U.S. patent application number 15/396970 was filed with the patent office on 2017-04-27 for high throughput detection of fusion proteins.
The applicant listed for this patent is ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE STATE OF AZ, ACTING FOR & ON BEHALF OF AZ STATE UN. Invention is credited to John Chaput, Joshua LaBaer, Mitch Magee, Ji Qiu, Sujay Sau, Justin Saul.
Application Number | 20170115299 15/396970 |
Document ID | / |
Family ID | 50728481 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170115299 |
Kind Code |
A1 |
Saul; Justin ; et
al. |
April 27, 2017 |
HIGH THROUGHPUT DETECTION OF FUSION PROTEINS
Abstract
A method and related microfluidic chip and kit for high
throughput detection of proteins of interest contained in a sample
is disclosed. The method comprises of specifically labeling fusion
proteins in a complex sample with fusion tag specific fluorophores
that specifically bind the fusion tags coupled to the proteins of
interest, and subjecting the sample to automated capillary
electrophoresis, wherein the presence of the proteins of interest
in the sample is detected by fluorescence signals associated with
the fusion tag specific fluorophores.
Inventors: |
Saul; Justin; (Mesa, AZ)
; Qiu; Ji; (Chandler, AZ) ; LaBaer; Joshua;
(Chandler, AZ) ; Magee; Mitch; (Chandler, AZ)
; Chaput; John; (Phoenix, AZ) ; Sau; Sujay;
(Mesa, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE STATE OF AZ,
ACTING FOR & ON BEHALF OF AZ STATE UN |
Scottsdale |
AZ |
US |
|
|
Family ID: |
50728481 |
Appl. No.: |
15/396970 |
Filed: |
January 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14073332 |
Nov 6, 2013 |
9535070 |
|
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15396970 |
|
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61724608 |
Nov 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/44791 20130101;
G01N 27/44726 20130101; B01L 3/5027 20130101; G01N 33/582
20130101 |
International
Class: |
G01N 33/58 20060101
G01N033/58; G01N 27/447 20060101 G01N027/447 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
DK093449 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A kit comprising: (a) a microfluidic chip adapted for use in a
method of high throughput detection of proteins of interest
contained in a sample, wherein the method comprises: (i) contacting
a sample not subjected to prior purification comprising
contaminating proteins without fusion tags and affinity tagged
fusion proteins of interest to a plurality of fluorescent labels
having specificity for the affinity tags, wherein, upon contacting,
fluorescent labels covalently bind the affinity tags to form
detectable protein complexes; (ii) subjecting the contacted sample
to microfluidic electrophoresis, wherein the presence of the
proteins of interest but not contaminating proteins in the
contacted sample is detected by fluorescence signals associated
with the affinity tags; (b) a fusion tag specific fluorophore; and
(c) a means for detection, wherein the presence of the proteins of
interest in the sample is detected by fluorescence signals
associated with the fusion tag specific fluorophores.
2. The kit of claim 1, wherein the fusion tag specific fluorophore
has an excitation and emission maxima near about 635 nm and about
700 nm, respectively.
3. The kit of claim 1, wherein the sample not subjected to prior
purification is a cell lysate.
4. The kit of claim 1, wherein the sample not subjected to prior
purification is a bacterial cell lysate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/073,332, filed on Nov. 6, 2013, now issued as U.S. Pat. No.
9,535,070, which claims the benefit of U.S. Provisional Application
61/724,608, filed Nov. 9, 2012, each of which is incorporated
herein by reference in their entireties for all purposes.
FIELD OF THE INVENTION
[0003] The present invention generally relates to protein
detection. In particular, the invention is related to methods and
devices for high throughput detection of fusion proteins with
fusion tag-specific fluorescent labels.
BACKGROUND
[0004] It is desired to have methods and devices of simultaneous
detection and purification of large numbers of proteins of interest
(POI). While methods currently exist to specifically detect
expression of fusion proteins, they are either not suitable for a
high throughput process, i.e. western blots, or are
non-qualitative, i.e., ELISAs.
