Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids

Article abstract of DOI:10.1021/ja061131o

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif wit

Full text of DOI:10.1021/ja061131o

Published on Web 06/30/2006
Regio- and Chemoselective Covalent Immobilization of Proteins through
Unnatural Amino Acids
Ce´cile Gauchet, Guillermo R. Labadie,† and C. Dale Poulter*
Department of Chemistry, UniVersity of Utah, 315 South 1400 East, Room 2020, Salt Lake City, Utah 84112
Received February 26, 2006; E-mail: poulter@chemistry.utah.edu
Scheme 1. Modification of Proteins with CaaX Recognition Motifs
Protein “chips” are useful for studying protein-ligand and
protein-protein interactions,1 including the detection of antibody-
antigen interactions,2 and permit high-throughput screening of
limited quantities of analytes in a microarray format.3 Devices with
covalently attached proteins1,4 are more robust than their non-
covalent counterparts.5 In addition, substantial enhancements in
sensitivity are seen when proteins are attached in a uniform
manner.4-6 Typically covalent immobilization is accomplished by
reactions that rely on nucleophilic moieties found in the side chains
of naturally occurring amino acids.1,7 We now report a general
approach for the regio- and chemoselective covalent immobilization
of soluble proteins on glass surfaces through an unnatural amino
acid created by post-translationally modifying a cysteine residue
with functional groups suitable for “click chemistry”8 or a
Staudinger ligation.9
Scheme 2. Preparation of Glass Slides for Protein Immobilization
by “Click Chemistry”
Protein farnesyltransferase (PFTase) catalyzes the alkylation of
the sulfhydryl moiety in the cysteine located in C-terminal CaaX
motifs, where X ) A, S, M, or Q, by farnesyl diphosphate (1) (see
Scheme 1).10 The reaction is general for any soluble protein bearing
a CaaX motif. We synthesized farnesyl analogues 2 and 3, both of
which are excellent alternate substrates for yeast PFTase with
catalytic efficiencies (1.6 and 0.58 µM-1 s-1) similar to that of 1
(0.76 µM-1 s-1).11 As model proteins for immobilization, we
engineered C-terminal CVIA motifs into green fluorescent protein
(GFP) and glutathione S-transferase (GST). Incubation of the
proteins with azide analogue 2 or alkyne analogue 3 gave GFP-
N3, GFP-C2, GST-N3, or GST-C2, respectively.
Azide-derivatized glass slides suitable for immobilization of the
complementary alkyne-derivatized proteins by “click chemistry”
were prepared as outlined in Scheme 2. The surface of amino-
activated glass slides was prepared in three steps.12 The amine
groups were first converted to the corresponding O-succinimidyl
carbamates, and PEG-containing amine linkers were then attached
through urea linkages, using a 5:1 molar ratio of 4 to 5.13 The slides
were then treated with ethanolamine to deactivate residual carbamate
groups. Phosphine-derivatized glass slides for Staudinger ligations
were prepared according to the procedure described by Raines and
co-workers.14
addition of Block It, followed by incubation for 5 h. The wells
were then washed five times with phosphate-buffered saline
containing 0.1% Tween 20 (PBST). For slides developed with
fluorescent antibodies to GFP and GST, a solution containing 4
µL of the appropriate antibody (4 µg/mL) was added to each well,
and the slide was incubated at 4 °C for 16 h. Fluorescence intensities
were measured by phosphorimaging.
The results of an immobilization experiment are summarized in
Figure 1. The concentrations of GFP-C2 and GST-C2 varied from
1 to 20 µM. No fluorescent signal was seen when the antibodies
were added to wells that did not contain GFP or GST proteins (wells
2c and 3c). To distinguish between specific and nonspecific binding,
GFP-F (wells 2a, 2b, 2d, 2e) and GST-F (wells 3a, 3b, 3d, 3e)
bearing a farnesyl group instead of the reactive alkyne moiety were
used as controls. Background fluorescence was seen for both of
the farnesylated proteins. The fluorescence signals for wells
containing proteins with covalently attached alkyne groups were
easily detected and significantly above background. Over a range
of 1-10 µM, the intensity of the signal increased by ∼50% for
each 2-fold increase in the concentration of the derivatized protein.
A maximum intensity was achieved at ∼20 µM protein. The
intensity of the signals did not increase when the time of the
immobilization reaction was extended from 2 to 7 h.
