Difference between revisions of "Part:BBa K3113051"

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−	<img src="https://2019.igem.org/wiki/images/e/ef/T--Munich--CD63Ser_TEM.png" width="100%" class="figure-img img-fluid rounded" alt="Histogramm of HEK: exosomes.">	+	<img src="https://2019.igem.org/wiki/images/9/91/T--Munich--Exo_MIN6_TEM.png" width="100%" class="figure-img img-fluid rounded" alt="Histogramm of HEK: exosomes.">
	<figcaption><b>Figure 4:</b>		<figcaption><b>Figure 4:</b>
−	Characterization of vesicles was performed by Transmission Electron Microscopy, TEM, (A and B) and Dynamik Light Scattering, DLS,  (C and D).  Grids with exosomes (A) and virus like particles (B) were negatively stained with uranyl acetate for TEM. The diameter for VLPs and exosomes was precisely determined with DLS. It was revealed that RNA loading increases the size of VLPs, whereas exosomes differ in size depending on the secreted organism.	+	Characterization of vesicles was performed by Transmission Electron Microscopy, TEM (A+B).  Grids with exosomes (A) were negatively stained with 1 % uranyl acetate for TEM. The diameter for VLPs and exosomes was precisely determined with DLS. It was revealed that the exosomes differ in size depending on the secreted organism. <br> A difference in size exosomes from MIN6-K8- and HEK293T-cells can be observed, but it is not statistical significant.
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	<img src="https://2019.igem.org/wiki/images/7/78/T--Munich--WBPurification.png" width="50%" class="figure-img img-fluid rounded" alt="Histogramm of HEK: exosomes.">		<img src="https://2019.igem.org/wiki/images/7/78/T--Munich--WBPurification.png" width="50%" class="figure-img img-fluid rounded" alt="Histogramm of HEK: exosomes.">
−	<figcaption><b>>Figure 5:Western Blot against CD63 and Internal His Tag</b> This compares in each case the native CD63, the CD63-Ser161-6xHis and the CD63-180-6xHis loaded with two different RNA binding proteins.	+	<figcaption><b>Figure 5:</b> The presence of His-tagged CD63 in the elutions of the purification was proven via western blotting. The top row shows the staining with the antibody mouse-anti-CD63 and goat-anti-mouse conjugated to horseradish peroxidase. The bottom row shows the staining with the antibody mouse-anti-internal-His-tag and goat-anti-mouse conjugated to horseradish peroxidase. Both rows show the same part of the blot at a size of around 70 kDa.
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Latest revision as of 02:26, 22 October 2019

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.

Usage

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.^[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]

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.

Both parts were put under the control of the mammalian constitutive promoter CAG (BBa_K3113000), contained the same kozak sequence (BBa_K3113003) before the CD63 ORF, and had the same polyadenylation signal motive (BBa_K3113004) at the end. When applying exosomes containing either the WT CD63 or our internally His-tagged CD63 on Ni-NTA columns, only the latter get purified efficiently (Figure 1). Thus, purification of exosomes does not happen due to unspecific interaction with the column material but is enabled by the His-tag on the extracellular loop. Moreover, the vesicle quality along the purification can be monitored with the HiBiT-tag inside the exosomes. Since the HiBiT signal of free His-CD63 outside exosomes is about 20 times lower than the total His-CD63 in the elution fraction (Figure 2), it can be concluded that most of it is still enclosed in exosomes. Therefore whole exosomes are eluted from the column using our improved BBa_K3113051.

Moreover, the vesicle quality along the purification can be monitored with the HiBiT-tag inside the exosomes. Since the HiBiT signal of free His-CD63 outside exosomes is about 20 times lower than the total His-CD63 in the elution fraction (Figure 2), it can be concluded that most of it is still enclosed in exosomes. Therefore whole exosomes are eluted from the column using our improved BBa_K3113051.

Transmission Electron Microscopy

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.

Sequence and Features

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.