[0005] At present, capillary electrophoresis instruments have been
adopted to assess protein purity and quantifying yield of hundreds
of samples within a few hours. Most of these methods lack the
capacity to detect specific POI, making them poorly suited for
screening for protein expression within complex samples, such as
cell lysates. Consequently, there are limited means to detect
fusion protein expression in a high throughput setting, despite the
increasing need. It is desirable to have the ability to detect
specific expression within lysates which will allow rapid
characterization of starting materials as part of a high throughput
protein production pipeline.
[0006] Needed in the art are methods and devices for high
throughput detection and purification of large numbers of proteins
of interest by using fusion tag-specific fluorescent labels and
electrophoresis techniques.
SUMMARY OF THE INVENTION
[0007] In one embodiment, the present invention discloses a method
for high throughput detection of proteins of interest with fusion
tag specific fluorophores that specifically bind the fusion tags
coupled to the proteins of interest, and subjecting the sample to
automated capillary electrophoresis, wherein the presence of the
proteins of interest in the sample is detected by fluorescence
signals associated with the fusion tag specific fluorophores.
[0008] In another embodiment, the present invention discloses a
method for high throughput detection, or assay of a protein, which
applies to the complex protein samples of a cell lysate.
[0009] In another embodiment, the present invention discloses a
method for high throughput detection, or assay of a protein, which
comprises a high throughput technique of automated capillary
electrophoresis technique, such as a microfluidic chip.
DESCRIPTION OF DRAWINGS
[0010] FIGS. 1A-1B are a schematic diagram depicting a general
structure of protein of interest (POI) expressed as fusion proteins
with an affinity tag (FIG. 1A) and the reaction of the affinity tag
binding to a fluorescent label (fluorophore) when folded correctly
(FIG. 1B).
[0011] FIGS. 2A-2B are a schematic diagram showing an example of
protein of interest (POI) expressed as HaloTag fusion proteins
including an affinity tag (FIG. 2A) and the reaction of HaloTag
covalently binding to a chloroalkane ligand when folded correctly
(FIG. 2B). The chloroalkane ligand can be functionalized with a
fluorescent label (fluorophore) for detection (FIG. 2B).
[0012] FIG. 3 is a schematic diagram showing that the fluorescent
label will remain bound to the HaloTag fusion protein after
denaturation and electrophoresis.
[0013] FIGS. 4A-4C depict sodium dodecyl sulfate polyacryamide gel
electrophoresis (SDS-PAGE) analysis visualized through the
fluorescent label (fluorophore) of Alexa660 (emission at 670 nm)
following 633 nm light excitation showing gel-based detection of
two HaloTag fusion protein examples of Halo-MAPK (FIG. 4A) and
HaIoTEV (FIG. 4B) in the presence of different concentrations of
Alexa660-Halo-ligand (for each category of the protein, the
concentrations of Alexa660-Halo-ligand increases from left to
right) in lysate.
[0014] FIGS. 5A-5B depict sodium dodecyl sulfate polyacryamide gel
electrophoresis (SDS-PAGE) analysis showing gel-based detection of
various fusion protein examples including HaloTag and the
fluorescent label (fluorophore) of Alexa660 (FIG. 5A) or Coomassie
(FIG. 5B). While that using Alexa660 showed distinctive detections
of various fusion proteins, the method using Coomassie showed less
distinctive detections of the targeting fusion proteins.
[0015] FIG. 6 is a systematic diagram showing a typical LabChip
setup from Caliper/PerkinElmer along with the standard experimental
protocol of the LabChip setup (adapted from PerkinElmer's website).
Automated capillary electrophoresis is the high throughput
alternative to traditional PAGE analysis. These instruments
commonly utilize a fluorescent protein stain that detects all
proteins non-specifically.
[0016] FIG. 7 is a schematic diagram showing the standard protein
detection protocol of LabChip setups where these setups commonly
utilize a fluorescent protein stain that detects all proteins
non-specifically.
