Intein-mediated biotinylation of proteins and its application in a protein microarray

Article abstract of DOI:10.1021/ja0265963

We report here the first example using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. With this novel array, proteins are site-specifically immobilized on the gla

Full text of DOI:10.1021/ja0265963

Published on Web 07/02/2002
Intein-Mediated Biotinylation of Proteins and Its Application in a Protein
Microarray
Marie-Laure Lesaicherre,† Rina Y. P. Lue,‡ Grace Y. J. Chen,†,‡ Qing Zhu,† and Shao Q. Yao*,†,‡
Department of Chemistry and Department of Biological Sciences, National UniVersity of Singapore,
3 Science DriVe 3, Singapore 117543, Republic of Singapore
Received April 19, 2002
DNA microarray is currently the method of choice for high-
throughput analysis of nucleic acids at their transcriptional level.
However, it has been shown that the mRNA expression level in a
cell does not correlate well with the abundance of proteins.1 To
gain more insights into protein functions, a number of technologies2
and binding chemistry3 have been developed to immobilize small
molecules,3a,b peptides,3c,d and proteins3e-g in a microarray for high-
throughput protein studies.4 In most cases, however, protein
immobilization was achieved via their nucleophilic residues,
Figure 1. MBP purification and biotinylation. (a) (1) Protein marker, (2)
uninduced cell extract, (3) induced cell extract, (4) flow-through from
resulting in random orientations of proteins on the glass surface.
Thus far, there has only been one report of site-specific attachment
of proteins on glass slides.5 Approximately 6000 yeast proteins were
expressed as his-tag fusions, spotted onto Ni-NTA functionalized
slides, and >80% were found to retain their full biological activities,
presumably as a result of site-specific immobilization which ensures
most proteins on the slide to be oriented correctly. However, the
binding between Ni-NTA and his-tag proteins is neither very strong
nor very stable and susceptible to interference by many commonly
used chemicals,6 making this immobilization method incompatible
with many protein screening assays. On the contrary, the biotin-
avidin interaction, one of the strongest noncovalent interactions
known,7 is stable toward a variety of harsh conditions8 and has
been widely used in standard biochemical assays for immobilization
purposes. The in vivo and in vitro biotinylations of proteins have
previously been reported,9 but with limited success due to low yields
and the nonspecific nature of the biotinylation reaction.9c In some
cases, biotinylation results in the addition of a long peptide to the
target protein, which may interfere with proper folding of the
protein.9d Furthermore, avidin is known to be toxic to cells, making
the expression of avidin-fused proteins a difficult task.10 Intein-
mediated expression of proteins has mostly been developed for
purification of fusion proteins on chitin columns11 and has also
found wide application in protein engineering where the expressed
protein ligation (EPL) strategy is utilized.11b,c Recently, this strategy
has been extended to modifications of proteins at their C-termini
with a number of chemical tags.12 We report here the first example
of using an intein-mediated expression system to express, purify,
and site-specifically biotinylate proteins, followed by immobilization
onto avidin-functionalized glass slides. Thus, we have created a
novel protein array on which site-specifically oriented proteins are
immobilized with a highly robust and extremely stable linkage
(Scheme 1) and are able to retain their full biological activities.
Three proteins of interest, namely MBP (maltose binding protein),
EGFP (enhanced green fluorescent protein), and GST (glutathione
S-transferase) were chosen as models and expressed in vivo as
column loading, (5) flow-through from column wash, (6) proteins bound
to chitin column before cleavage, (7) flow-through from quick flush of
cleavage agent, (8-9) first two elution fractions after overnight incubation
at 4 °C with 1, (10) remaining proteins bound to chitin column after
cleavage. (b) Western blotting of biotinylated MBP using Streptavidin-HRP
for detection.
Scheme 1. Intein-Mediated Expression, Purification, Biotinylation,
and Immobilization of Proteins
fusion proteins with an intein tag (intein fused to chitin binding
domain) at their C-termini. The proteins were purified and
biotinylated, in a single step, by first loading the crude cell lysate
onto a column packed with chitin beads and then flushing the
column with biotinylated cysteine, 1, to obtain the C-terminally
biotinylated proteins (Scheme 1), which were unambiguously
confirmed by SDS-PAGE and western blotting (Figure 1). On the
basis of SDS-PAGE, the biotinylation reaction took place with 90-
95% efficiency, generating proteins in sufficient purity (>95%)
which were spotted directly, without any further treatment, onto
an avidin-functionalized slide to obtain the corresponding protein
array.
* Address correspondence to this author. E-mail: chmyaosq@nus.edu.sg.
