Covalent and selective immobilization of fusion proteins

Article abstract of DOI:10.1021/ja034145s

A general method for the covalent immobilization of fusion proteins is presented. The approach is based on the unusual mechanism of the human O₆-alkylguanine-DNA alkyltransferase, which irreversibly transfers the alkyl group from its substrate,

Full text of DOI:10.1021/ja034145s

Published on Web 06/10/2003
Covalent and Selective Immobilization of Fusion Proteins
Maik Kindermann,†,‡ Nathalie George,†,‡ Nils Johnsson,§ and Kai Johnsson*,†,‡
Institute of Molecular and Biological Chemistry and Institute of Biomolecular Sciences, Swiss Federal Institute of
Technology, CH-1015 Lausanne, Switzerland, and Institute of Toxicology and Genetics, Forschungszentrum
Karlsruhe, D-76021 Karlsruhe, Germany
Received January 13, 2003; E-mail: kai.johnsson@epfl.ch
Protein microarrays, microbeads, and protein sensor chips play
an increasingly important role for characterizing the functions and
interactions of proteins.1 These solid-phase-based assays require
the immobilization of proteins in their native state. Current
immobilization techniques rely either on adsorption, on direct
covalent immobilization of purified proteins to chemically activated
surfaces, or on their expression as a fusion to a polypeptide that
mediates the immobilization on an appropriately derivatized
surface.2 Here, we report on a general method for immobilization
of proteins that are genetically linked to a mutant of the human
DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT).
The high specificity and covalent nature of the approach make it
an attractive alternative to all currently used protocols for the
immobilization of fusion proteins.
The DNA repair protein hAGT (207 residues) transfers the alkyl
group from its substrate, O6-alkylguanine-DNA, to one of its
Figure 1. Immobilization of hAGT fusion proteins. (A) hAGT-based DNA
repair; (B) structure of 1; (C) covalent immobilization of hAGT fusion
proteins on surfaces displaying 1.
cysteine residues (Figure 1A).3 We have recently shown that O6-
benzylguanine (BG) substituted at the 4-position of the benzyl ring
can be used to specifically label N- or C-terminal hAGT fusion
proteins with different labels in vivo and in vitro.4 On the basis of
these observations, we reasoned that surfaces displaying BG
derivatives of the type 1 should result in the selective immobilization
of hAGT fusion proteins (Figure 1B, C).
Compound 1 was synthesized from readily available materials
(Supporting Information). The primary amino group of 1 should
allow its immobilization on a wide variety of different surfaces
displaying activated carboxyl groups. A flexible tetraethylene glycol
was chosen as a linker between the O6-benzylguanine and the
terminal amino group to maximize the accessibility of immobilized
Figure 2. SPR data for the immobilization of GST-GEhAGT on BG-covered
1 toward hAGT fusion proteins. In all of the following experiments,
sensor chips. The flow rate in all experiments is 1 µL/min. HBS: 10 mM
a recently generated hAGT mutant, GEhAGT, with nearly 20-fold
increased activity against BG was used.5 The second-order rate
constant for the reaction of GEhAGT with BG derivatives of the
type 1 in solution was determined to be 5000 s-1 M-1 (Supporting
Information).
HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween 20. (1) GST-GEhAGT (0.1
mg/mL); (2) GST-GEhAGT (0.1 mg/mL), preincubated with BG (40 µM);
(3) anti-GST antibody (0.1 mg/mL) after incubation of a chip with GST-
GEhAGT as in lane 1; (4) anti-GST antibody (0.1 mg/mL) directly on BG-
covered sensor chip.
To demonstrate a selective immobilization of hAGT-X where
X can be any protein, 1 was coupled to commercially available
carboxymethylated dextran chips suitable for surface plasmon
resonance (SPR) experiments.6 Using SPR, both the immobilization
of a protein onto the sensor chip as well as its subsequent
interactions with other proteins or compounds can be monitored
directly. The carboxyl groups of the carboxymethylated dextran
were transiently transformed into N-hydroxysuccinimide (NHS)
esters and subsequently reacted with 1. On average, immobilization
of 1 led to a SPR signal of 2000 resonance units. Next, the
immobilization of a fusion between glutathione S-transferase and
GEhAGT (GST-GEhAGT) was investigated. GST-GEhAGT was flown
over a BG-covered sensor chip, and binding was followed by SPR.
Binding of GST-GEhAGT led to an increasing SPR-signal over time,
and bound protein could not be removed by washing with buffer
(Figure 2). Prior incubation of GST-GEhAGT with BG completely
prevented its binding to the surface of the sensor (Figure 2). The
immobilization of GEhAGT on chips displaying BG followed pseudo
first-order kinetics, and the rate constant for immobilization was
measured to be 225 s-1 M-1 (Supporting Information).
To demonstrate that immobilized GEhAGT fusion proteins can
be used for the analysis of protein-protein interactions, anti-GST
antibody was flown over GST-GEhAGT derivatized sensor chips.
A strong signal from bound antibody was detected (Figure 2),