[0017] FIG. 8 is a schematic diagram showing the protocol of the
present invention with LabChip applied to fusion tag detection
where specifically labeled fusion proteins lead to a specific
detection only on the labeled samples on LabChip.
[0018] FIG. 9 depicts LC90 fluorescent HaloTag ligand detection in
the presence of Bovine serum albumin (BSA; .about.65 kDa) using the
LabChip setup. Since BSA does not react with the HaloTag ligand,
the observed BSA signal is constant with varying
Alexa660-Halo-Ligand concentrations.
[0019] FIG. 10 depicts LC90 fluorescent HaloTag ligand detection on
the fusion protein of HaIoTEV using the LabChip setup. Due to the
formation of fusion protein of HaIoTEV, the observed signal of
HaIoTEV increases with the increasing concentrations of Halo
ligand.
[0020] FIGS. 11A-11C depict 32 proteins expressed as N-terminal
HaloTag fusion proteins in HeLa cell lysate and incubated with
HaloTag-Alexa660 ligand. Expression reactions were denatured and
separated by PAGE. (A) Coomassie stain of 1 uL expression
reactions; (B) In-gel fluorescence of the same gels prior to
Coomassie staining; (C) LabChip virtual gel of collection.
DESCRIPTION OF THE INVENTION
[0021] Before the present materials and methods are described, it
is understood that this invention is not limited to the particular
methodology, protocols, materials, and reagents described, as these
may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by any later-filed
nonprovisional applications.
[0022] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. The terms "comprising" and variations
thereof do not have a limiting meaning where these terms appear in
the description and claims. Accordingly, the terms "comprising",
"including", and "having" can be used interchangeably.
[0023] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0024] As used herein, the term "fusion protein" refers to proteins
created through the joining of two or more genes which originally
coded for separate proteins. Translation of this fusion gene
results in a single polypeptide with functional properties derived
from each of the original proteins. Recombinant fusion proteins may
be created artificially by recombinant DNA technology for use in
biological research or therapeutics. Often for affinity
purification, the gene for an affinity tag is combined with the
gene for a protein of interest, in order to express the protein of
interest fused to an affinity tag.
[0025] As used herein, the term "high-throughput detection" or
"high-throughput screening" refers to a method for scientific
experimentation especially used in drug discovery and relevant to
the fields of biology and chemistry. Using robotics, data
processing and control software, liquid handling devices, and
sensitive detectors, high-throughput screening may allow a
researcher to quickly conduct millions of chemical, genetic or
pharmacological tests. Through this process one can rapidly
identify active compounds, antibodies or genes which modulate a
particular biomolecular pathway.
[0026] The results of these experiments may provide starting points
for drug design and for understanding the interaction or role of a
particular biochemical process in biology.
[0027] As used herein, the term "enzyme-linked immunosorbent assay
(ELISA)" refers to a test that uses antibodies and color change to
identify a substance. ELISA is a popular format of a "wet-lab" type
analytic biochemistry assay that uses a solid-phase enzyme
immunoassay (EIA) to detect the presence of a substance, usually an
antigen, in a liquid sample or wet sample. The ELISA may be used as
a diagnostic tool in medicine and plant pathology, as well as a
quality-control check in various industries.
[0028] As used herein, the term "affinity label" refers to a
substance, molecule or biomolecule, which binds rapidly, and
irreversibly to the protein of interest to form a "fusion tag" or a
"affinity tag", by a means, e.g., expression, and which is further
capable of specifically binding to a fluorescent label.
[0029] As used herein, the terms "fluorescent label",
"fluorophore", "dyes", and "fluorescent molecule" have the same or
similar meaning in the invention and are interchangeable throughout
the invention. For example, the term "fluorophore", "fluorochrome",
or "chromophore", as used herein, refers to a fluorescent chemical
compound that can re-emit light upon light excitation. Fluorophores
may typically contain several combined aromatic groups, or plane or
cyclic molecules with several .pi. bonds.