Tel: 65-68741683. Fax: 65-67791691.
A protein array was generated with the biotinylated EGFP, MBP,
and GST and probed with Cy3-anti-EGFP, Cy5-anti-MBP, and
† Department of Chemistry.
‡ Department of Biological Sciences.
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10.1021/ja0265963 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
harsh washing conditions and then detected with FITC-labeled anti-
GST for any loss of GST on the surface. No loss of GST was
observed even after the slide had been treated with (1) 1 M acetic
acid at pH 3.3, (2) 60 °C water, and (3) 4 M GuHCl for a prolonged
time (see Supporting Information), in sharp contrast with that of
his-tag proteins on a Ni-NTA slide.13b
Findings described here present a new strategy for site-specific
protein biotinylation and immobilization on a glass surface,
generating a novel protein array on which proteins are oriented
optimally and able to retain their native activity suitable for
subsequent biological screenings. The advantage of avidin/biotin
linkage over his-tag/Ni-NTA strategies for protein immobilization
is highlighted by its ability to withstand a variety of chemical
conditions, which may make this new protein array compatible with
most biological assays.
Figure 2. Site-specific immobilization of biotinylated, functionally active
proteins onto avidin slides. (a) EGFP, MBP, and GST were individually
detected with Cy3-anti-EGFP (green), Cy5-anti-MBP (red), and FITC-anti-
GST (blue), respectively; (b) specific detection of all three proteins with a
mixture containing all three antibodies; (c) fluorescence from the native
EGFP; (d) specific binding between GST and its Cy3-labeled natural ligand,
glutathione. No binding between glutathione and EGFP/MBP was observed
(data not shown).
FITC-anti-GST, respectively. Three corresponding nonbiotinylated
proteins were also spotted onto the same slide, as controls, and the
array was incubated with either individual antibodies (Figure 2a)
or a mixture of all three antibodies (Figure 2b). Only specific
binding between the biotinylated proteins and their corresponding
antibodies was observed, regardless of the presence of other proteins
(Figure 2a) and antibodies (Figure 2b), indicating the specific
immobilization and versatility of this new protein array. Further-
more, no fluorescence signal was observed with the nonbiotinylated
control proteins (data not shown), confirming the essence of
biotinylation for protein immobilization.
The most critical issue in generating a protein array is to ensure
that proteins maintain their native activity, as it is previously known
that proteins tend to denature on glass surfaces.3e To confirm that
biotinylated proteins immobilized on the avidin slide retain their
proper foldings, the native fluorescence of EGFP on the slide was
monitored (Figure 2c). No loss of fluorescence intensity was
observed after prolonged incubation at 4 °C, suggesting that folding
of the protein was properly maintained on the slide. In a separate
experiment, a slide immobilized with EGFP, MBP, and GST was
incubated with Cy3-labeled glutathione, a known natural ligand of
GST (Figure 2d). The result showed exclusive binding between
GST and glutathione, further indicating full retention of the native
GST activity. Furthermore, all data gathered thus far indicates that
the presence of avidin as a molecular layer between the immobilized
proteins and the glass surface also serves to minimize nonspecific
absorption of proteins.13a Further improvement may be readily made
by using streptavidin as the immobilization agent on the slide in
place of avidin, which is a glycoprotein and known to have higher
nonspecific binding characteristics.14
Thus far, the only reported method for site-specific attachment
of proteins in a microarray has been the immobilization of his-tag
proteins on slides functionalized with Ni-NTA.5 However, the
binding between his-tag proteins and the Ni-NTA complex is not
very strong and is incompatible with many common chemicals such
as DTT, SDS, EDTA, etc. The binding is also depleted outside the
4-10 pH range or when the buffer contains high concentrations of
common salts. On the contrary, the binding between biotin and
avidin is one of the strongest known (dissociation constant ≈ 10-15
M) and is stable under most stringent conditions.7 Avidin is also
extremely stable,8 making it an ideal agent for slide functionaliza-
tion. In addition, the interaction between avidin and biotin is
instantaneous, hence requiring no incubation for protein im-
mobilization. To confirm the benefit of the avidin-biotin linkage,
slides immobilized with GST were first subjected to a number of
Acknowledgment. Funding was provided by the National
University of Singapore (NUS) and the National Science &
Technology Board (NSTB) of Singapore.
Supporting Information Available: Detailed protocols of protein
expression, purification, biotinylation and immobilization, chemical
synthesis of compound 1, and microarray related experiments (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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