Difference between revisions of "Part:BBa K3606000"

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See detailed characterization of our signal-peptide guided CaAP secretion system in <html>< a href="https://2020.igem.org/Team:Fudan" target="https://parts.igem.org/Part:BBa_K3606038">BBa_K3606038</ a> and <html>< a href="https://2020.igem.org/Team:Fudan" target="https://parts.igem.org/Part:BBa_K3606035">BBa_K3606035</ a>
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See detailed characterization of our signal-peptide guided CaAP secretion system in <html>< a href="https://2020.igem.org/Team:Fudan" target="_blank">BBa_K3606038</ a> and <html>< a href="https://2020.igem.org/Team:Fudan" target="_blank">BBa_K3606035</ a>
  
 
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Revision as of 03:39, 28 October 2020


CaAP, Calcium Absorption Peptide

CaAP(Calcium Absorption Peptide) is an artificially designed peptide to promote calcium absorption of intestinal epithelial cells.

Usage and Biology

CaAP(Calcium Absorption Peptide) is designed for facilitating calcium absorption in the intestinal digestive process. The sequence design of CaAP is based on calcium-binding peptides, a class of oligopeptides capable of chelating calcium, which have been found in hen egg yolk, cow milk casein, whey, soy, wheat germ and tilapia fish, etc. They have drawn a great deal of attention from researchers for their ability to promote calcium uptake in Caco-2 cells, indicating that they could serve as a novel kind of nutritional supplementary with high efficiency to help address the calcium deficiency problem and elevate calcium bioavailability, especially for the elderly. Moreover, the biosafety of the peptides used in our design can be guaranteed as the MTT assay have shown their absence of cytotoxicity.

The whole sequence of CaAP contains 5 different calcium-binding peptides: GPAGPHGPVG, FDHIVY, YQEPVIAPKL[1]., NDEELNK[2]., and DHTKE[3]., which have been shown to enhance calcium transport into Caco-2 cell monolayers prominently as compared with the calcium alone. Among these peptides, GPAGPHGPVG, FDHIVY, YQEPVIAPKL and NDEELNK function by forming calcium-peptide complex through Asp and Glu residues. As for DHTKE, which also facilitates the calcium influx into the cytoplasm of intestinal epithelial cells through the calcium chelation, it interacts with the bivalent calcium ion by carboxyl oxygen and amino nitrogen of Asp and Glu as well as the imidazole nitrogen atoms of His residue. <p>To efficiently express CaAP in the engineered strains(E.coli) and allow them to play a role when secreted into the extracellular space, sequences of the 5 mentioned oligopeptides are linked together through FR junctions(Phe-Arg). Hence, they could be expressed in the form of a whole peptide in the engineered bacteria and later cleaved into functional oligopeptides in the presence of digestive enzymes in the intestinal lumen.And we also added 6x-His tag to the C terminal of CaAP for its purification and characterization.

Moreover,to promote calcium bioavailability, CaAP needs to be expressed in our probiotics and subsequently secreted into the extracellular space. Therefore, we linked 5 signal peptides which belong to the Type II secretion system in E.coli., to the N terminal of CaAP, respectively.

The 5 secretion peptides are listed below:

Signal Peptide Number for Registry
NSP4 BBa_K3606042
OmpA BBa_K3606043
DsbA BBa_K3606030
PelB BBa_K208004
PhoA BBa_K808028
Figure 1. The sequence design of CaAP(Calcium Absorption Peptide)

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 78
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

See detailed characterization of our signal-peptide guided CaAP secretion system in < a href="https://2020.igem.org/Team:Fudan" target="_blank">BBa_K3606038 and < a href="https://2020.igem.org/Team:Fudan" target="_blank">BBa_K3606035 ===References===
  1. ↑ Three Newly Isolated Calcium-Chelating Peptides from Tilapia Bone Collagen Hydrolysate Enhance Calcium Absorption Activity in Intestinal Caco-2 Cells. Wanwen Liao, Hui Chen, Wengang Jin, Zhennai Yang, Yong Cao, and Jianyin Miao. Journal of Agricultural and Food Chemistry 2020 68 (7), 2091-2098.
  2. ↑ In vitro digestion profile and calcium absorption studies of a sea cucumber ovum derived heptapeptide–calcium complex. Pengbo Cui, Songyi Lin, Ziqi Jin, Beiwei Zhu, Liang Song and Na Sun. Food Funct., 2018,9, 4582-4592.
  3. ↑ An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide–Calcium Complex. Na Sun, Ziqi Jin, Dongmei Li, Hongjie Yin, and Songyi Lin. Journal of Agricultural and Food Chemistry 2017 65 (44), 9782-9789.