[0030] Fluorophores may sometimes be used alone, as a tracer in
fluids, as a dye for staining of certain structures, as a substrate
of enzymes, or as a probe or indicator (when its fluorescence is
affected by environment such as polarity, ions, et. al.). But more
generally fluorophore may be covalently bonded to a macromolecule,
serving as a marker (or dye, or tag, or reporter) for affine or
bioactive reagents (antibodies, peptides, nucleic acids, et. al.).
Fluorophores may notably be used to stain tissues, cells, or
materials in a variety of analytical methods, i.e. fluorescent
imaging and spectroscopy.
[0031] In one embodiment, the present invention relates to proteins
of interest (POI) expressed as fusion proteins including an
affinity tag, which further specifically binds to a fluorescent
label. FIG. 1 shows a general concept of fusion protein expression
with an affinity tag. The POI were initially expressed as fusion
proteins including an affinity tag. The expression of such fusion
proteins employs techniques of molecular biology that are well
known to those skilled in the art and consist of, e.g., recombinant
protein expression and any other suitable techniques.
[0032] The affinity tag may be any molecules or biomolecules which
are capable of specifically linking to one fluorescent molecule as
the fluorophore (fluorescent label). Specifically, the affinity tag
may be proteins having suicidal enzymatic activity, also called
suicide enzymes. Suicide enzymes are proteins whose enzymatic
activity is modified by specific mutations allowing them to bind a
substrate rapidly, covalently, and irreversibly, as these enzymes
can each bind only one fluorescent molecule, and the further
binding activity of the enzyme is blocked by the binding of the
substrate.
[0033] The fluorophore or fluorescent label may be any fluorescent
molecules or dyes. Specifically, the fluorophore or fluorescent
label may be a dye compatible with the fluorescent detection
equipment being used. Some examples of these dyes may be seen
below. All of these dyes may be commercially available as
fluorescent substrates for fusion tags, and each binds covalently
to a different tag. Most developed fusion tags may have many
fluorescent variations of the ligand to cover a broad portion of
the UV spectrum. Any suitable fluorescent molecules or dyes may be
used for the present invention. In a preferred embodiment of the
present invention, a microfluidic system such as LabChip systems
(available from Caliper) may be equipped with a 635 nm excitation
light source, and a 700 nm emission filter, making dyes with
excitation and emission maxima near these wavelengths ideal choices
when selecting a fluorophore for use on a LabChip system. Any
fluorescent molecules or dyes having suitable excitation and
emission maxima near the required wavelength may be used for the
present invention.
[0034] For example, an Alexa660 fluorophore meets these criteria,
although other fluorophores are likely to work as well. Examples of
available fluorescent dyes or fluorophores may include those
covalently bind to fusion tags. Dyes or fluorophores with
excitation and emission maxima near 635 and 700 nm are expected to
be compatible with LabChip systems.
[0035] The examples of fluorophores may include HaloTag Alexa Fluor
488 Ligand as shown below (available from PROMEGA). HaloTag Alexa
Fluor 488 Ligand has maxima excitation and emission at 499/518 nm
(ex/em). As comparison, the excitation and emission maxima for
Alexa660 are 654/690 nm (ex/em).
##STR00001##
[0036] The examples of fluorophores or dyes may also include
SNAP-Cell 505 (available from NEB), as shown below. SNAP-Cell 505
has its excitation and emission maxima at 504/532 nm (ex/em). The
structure of SNAP-Surface Alexa Fluor 647 (not shown) and its
excitation and emission maxima are 652/670 nm (ex/em).
##STR00002##
[0037] The examples of fluorophores or dyes may also include
CLIP-Surface 647 (available from NEB), and it has its excitation
and emission maxima at 635/650 nm (ex/em).
##STR00003##
[0038] The examples of fluorophores or dyes may further include
Lumio-Red (available from LIFE TECHNOLOGIES), and it has excitation
and emission maxima at 593/608 nm (ex/em).
