Coding

Part:BBa_K3113072

Designed by: Sarah Brajkovic   Group: iGEM19_Munich   (2019-10-13)
Revision as of 22:45, 21 October 2019 by SarahiGEMMUC (Talk | contribs)


CD63_Asn180_6xHis

CD63 is a tetraspanin found in exosomes. This version has a His-tag loop cloned into the large extracellular loop at N180 for purification of exosomes.

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 Asn180 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.[4] BBa_K3113051 is thus an improvement from iGEM 2018 XJTLU-China's part BBa_K2619003[5], which just contains the human CD63 sequence.


Figure 1:CD63 with a polyhistidine integrated in the large extracellular loop


Biology

CD63 is a Tetraspanin. Tetraspanins are a superfamily of cell surface-associated membrane proteins with four transmembrane domains. CD63 was the first characterized Tetraspanin. 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.[6]

Characterization


Western Blot

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.

Histogramm of HEK: exosomes.
Figure 5:Western Blot against CD63 and Internal His Tag This compares in each case the native CD63, the CD63-Ser161-6xHis and the CD63-180-6xHis loaded with two different RNA binding proteins.


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]

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. ↑ Alves, N.J., Turner, K.B., DiVito, K.A., Daniele, M.A., and Walper, S.A. (2017). Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant. Res. Microbiol. 168, 139–146.
  5. ↑ https://parts.igem.org/Part:BBa_K2619003
  6. ↑ 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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