His-tagged CD63 for Exosome Purification
The part we have based our improvement on was submitted by iGEM 2018 XJTLU-China's team with the registry number BBa_K2619003. It consists of the human CD63 sequence, a transmembrane protein considered to be one of the main exosomal specific markers. We added a 6xHis-tag with a short glycine-serine linker after position Ser161 to create BBa_K3113051, which enables Ni-NTA affinity purification of exosomes for the first time.
Improvement and Protein Engineering
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 with a short glycine-serine linker to the large extracellular loop of the exosomal marker CD63. We based the selection of the incorporation sites on a structural model for CD63 generated with swissmodel.expasy.org and based on the structure of the related tetraspanin CD81. We modelled our selection for the incorporation of the 6xHis-tag on accessibility as well as flexibility and we have taken into account to keep glycosylation sites as well as potential disulfide bonds intact. To ensure minimal interference with the correct folding of CD63, not only a polyhistidine tag was incorporated but also a a short glycine-serine linker. We looked into the structure and found several accessible and flexible sites for the incorporation of the affinity tag. The sites around the amino acids Ser161 and Asn180 seemed suitable. To our knowledge, Ni-NTA affinity chromatography has not been previously 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, which just contains the human CD63 sequence.
Experimental Data
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 gets purified effectively (Figure 1). Thus, purification of our bioengineered exosomes does not happen due to unspecific interaction with the column material but is enabled by the His-tag incorporated in the large 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-tagged CD63 outside exosomes is about 20 times lower than the total His-tagged 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. For more details on checking the quality of vesicles with the HiBiT assay, read more on our measurement page.
Protein Engineering
We tested also another extracellular loop-position of CD63 for the implementation of the polyhistidine-tag. Those engineered CD63 proteins have the polyhistidine-tag at the Asn180 (BBa_K3113072).
Also for this CD63-Asn180-6xHis we were able to show, that the purification is effectively compared to native CD63 (Figure 3).
Testing 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.