##STR00004##
[0039] In one embodiment, the affinity tag may be SNAP-tag
(marketed by COVALYS), a mutant of an alkylguanine-DNA
alkyltransferase, and one of the substrates for SNAP-tag is
benzylguanine.
[0040] In one preferred embodiment of the present invention, the
affinity tag may be HaloTag (marketed by PROMEGA), a mutant of a
dehalogenase, and some of substrates for HaloTag are compounds of
the chloroalkane family. The native enzyme of HaloTag is DhaA, a
monomeric protein (MW.apprxeq.33 KDa), which breaks carbon halogen
bonds in aliphatic halogenated compounds. Upon nucleophilic attack
of Asp106 in the enzyme carbon-halogen bonds of chloroalkane, an
ester bond is formed between fluorophore HaloTag ligand and the
enzyme. HaloTag may contain a critical mutation from His272 to Phe
in the catalytic triad so that the produced ester bond between the
enzyme and the ligand can not be further hydrolyzed (Technical
manual, Promega, "HaloTag Interchangeable Labeling Technology";
http://www.promega.com, 2006). FIG. 2 shows that HaloTag may be
used as an affinity tag for the expression of proteins of interest
(P01) into HaloTag fusion proteins (FIG. 2A). As shown in FIG. 2B,
HaloTag in the fusion proteins may further covalently bind a
chloroalkane fluorophore HaloTag ligand by cleaving the
carbon-chloride bond in the ligand. The binding process between
fusion proteins and fluorescent HaloTag ligand requires that
HaloTag be correctly folded.
[0041] Further, as shown in FIG. 3, the resulting fluorescent label
having a covalent bond between HaloTag in the fusion proteins and
the ligand may remain bound to the HaloTag fusion protein after
denaturation and electrophoresis. These observations indicate that
binding between the ligands and the fusion tag is permanent,
allowing denaturation of the protein to perform electrophoresis and
assay molecular weight.
[0042] In one embodiment, the present invention relates to methods
of detection of HaloTag fusion proteins in lysate. The HaloTag
fusion proteins including a fluorescent label may be detected by
using a gel-based detection technique. Any gel-based detection
techniques may be suitable for the present invention. In one
preferred embodiment, the gel-based detection techniques may be
SDS-PAGE. FIG. 4 shows a graph of sodium dodecyl sulfate
polyacryamide gel electrophoresis (SDS-PAGE) analysis on the
detection of HaloTag fusion proteins in lysate. Two HaloTag fusion
proteins were investigated including Halo-MAPK (FIG. 4A) and
HaIoTEV (FIG. 4B). Halo-MAPK (FIG. 4A) includes a HaloTag protein
(.about.35 kDa) and a MAPK protein (.about.40 kDa), showing a total
molecular weight of .about.75 kDa. HaIoTEV (FIG. 4B) includes a
HaloTag protein (.about.35 kDa) and a TEV protease (.about.45 kDa),
showing a total molecular weight of .about.80 kDa. Alexa660 was
used as the fluorescent label (fluorophore) for both fusion
proteins of Halo-MAPK and HaIoTEV. The fusion proteins were
visualized through the fluorescent label (fluorophore) of Alexa660
(emission at 670 nm) following 633 nm light excitation.
[0043] The HaloTag fusion proteins in a complex protein mixture,
may be detected directly by the gel-based techniques. As shown in
FIG. 4, the fusion protein of Halo-MAPK (1:10 lysate) was clearly
visualized, which was confirmed with the total molecular weight
mark at .about.75 kDa. Further, with increasing concentrations of
Alexa660-Halo-ligand (from left to right), the fusion protein of
Halo-MAPK showed an increasing intensity of detection. As a
comparison, in the absence of the corresponding DNA, lysate
Halo-MAPK can not be detected.
