Difference between revisions of "Part:BBa K3113072"
 
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==Experimental Characterization by AFCM-Egypt==
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In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P4) including CD63 an L7Ae, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
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We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P4).
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<h2>Characterization</h2>
 
<h2>Characterization</h2>

Latest revision as of 20:56, 11 October 2023


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

CD63“ width=


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 was the first characterized tetraspanin. It is abundantly represented in late endosomes and lysosomes, as well as exosomes. The gene, coding for CD63, is located on the human chromosome 12q13. Although the intracellular function of CD63 remains to be elucidated, a number of studies performed in different cell types implicate a role for CD63 in intracellular transport of other proteins.[6]

Characterization By Mutational Landscape by AFCM-Egypt

In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (D230N) shows the highest epistatic fitness, while the lowest score was associated with the mutation (V96S).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of CD63.

Literature Characterization by AFCM-Egypt

PCR was used to amplify the fragment of the outside membrane of SjCD63. The resulting cDNA was cloned into the pET-28a vector to produce the recombinant protein. The recombinant plasmids were then transformed into E. coli BL21 competent cells. The His-tagged CD63 recombinant protein was expressed in E. coli and purified using a Ni column

SDS-PAGE of the purified recombinant protein showed a single band of the expected size, indicating that the purified CD63 protein was of high purity . The purified recombinant SjCD63 was further confirmed by mass spectrometry. In summary , a combination of PCR, cloning, expression, and purification techniques was used to produce a high-purity recombinant SjCD63 protein.

Experimental Characterization by AFCM-Egypt

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P4) including CD63 an L7Ae, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.





We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P4).


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. We can see a comparison between 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 polyhistidine-tag engineered by us, works. We analysed four different fractions (load, flow-through, wash and elution) with Promega's HiBiT Extracellular Detection 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 individual fractions were analysed using the HiBiT assay. To test the purification efficiency, 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 below has been derived 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 whether our engineered CD63-Ser161-His works in other cell lines, we purified 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.