Biomedical Engineering Reference
In-Depth Information
26.3.4 Biotin-Avidin Fusion Model
pure due to the fact that the chemical yield of every
reaction that adds an additional amino acid to the peptide
chain is not 100%.
S
alik et al. have shown a method for assessing the potential
for protein delivery without the need to go through all the
steps needed to clone, express, and purify using prokaryotic
expression methods. They have a method that is based on the
use of a biotin-tagged peptide, synthesized through conven-
tional solid-phase peptide synthesis (SPPS), where one of
the amino acids in the peptide sequence is tagged with
biotin, thus creating a model for a peptide protein fusion.
Delivery is limited to just two proteins, avidin and strepta-
vidin, which bind very strongly to biotin (with a dis-
association constant of about 10
14
mol/L). This method
is perhaps most beneficial if one wishes to assess and
compare the ability of different CPPs to deliver tightly
bound (mimicking fused) protein cargo. The technical
details for this strategy can be found in the paper [18].
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26.3.7 Purification Tag
A histidine tag is a hexahistidine (six consecutive histidines)
motif that is usually placed at the C- or N-terminus of a
recombinant protein. It is used for purification of proteins
expressed recombinantly in E. coli. The least cumbersome
way to add a histidine tag is to utilize a vector that already
has the coding sequence right after the start codon or directly
before the stop codon. There are commercially available
vectors from many manufacturers.
The hemagglutinin tag is another purification tag with the
amino acid sequence YPYDVPDYA that works in a similar
fashion as the hexahistidine tag.
Histidine- and hemagglutinin-tags are not the only tags
employed to purify recombinant proteins. An example of
another tag that has had widespread use is the FLAG-tag, it
has recently been employed for CPP-fusion proteins [81].
The FLAG-tag is a polypeptide tag (DYKDDDDK) that can
be used in much the same way as the His6-tag; in addition, it
has the added benefit that there are antibodies available for
the tag, which makes it possible to use FLAG-tagged
proteins in conjunction with antibody-based assays.
26.3.5 Expressed Protein Ligation
Yu et al. have described a method called expressed protein
ligation [79] for preparation of fusion proteins. Fusion pro-
teins expressed in E. coli can be contaminated by endotoxins;
this contamination can induce a multitude of unwanted
physiological responses ranging from fever to death. The
endotoxin problem is worsened by the fact that endotoxins are
negatively charged and the TAT peptide and oligoarginine
peptides are positively charged, thus making the separation of
endotoxins from fusion proteins of TAT and other arginine-
rich peptides potentially difficult. The peptides CG-R12
[NH2-Cys-Gly-(Arg)
12
-amide] and CG-dR12 [NH2-Cys-
Gly-D(Arg)
12
-amide] were synthesized using Fmoc solid-
phase peptide synthesis using a Rink amide resin using the
standard protocol of a Shimadzu PSSM-8 peptide synthesizer.
For the preparation of FITC-GC-R12 and FITC-R12, a
g
-aminoburyl moiety was employed as a connecting linker
between N-terminal FITC and the arginine peptides. Avector
with eGFP, a mini-intein, and chitin binding domain (CBD)
was prepared using a commercial system (IMPACT, New
England Biolabs, Ipswich, MA). The intein-CBD domain
allows for purification using a chitin bead column followed
by on-column cleavage to generate a thioester of eGFP. This
product can then be covalently linked with the cysteine in the
CG-R12 peptide. The method has the additional benefit over
His6-tagged fusion proteins that the purification tag is
removed before the chemical ligation.
26.3.8 Affinity Chromatography
The use of affinity for purification of proteins has been a
method used since at least the end of the 1960s [82]. Here a
protein's affinity for a substrate or an inhibitor is utilized.
The substrate is attached to a cross-linked porous polymer
contained in a column. The proteins that do not interact with
the material in the column will pass through immediately,
whereas the proteins that have affinity for the substrate that
is bound in the column will be retarded. Elution is achieved
by changing the pH or ion strength or by adding a competi-
tive inhibitor in solution.
The use of histidine-tagged proteins expressed recombi-
nantly in E.coli simplifies the process of finding a suitable
substrate for the protein that is to be purified. The histidine
tag has a high affinity for metal ions and can be purified
using metal-chelate affinity chromatography [83]. The pro-
tein of interest binds to the column via interactions with the
metal ions and can then be eluted using a solution of EDTA,
ammonium chloride, imidazole, histidine, glycine, or high
levels of competing metal ions, the most common being
imidazole. Affinity chromatography using histidine-tagged
proteins usually yields relatively pure products. Commercial
kits for purification of histidine-tagged proteins are available
from a number of different manufacturers. Affinity purifi-
cation using a histidine tag in combination with a hemag-
glutinin tag, which utilizes
26.3.6 Purification
Recombinantly expressed proteins, fusion proteins, and
synthetically produced peptides need to be purified.
Recombinant proteins exist only in a mixture together
with the endogenous proteins of the host organism. Peptides
that are synthesized manually or using a synthesizer are not
a
similar
affinity-based
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