Difference between revisions of "Part:BBa K3195002"

Revision as of 11:46, 18 October 2019

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 C-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, an C-terminal 6XHis-tag were added.
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 three variables in order to determine the optimal protein expression conditions:1. Induction strategy 2. Temperature and time for induction 3. E.coli strain used for protein production. Bacteria starter obtained by incubation at 37°C and harvested by centrifugation. The results are in Figure 1.

Figure 1. 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

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