[0044] Alternatively, HaloTag fusion proteins may be detected after
the fusion proteins were purified. As shown in FIG. 4, the purified
HaIoTEV protease was clearly visualized, which was confirmed with
the total molecular weight mark at .about.80 kDa. Further, with
increasing concentrations of Alexa660-Halo-ligand (from left to
right), the fusion protein of HaIoTEV showed an increasing
intensity of detection.
[0045] Further, gel-based detection techniques on HaloTag fusion
proteins in lysate may show suitable sensitivity of detection with
various molecular weight (size) of the fusion proteins. Reference
is now made to FIG. 5, which shows sodium dodecyl sulfate
polyacryamide gel electrophoresis (SDS-PAGE) analysis on HaloTag
fusion protein examples, having various molecular weights. As shown
in FIG. 5A, when Alexa660 was used as the fluorescent label, the
HaloTag fusion proteins, having molecular weight in the range of
30-90 kDa, were distinctively detected. Visualized through the
fluorescent label (fluorophore) of Alexa660 (emission at 670 nm)
following 633 nm light excitation, the HaloTag fusion proteins
showed distinct bands of detection corresponding to their molecular
weights. As shown in FIG. 5B, total protein staining with coomassie
reveals the high protein complexity of the lysate and the
relatively low abundance of the HaloTag fusion proteins. The
fluorescent analysis much more clearly indicates successful
expression of the fusion proteins than coomassie staining (FIG.
5A).
[0046] Various protocols of gel-based detection on fusion proteins
in lysate have already been published or marketed using gel-based
fluorescent detection of covalently bound fluorophores to affinity
tags. The commercially available products include, e.g., HaloTag
from Promega, Lumio Tag from Life Technologies, and SNAP/CLIP Tags
from NEB. Nevertheless, the gel-based detection protocols show many
limitations. For instance, gel-based detection protocols are not
high throughput technologies. Consequently, these gel-based
detection protocols are commonly applied to small scale
problems.
[0047] While there is an increasing need for high throughput
detection of fusion proteins in lysate, automated capillary
electrophoresis represents one of the potential high throughput
alternatives to traditional PAGE analysis. Caliper is the primary
manufacturer of automated capillary electrophoresis
instruments/chips. Caliper's LabChip systems are the industry
standard for high throughput expression/purification
characterization. LabChip, as a microfluidic lab-on-a-chip
technology, represents a significant advancement in laboratory
experimentation, bringing the benefits of miniaturization,
integration and automation to numerous research-based industries.
Nevertheless, LabChip instruments utilize a fluorescent protein
stain that detects all proteins non-specifically.
[0048] FIG. 6 shows a typical LabChip setup from
Caliper/PerkinElmer along with the standard experimental protocol.
As shown in FIG. 6, a standard LabChip protein separation protocol
includes: 1) Denatured, unstained, unlabeled protein sample is
sipped into chip (.about.170 nL); 2) Sample is mixed with a
quantification marker (1:2 ratio); 3) Sample enters gel matrix and
mixes with LC90 fluorescent dye (3 and 8; +/-); 4) Proteins in
sample undergo electrophoretic separation (7 and 10; +/-); 5)
Sample is diluted with gel matrix (dye-free); 6) Sample is analyzed
with 635/700 nm fluorometer.
[0049] Although automated capillary electrophoresis such as using
LabChip is the potential high throughput alternative to traditional
PAGE analysis, the commonly available instruments of LabChips
utilize a fluorescent protein stain that detects all protein
non-specifically. As shown in FIG. 7, due to the non-specific
binding ability of the fluorescent protein stain, after
denaturation and loading the samples of proteins into the chip, all
the proteins including proteins of interest (POI) and all other
proteins would be labeled with the non-specific fluorescent protein
stain. Consequently, in the resulting spectra, the signal of POI
would be buried in the signals from the other undesired
proteins.
