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Cloning strategies

IdeS·PtA-Z fusion protein

Information here.

CPP fusions

As described in our project introduction, one of our aims was to target proteins to keratinocytes and melanocytes in human skin using cell-penetrating peptides. As we didn't know which of (or if) our CPPs would be most efficient in protein delivery, we set out to test all three in parallel.

At least two of our CPPs (LMWP and Tp10) have not previously been tested using recombinant technique, i.e. in protein fusions. Although these have been shown to successfully deliver protein cargo into cells and/or skin, these proteins have been chemically conjugated to the CPPs,
Cloning strategy for proteins fused to CPPs
Cloning strategy for proteins fused to N-part CPPs
usually to amino acid side chains. We were therefore unsure as to whether membrane and skin penetration would be most effective when fusing the CPPs to our proteins' N- or C-termini; we also discussed that this may possibly vary for different proteins, as different cargo properties may interact and possibly disturb our CPPs. In relation to this, we also discussed the probability of the CPPs disturbing the enzymatic activity of our fused cargo proteins, and whether this risk would differ depending on to which side of the protein (N- or C-terminus) the CPP is fused.

As time is very limited in the iGEM competition, and as we would not be able to test fusion to one side at the time, we decided to fuse the CPPs to both sides of our proteins, and draw our own conclusions by investigating both CPP and protein activity.

Another factor we took into consideration was the additional amino acids added to parts adapted to the [Freiburg assembly standard]. Due to the small size of our CPPs (11-21 a.a.) we thought that even a very small variation to the amino acid sequence could affect their function. For our N-terminal protein fusions we therefore designed N-part versions of our CPPs, which prevents the addition of the two extra amino acids in the CPP N-terminal (Ala & Gly) by removing the standard-specific NgoMIV restriction site.

Finally, we also attached a 6x His tag to each one of our protein fusions to enable antibody detection and protein purification.

The figures to the right illustrate the cloning strategy for our CPP·protein fusions, and insertion into the IPTG-inducible protein expression pEX (BBa_K243033). Tables below show an overview of our many CPP·protein fusion combinations.

C-terminal CPP fusions
Purification tag Protein CPP

His-tag BioBrick.png
BFGF BioBrick.png

SOD1 BioBrick.png
IdeS BioBrick.png
ProteinA BioBrick.png

TAT BioBrick.png

LMWP BioBrick.png

Tp10 BioBrick.png

N-terminal CPP fusions
N-part CPP Protein Purification tag
NTAT BioBrick.png

NLMWP BioBrick.png

NTp10 BioBrick.png

BFGF BioBrick.png

SOD1 BioBrick.png
IdeS BioBrick.png
ProteinA BioBrick.png

His-tag BioBrick.png

The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/