Coding

Part:BBa_K3113072

Designed by: Sarah Brajkovic   Group: iGEM19_Munich   (2019-10-13)
Revision as of 23:47, 21 October 2019 by Johanna Munich2019 (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 2: 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.

Purification

We were able to show, that your polyhistidine-tag works. We analysed the four different fractions (load, flow-through, wash and elution) with Promega's HiBiT-Luciferase assay. The HiBiT-protein was therefore fused to the intracellular site of CD63.

Histogramm of HEK: exosomes.
Figure 3: Purification of exosomes containing the engineered CD63-Ser161-6xHis. The bioengineered CD63 containing a polyhistidine sequence in the large extracellular loop (CD63-6xHis) can be purified using Ni-NTA Agarose beads. In this set-up, the exosomes were purified from transfected HEK293T cells and analysed the individual fractions using the HiBiT assay To test the efficacy of the purification, we compared the results for the CD63-6xHis to the values of unspecific binding of wild-type CD63 to the Ni-NTA beads. The data shown in the figure derive from three independent purification experiments. Using unpaired two-tailed t-test, it can be shown that more HiBiT-Signal can be found in the wash for wild-type CD63 than for the bioengineered CD63-6xHis (p < 0.05, n(CD63)=2, n(CD63-His-L7Ae)=2, n(CD63-His-MCP)=2). For the elution, more HiBiT-signal can be found in CD63-6xHis-MCP samples than in wild-type CD63 samples (p < 0.05, n(CD63)=2, n(CD63-His-L7Ae)=2, n(CD63-His-MCP)=2).

Expression in MIN6-K8 cells

To test wether our engineered CD63-Ser161-His works also in other cell lines, we perfomed a purification on supernatant of transfected MIN6-K8 cells (Figure 4). We were able to purify exosomes containing the engineered CD63 from MIN6-K8 cells.

Histogramm of HEK: exosomes.
Figure 4: Ni-NTA affinity purification of exosomes from MIN6-K8 monitored via the HiBiT-tag on CD63. Tested were supernatants containing exosomes with transfected WT CD63 or a His-tagged CD63 version. The tag is located in the large extracellular loop at position Ser180. Data from one purification experiments.


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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