[0050] In one preferred embodiment, the present invention relates
to high throughput detection methods and devices by combining a
high throughput automated capillary electrophoresis technique such
as the LabChip technology with fusion tag detections. The tag
molecule may be any molecules or biomolecules, which are capable of
specifically binding to a fluorescent molecule. The examples may
include SNAP-tag, CLIP, Lumio Tag, or HaloTag. Even though HaloTag
has been described during the following examples and embodiments, a
person having ordinary skill in the art will understand that any
other tag molecules may be used for the present invention.
[0051] Further, even though LabChip technology has been used during
the following examples and embodiments, a person having ordinary
skills in the art will understand that any high throughput
automated capillary electrophoresis techniques may be used for the
present invention.
[0052] As shown in FIG. 8, an additional step of mixing the
proteins with non-fluorescent protein stains is not needed for the
fusion protein detection protocol, as the fusion protein including
POI has been specifically labeled. Thus, after denaturation and
loading the proteins into the chip, only the labeled fusion protein
including POI will be detected as it is the only
fluorescence-labeled protein. Consequently, the resulting spectra
would only exhibit the signal of the desired protein as shown in
FIG. 8. The much cleaner signal detection corresponding to POI
enables the present invention as a high throughput detection
technique.
[0053] To further confirm the feasibility of a combination of
LabChip with fusion tag detection technique, a controlled
experiment was conducted for the detection of Bovine serum albumin
(BSA) in the presence of LC90 fluorescent HaloTag ligand
(Alexa660-Halo-ligand). As BSA lacks a HaloTag fusion, BSA is not
expected to react with the fluorescent HaloTag ligand. As shown in
FIG. 9 the peak detected at 65 kDa is consistent with the molecular
weight of BSA. Further, the intensity of BSA peak at 65 kDA (FIG.
9; left) and the signal in Gel (FIG. 9; right) remain unchanged
with the increasing concentrations of Alexa660-Halo-Ligand. These
observations confirm the unreactivity between a non HaloTag fusion
protein and HaloTag ligand.
[0054] To confirm that a combination of LabChip with fusion tag
protocols will qualitatively and quantitatively assay and detect
proteins in complex samples using high throughput LabChip
technique, a detection experiment for the fusion protein of HaIoTEV
was conducted. FIG. 10 shows the spectra (left) and the
corresponding gel signals (right) of a LC90 fluorescent HaloTag
ligand detection on the fusion protein of HaIoTEV using the LabChip
setup. As discussed above, HaIoTEV includes a HaloTag protein
(.about.35 kDa) and a TEV protease (.about.45 kDa), showing a total
molecular weight of .about.80 kDa. Alexa660 was used as the
fluorescent label (fluorophore) for HaIoTEV. As shown in FIG. 10
(left), the fusion protein of HaIoTEV was detected at 80 kDa,
consistent with its total molecular weight. More importantly, as
the concentration of Alexa-Halo-Ligand increases, the intensity of
the HaIoTEV peak at 80 kDa was enhanced proportionally (FIG. 10;
left). These observations indicate that the combination of LabChip
with fusion tag protocols can not only qualitatively but also
quantitatively assay and detect proteins. The corresponding gel
signals (FIG. 10; right) are consistent with these
observations.
[0055] Further, as the observed fusion proteins show sharp and
narrow peaks during these detections (FIGS. 9 and 10), the present
invention of a combination of LabChip with fusion tag protocols can
be applied to assay, detect, and purify proteins in complex samples
such as lysates. Fusion proteins in expression mixtures can be
qualitatively and quantitatively detected without prior
purification in a high throughput fashion. The high throughput
capability of the LabChip techniques enables the present invention
to detect large numbers of proteins of interest (POI), thus
allowing for a collection of proteins to be detected with a unified
method rather other labor-intensive methods such as an individual
method for each POI.
[0056] Although companies such as Caliper have described an immune
precipitation technology, the present invention have many
advantages over the previous technology. This approach allows for
rapid high throughput screening of expression without the need for
antibodies. The present approach may also be applied to strong
non-covalent bond interactions such as that between biotin and
streptavidin tag as long as the binding strength can withstand
downstream analysis protocols.
