BTN a 000114004 o 190685a

Features

BioSpotlight
Playing tag with proteins
Non-chromatographic methods for protein purification are
attractive in light of the expense and time required for chromatography. Perhaps the most promising of these approaches has
been the use of protein or peptide stimulus-responsive tags that
allow the rapid and reversible precipitation of target proteins
from complex solutions. After isolation of the fusion protein,
the tag can be removed by inducing a coupled self-cleaving intein
domain or through treatment with a site-specific protease.
A number of these tags, however, require heat or high salt
treatment to induce their precipitation, which can be potentially deleterious for some target proteins. In this month’s issue of
BioTechniques, S. Banta and colleagues at Columbia University
(New York, NY) describe a gentler approach using a synthetic
peptide tag they developed that can be reversibly precipitated
in response to calcium. The new tag came about as a serendipitous discovery during the authors’ research into repeat scaffolds
for stimulus-responsive protein engineering applications. The
calcium-responsive repeat-in-toxin (RTX) domain is found in
various bacterial proteins secreted through the type I system
and consists of repeats of a nine-amino-acid sequence. After

Vol. 54 | No. 4 | 2013

achieve increased solubility, so LaCava
Cto
and his team tested alternative substitu-

NH2

Competitive Displacement of SpA

OH

NH
O

NH

N
O

H
N

O

H
N

HN

H
N

HN
O

O

HN

NH2
OH

O

O
S

O

O

B
Elution Reagent Solubility
3
2.73
2.36
2

2.55

2.22

1.18

1

RT
0.86

4 ºC

Reagent

PEGylOx structure and effectiveness as an elution reagent

180

PEGylOx

25

See20 “Improved native isolation of
endogenous
protein-A tagged protein
15
complexes”
on
page 213.
10
5

Written by Patrick Lo, Ph.D., and Nathan
0
Blow,
Ph.D.10
20
30
40

50

x
lO
Gy

Gy

lO

x
RPE

PE

oO

x

0

% SpA Retained

50

0

NH

O

NH

NH

N
H
N

N
H

O

HN

O

O

Buffer
PEGylOx
BioOx

25
00

S

00

O

20

O

NH

HO

00

O

PEGylOx

15

O
O

100 at that same N-terminal portion
tions
90 peptide, identifying an additional
of the
80
polyethylene
glycol (PEG) moiety of
four70 unit lengths that was most effective

60
in increasing
solubility. Comparisons
PEGylOx IC 50 166 µM
50
between
Bio-Ox
the newly generated
BioOx ICand
50 159 µM
40
peptide
PEGylOx showed that PEGylOx
30 effectively release 60%-85% of two
could
test20protein complexes within 15 minutes
10
while
Bio-Ox was unable to release either
0
of these
complexes within that time
period. The authors also demonstrated
Concentration of Reagent (μM)
that, similar
to the original Bio-Ox,
PEGylOx could be removed from a
sample using a simple spin column with
D
a 40kDa molecular weight cutoff for

downstream
assays. This
newly improved
Competitive
Displacement
of SpA
40
peptide
should prove highly advantageous
to the
35 large number of researchers taking
advantage
of SpA-tags in their affinity
30
isolation workflows.

10
00

O

% SpA Retained

O

Bi

Affinity isolation of protein complexes can
be greatly enhanced by speed–the faster the
time between isolation and a downstream

assay, the better the chance everything is
completely recovered in a native state.
One widely used affinity isolation system
is based on the interaction between S.
aureus Protein-A (SpA) and immunoglobulin G (IgG). Here, a bait protein is
tagged with SpA and interacting partners
are isolated using either competitive elution
or cleavage. When it comes to cleavage, the
reaction may not be uniform and also tends
to be rather slow. On the other hand, while
competitive elution is more uniform, it also
requires several hours of incubation. In this
issue of BioTechniques, John LaCava and
his colleagues from Rockefeller University
(New York, NY) describe the design of
an improved reagent for the competitive
elution of SpA tagged native complexes
that works within 15 minutes under very
mild conditions. Previously, the authors
described a modified peptide generated
from the Fc fragment of IgG called Bio-Ox
that requires a 2-hour or longer incubation
for competitive elution. They reasoned
that by increasing the solubility of this
peptide, it might be possible to reduce
the elution time. The original Bio-Ox
peptide was modified at the N terminus

See “A designed, phase-changing RTX-based peptide for
efficient bioseparations” on page 197.

A

Concentration (mM)

Biochemistry’s 15-minute
workout

designing a consensus RTX repeat sequence, the authors found
that constructs containing multiple repeats of the consensus
unit fused to the C-terminus of maltose-binding protein (MBP)
caused its precipitation in the presence of calcium. Precipitation
was most efficient for constructs with 13 or 17 consensus repeats
and was easily reversed upon the addition of EGTA. Of several
cations tested, only calcium could induce precipitation when the
17-repeat tag was fused to various target proteins. High yields of
all the fusion proteins were obtained, and the functionality of
the fused target proteins was also retained. To increase the utility
of this tag, an enterokinase cleavage site was engineered between
the tag and the target protein. Cleavage of the isolated fusion
protein with enterokinase, followed by calcium-induced precipitation of the tag, left the purified target protein in solution. This
new calcium-precipitable tag should prove to be a welcome and
valuable new method for the rapid and selective purification of
recombinant proteins.

Time of Elution (min)

BioTechniques 54:180 (April 2013)
doi10.2144/000114004

www.BioTechniques.com

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