One microliter samples of solutions of GFP-C2 or GST-C2 in
3:1 (v/v) water:glycerol, containing 1.7 mM Cu+, were spotted into
wells on a silicone-masked azido-derivatized glass slide. The slides
were maintained at 4 °C in a humidified chamber for 2 h before
The antibodies could be removed by treatment with an acidic
saline solution for 2 h at 80 °C. Incubation of the stripped slides
with anti-GFP for 16 h at 4 °C only gave signals corresponding to
locations of immobilized GFP. A second cycle of stripping,
followed by incubation with anti-GST only gave signals at the
locations of immobilized GST. Thus, GFP and GST remain attached
† Present address: IQUIOS-CONICET, Universidad Nacional de Rosario,
Suipacha 531, 2000, Rosario, Santa Fe, Argentina.
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J. AM. CHEM. SOC. 2006, 128, 9274-9275
10.1021/ja061131o CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Fluorescence intensities of GFP-N3 (green) immobilized by
Staudinger ligation and the GFP-F (yellow) control after storage in buffer.
Figure 1. Plate: Fluorescence intensities of immobilized proteins and
controls. GFP-C2: 1 µM (2f), 5 µM (1e, 1f), 10 µM (1c, 1d), 20 µM (1a,
1b). GFP-F: 15 µM (2a, 2b, 2d, 2e). GST-C2: 1 µM (3f), 5 µM (4e, 4f),
10 µM (4c, 4d), 20 µM (4a, 4b). GST-F: 15 µM (3a, 3b, 3d, 3e); GFP
antibody (2c); GST antibody (3c). Plot: Average values of relative
fluorescence intensity versus protein concentration. GFP proteins (red); GST
proteins (blue); controls (green).
protein to a surface by “click chemistry” or an ω-azide for
Staudinger ligations. The triazole and amide linkages produced by
the respective reactions are chemically robust.
Acknowledgment. The authors are grateful to Prof. R. T. Raines
for a sample of protected phosphinothiol and helpful discussions.
This project was supported by NIH Grant GM 21328.
to the slides under conditions sufficiently stringent to disrupt
interactions between the immobilized proteins and their respective
antibodies.
In another experiment, GFP-C2 (25-100 µM) was immobilized
by the “click” procedure, and the slide was analyzed directly by
phosphorimaging without conjugation with fluorescent anti-GFP.
A fluorescent signal was detected after the slide had been thoroughly
washed with PBST. The slide was then allowed to stand in
phosphate buffer (pH 7.0) at 4 °C for 2 days. After 2 days, the
signal had only diminished by 22%. Thus, GFP retained its native
fold during the immobilization and subsequent storage in buffer.
Staudinger ligations were performed with GFP-N3 and GST-
N3 using slides derivatized with diphenylphosphine groups.14
Preliminary experiments using an aqueous buffer for the im-
mobilization reactions gave slides with high backgrounds for
GFP-F and GST-F visualized by fluorescent antibodies. Lower
backgrounds were achieved when the ligation was carried out in
50:1 DMF/water. We were concerned that these conditions were
not compatible with preserving the native fold of proteins. GFP-
N3 was immobilized by the Staudinger ligation. The slides were
stored in buffer and visualized by direct imaging of the GFP
fluorophore over a period of 6 days. The fluorescence intensity of
spots corresponding to immobilized GFP-N3 and the GFP-F
control decreased during the first 4 days; however, the difference
between the intensities for immobilized GFP and the control
remained constant (see Figure 2). After 4 days, signals for the
control samples were reduced to background levels, whereas the
signal for covalently bound GFP remained constant.
Supporting Information Available: Procedures for synthesis of
linkers, preparation of slides, cloning, protein expression and purifica-
tion, derivatization and microarray-related experiments. This material
is available free of charge via the Internet at http://pubs.acs.org.
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In summary, we have developed a new strategy of the regio-
and chemoselective immobilization of proteins on surfaces through
an unnatural amino acid created post-translationally at the cysteine
residue in a C-terminal CaaX motif. The CaaX recognition motif
can be easily appended to the C-terminus of a cloned protein by a
simple modification of the corresponding gene. Virtually any soluble
protein or peptide bearing the CaaX motif is a substrate for PFTase.
The cysteine residue can then be modified with farnesyl analogues
bearing either an ω-alkyne moiety useful for immobilization of the
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