Scar

Part:BBa_K5384002

Designed by: Si Cheng   Group: iGEM24_HBUT-Wuhan   (2024-08-23)


DDDDK

Enterokinaase (EK) is a heterodimeric serine proteolytic enzyme found in mammalian duodenum. It can efficiently and specifically recognize asp-asp-asp-lys (DDDDK) sequences in proteins, and hydrolyze the lysine at the C-terminal of peptide bonds. It is very suitable for enzyme digestion of genetically engineered fusion protein, and is specially designed for cleavage of recombinant fusion protein. This characteristic makes EK widely used in the field of biomedicine.
Figure 1 Three dimensional structure of enterokinase

Usage and Biology

DDDDK enterokinase is a tool enzyme. Enterokinase specifically cleaves peptides or proteins containing recognition sequences and is commonly used in biotechnology to perform specific cleavages of fusion proteins to release the protein of interest. DDDDK enterokinase is highly effective and specific. In protein structure and function studies, it is used to accurately cleave the target protein from the fusion protein for subsequent analysis and experimentation. In the biopharmaceutical process, it can be used to prepare protein drugs with specific activities. The enterokinase recognition site on the plasmid serves as a recognition site, allowing the enterokinase to act on the induced proteins.[1,2]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Application

The low-cost and high-efficiency one-step protein separation method of histidine (His) labeling at the end of the protein and affinity purification of Nickel metal chelating column is also widely used in the separation and purification of rEK, with a yield of more than 50%. For the His-tag site, Choi et al. found that the enzyme activity of rECL was not changed after the C-terminus was His-labeled, but the enzyme activity was lost after the N-terminus was labeled by the Nickel metal chelating column.[3] In this project, we designed a DDDDK site between His-tag and Vg segment, which can be recognized and cleaved by EK.

References

[1]ZHANG Xianghui,TAN Shuhua,LI Taiming. Research progress on the characteristics of enterokinase and its genetic engineering. CNKI, 2005

[2]Xu Liang, Wang Wenjing, Zu Xiangyang, Wang Xiaochun. Characteristics of enterokinase digestion of fusion protein Trx-Exendin-4. 《CNKI; WanFang", 2012

[3]Cao Zhifei, Li Xinping, Fan Kai, Li Jing, et al. Expression and purification of recombinant bovine enterokinase light chain gene in Pichia pastoris. 《CNKI; WanFang》,2006

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Categories
//cds/enzyme
Parameters
biology