whereas no binding of the antibody was observed on BG-covered
sensor chips lacking the attached GST-GEhAGT (Figure 2). As
expected, the ratio of bound antibody to immobilized GST-GEhAGT,
† Institute of Molecular and Biological Chemistry, Swiss Federal Institute of
Technology.
‡ Institute of Biomolecular Sciences, Swiss Federal Institute of Technology.
§ Institute of Toxicology and Genetics.
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C O M M U N I C A T I O N S
steadily increased over a time period of 35 min (Figure 3A). The
immobilized species was identified as GST-GEhAGT as it was
recognized by anti-GST antibody on the chip (Figure 3A). Im-
mobilization was specific because preincubation of the E. coli
extract with BG (100 µM) decreased the SPR signal by a factor of
50 (Figure 3A). Furthermore, no signal was observed from extracts
of E. coli expressing only GST without the attached hATG
(Supporting Information).
The results demonstrate a number of major advantages of the
hAGT-mediated immobilization procedure. (1) The attachment to
the surface is very gentle, and the fusion protein is exclusively
coupled via the hAGT moiety, leaving the protein of interest
accessible for interactions with other molecules. (2) The attachment
to the surface is covalent. This ensures that the protein remains
linked to the solid phase under a variety of different conditions.
(3) The immobilization on BG-covered surfaces is specific, enabling
the immobilization of hAGT fusion proteins without time-consum-
ing purification steps. BG shows no significant reactivity against
proteins other than hAGT. This makes it superior to the suicide
inhibitor 4-nitrophenyl phosphonate as an immobilization device,
because 4-nitrophenyl phosphonates are known to react with a
variety of proteases, lipases, and esterases.2d,8 (4) hAGT as a fusion
tag can be used both for the covalent immobilization and the
attachment of a variety of chemical or spectroscopic probes.4 This
versatility makes possible the use of once constructed hAGT fusion
proteins for very different investigations. All features combined
should establish hAGT fusions as unique tools in functional
proteomics.
Figure 3. Immobilization of hAGT fusion proteins out of cell extracts.
(A) SPR data for the immobilization of GST-GEhAGT on BG-covered sensor
chips out of cell extracts. HBS: 10 mM HEPES, pH 7.4, 150 mM NaCl,
1% Tween 20. (1) Lysate (10 mg/mL total protein); (2) lysate (10 mg/mL
total protein) preincubated with BG (100 µM); (3) anti-GST antibody (0.1
mg/mL) after incubation of a chip with cell extract as in lane 1. (B) Lane
1: SDS-PAGE of cell extract of Xl1-Blue expressing GST-GEhAGT used
in (A) and subsequent staining with Coomassie Blue.
as calculated from the measured resonance units, decreased with
increasing surface density of immobilized GST-GEhAGT. GST-
GEhAGT densities as low as 0.7 ng/mm2 gave a 1:1 ratio of antibody
bound per GST-GEhAGT, whereas the ratio decreased to 1:6 at
densities of 17 ng/mm2. The density of the immobilized hAGT
fusion protein can be readily adjusted by controlling the reaction
time on the chip. Furthermore, chips covered with GST-GEhAGT
and anti-GST antibody were flushed with 10 mM HCl to remove
any noncovalently bound protein. Subsequent application of new
anti-GST antibody to this chip resulted again in a specific binding
signal, indicating that most of the immobilized GST-GEhAGT is
indeed covalently bound (Supporting Information). The intensity
of this signal was decreased by 30% as compared to the first
incubation with antibody. The observed decrease in the binding of
antibody after the HCl washing can be explained in two ways: (i)
GST forms a dimer, and not necessarily both halves of a GST-
GEhAGT dimer are covalently bound to the sensor chip; (ii) the
treatment with 10 mM HCl might denature a fraction of the GST,
thereby reducing the subsequent binding of the antibody.
To demonstrate that the hAGT-based immobilization does not
interfere with the biological activity of the protein of interest, we
measured the GST activity of GST-GEhAGT after its immobilization.
GST-GEhAGT was immobilized on agarose beads displaying 1, and
GST activity was measured by incubating the beads with glutathione
and 1-chloro-2,4-dinitrobenzene (CDNB). GST catalyzes the ad-
dition of glutathione to CDNB, a reaction that can be followed at
345 nm.7 Incubating agarose beads displaying immobilized GST-
GEhAGT with a solution of glutathione and CDNB led to rapid
product formation, while agarose beads displaying no GST-GEhAGT
showed no such activity, demonstrating that GST-GEhAGT at least
partially retains its activity after immobilization (Supporting
Information).
Acknowledgment. This work was financed by the Swiss
National Science Foundation, the Bundesamt fuer Berufsbildung
und Technologie (CTI project 6255.1), and the Bundesamt fuer
Bildung und Wissenschaft (project X-TB). We thank Christoph
Bieri, Kirstin Leufgen, and Horst Vogel for advice and support.
Supporting Information Available: Experimental details (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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