be found in the animal cell fluid of digestive tract, brain, eye, heart, liver and other tissues^[1].According to the difference of serine residues, metal ions and cysteine residues in the active center of carboxypeptidase, carboxypeptidase is divided into serine carboxypeptidase, metal carboxypeptidase and cysteine carboxypeptidase. Carboxypeptidase Z/N is a kind of cysteine carboxypeptidase found in amphioxus (Branchiostoma)^[2] which contains cysteine in its catalytic functional domain.This enzyme removes N-terminal Arg residues from substrates, as do many of the other members of the gene family^[3]. However it also contains a metallocarboxypeptidase domain. That's why it is called carboxypeptidase Z/N not carboxypeptidase Z or carboxypeptidase N.
BBa_K3195002 was created for mass expression of this enzyme in E.coli(BL21). We remove the signal peptide and optimize its condons. Moreover, we added GST-Tag at C-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 8(in red) was degraded by carboxypeptidase Z/N. The sample 12 was degraded by subtilisin-like protease.

In our project, we characterised this BioBrick by expressing it in E.coli(BL21). However, this BioBrick can be implemented in any host expression system by cloning it into an appropriate vector.

Characterization

Expression

Optimal Conditions
The biobrick was cloned in pGEX-6P-1 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: 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 4.2mg.

Figure 3. Coomassie blue staining. Reducing-PAGE and WB 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. GLY-90, CYX-92 and LEU-261 were found in carboxypeptidase Z/N.

Figure 4. The three dimension prediction of carboxypeptidase Z/N.

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 subtilisin-like protease , they can decompose microcystin RR.

References

[1]Ahn, S. J., Kim, N. Y., Jeon, S. J., Sung, J. H., Je, J. E., Seo, J. S., … Lee, H. H. (2008). Molecular cloning, tissue distribution and enzymatic characterization of cathepsin X from olive flounder ( Paralichthys olivaceus). Comparative Biochemistry and Physiology, Part B, 151(2), 203–212. https://doi.org/10.1016/j.cbpb.2008.07.001
[2]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
[3]Reznik, S. & Fricker, L. CMLS, Cell. Mol. Life Sci. (2001) 58: 1790. https://doi.org/10.1007/PL00000819

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1813
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 673
  Illegal XhoI site found at 1939
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1906
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 85