Coding

Part:BBa_K3113051

Designed by: Sarah Brajkovic, Joshua Hesse   Group: iGEM19_Munich   (2019-09-05)
Revision as of 20:38, 20 October 2019 by Theresakeil (Talk | contribs)


CD63_Ser161_6xHis

To facilitate the purification of intact exosomes, we engineered the exosomal marker CD63 by integrating a polyhistidine repeat into the large extracellular loop of the tetraspanin. Based on SWISS-MODEL, a protein structure homology-modelling server, we were able to model CD63 and to search for sites within CD63 that are accessible.


CD63 structure based on CD81; added His-tag in red

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]

Usage

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This part was designed to allow the purification of exosomes via affinity chromatography. The exosomal marker protein CD63 belongs to the family of tetraspanins and is therefore composed of four alpha-helical transmembrane domains with two extracellular loops. Both the N- and the C-terminus point towards the inside of exosomes, rendering terminal His-tagging useless for affinity purification of exosomes. Therefore, we innovatively fused a 6xHis-tag to an external loop of the exosomal marker CD63. Specifically, the 6 histidines were added after position Ser161 based on a structural model for CD63[1] generated with swissmodel.expasy.org[2] and based on the structure of the related tetraspanin CD81[3]. To our knowledge, Ni-NTA affinity chromatography has not been previously been used to purify exosomes, it has only been applied to other His-tagged membrane structures (Alves et al 2017). BBa_K3113051 is thus an improvement from iGEM 2018 XJTLU-China's part BBa_K2619003[4], which just contains the human CD63 sequence.

Figure 2:CD63


Biology

CD63 is a Tetraspanine. Tetraspanins are a superfamily of cell surface-associated membrane proteins with four transmembrane domains. CD63 was the first characterized Tetraspanine. It is abundantly present in late endosomes and lysosomes as well as exosomes. The gene is located on the human chromosome 12q13. Although the intracellular function of CD63 remains to be established, a number of studies performed in different cell types implicate a role for CD63 in intracellular transport of other proteins.[5]

Characterisation


Purification

We performed a Ni-NTA affinity chromatography to purify our modified exosomes from the supernatant of HEK293T and Min6-K8 cells. Afterwards, a HiBit assay was performed with the Flow-through, wash and elution phases to measure the content of CD63-Ser161-6xHis which was fused to a HiBit split luciferase.

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Figure 1: Ni-NTA affinity purification of exosomes monitored via the HiBiT-tag on CD63. Tested were exosomes containing WT CD63 and His-tagged CD63 between positions Ser161 and K162. Data from three independent purification experiments.


WesternBlot

To confirm the HiBit data of the purification a Western Blot was performed with the elution phases of the CD63-6xHis constructs as well as the concentrated SN of exosomes with natural CD63.


References


  1. ↑ https://2019.igem.org/wiki/images/0/02/T--Munich--CD63_Structure_pdf.pdf
  2. ↑ https://swissmodel.expasy.org
  3. ↑ https://swissmodel.expasy.org/templates/5tcx.1
  4. ↑ https://parts.igem.org/Part:BBa_K2619003
  5. ↑ Trafficking and function of the tetraspanin CD63 Cell Microscopy Center, Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands Received 16 September 2008, Accepted 23 September 2008, Available online 7 October 2008.
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