[0057] Additionally, the current approach directly labels fusion
proteins with a fusion tag specific fluorophore, and the present
invention uses a capillary electrophoresis instrument to
specifically detect fusion proteins within complex samples. The
present invention also utilizes fusion tags that are able to bind
to their specific ligands with high affinity, such as SNAP-tag,
CLIP-tag, Halo Tag, Lumio Tag, and others.
[0058] In terms of comparing to existing technologies (e.g.,
proteinsimple.TM.), the present invention offers at least the
following advantages: (a) the present invention can be used on an
unmodified LabChip GX or GXII system, and there is no need to
purchase a new instrument dedicated to high throughout specific
protein detection; (b) the present invention is much faster
(.about.1 hour per plate) than, e.g., the proteinsimple.TM.'s
system (up to 19 hours per plate); (c) the present invention
doesn't require antibodies; and (d) the present invention can be
used in conjunction with the normal operation of LabChip's protein
detection kits, which is thus capable of comparing specific fusion
protein signals with total protein signals.
[0059] In one aspect, the present invention discloses a method for
high throughput detection of proteins of interest contained in a
sample. The method comprises the steps of (a) contacting a sample
comprising proteins of interest coupled to fusion tags with fusion
tag specific fluorophores that specifically bind the fusion tags
coupled to the proteins of interest; and (b) subjecting the sample
to automated capillary electrophoresis, wherein the presence of the
proteins of interest in the sample is detected by fluorescence
signals associated with the fusion tag specific fluorophores.
[0060] The present invention may generally be applied to high
throughput detection of proteins of interest contained in any
suitable samples. In one specific embodiment in the present
invention, the sample may be a cell lysate, more specifically, a
bacterial cell lysate.
[0061] Any suitable fusion tag may be used for the present high
throughput detection of proteins of interest contained in a sample.
In one specific embodiment, the suitable fusion tag may be a fusion
tag protein.
[0062] In one embodiment, the fusion tags may specifically link to
fusion tag specific fluorophores, which may be specifically coupled
to the proteins of interest. Any suitable means of linking may be
used to connect the fusion tags, fusion tag specific fluorophores
and the proteins of interest. In one specific embodiment, the
fusion tags, fusion tag specific fluorophores and the proteins of
interest may be linked through chemical bonds, such as covalent
bonds. Any suitable chemical bonds may be used for the present
invention.
[0063] In one embodiment of the present invention, any fluorophores
may be used as the fusion tag specific fluorophores. In one
specific embodiment, the fusion tag specific fluorophore may have
an excitation and emission maxima near the specifically required
wavelength, such as about 635 nm for excitation and about 700 nm
for emission.
[0064] In one specific embodiment of the present invention, the
suitable fusion tag may be a fusion tag protein which are
commercially available, such as SNAP-tag, CLIP-tag, Lumio tag, or
HaloTag.
[0065] In one embodiment of the present high throughput detection
of proteins of interest contained in a sample, the automated
capillary electrophoresis may be carried out on a microfluidic
chip.
[0066] In one aspect, the present invention provides a microfluidic
chip for use in a method of high throughput detection of proteins
of interest contained in a sample according to any of the above
methods.
[0067] In another aspect, the present invention provides a kit. The
kit comprises a microfluidic chip for use in a method of high
throughput detection of proteins of interest contained in a sample
according to any of the above methods; a fusion tag specific
fluorophore; and a means for detection, wherein the presence of the
proteins of interest in the sample is detected by fluorescence
signals associated with the fusion tag specific fluorophores.
[0068] In one specific embodiment of the kit, the fusion tag
specific fluorophore has an excitation and emission maxima near
about 635 nm and about 700 nm, respectively.
[0069] EXAMPLES
[0070] Example 1
Formation of Fusion Proteins Containing Affinity Tag and Proteins
of Interest
[0071] HaloTag was used as an example affinity tag for all the
Examples. However, a person having ordinary skill in the art will
understand that any suitable affinity tags will work for the
purpose of the present invention. Proteins of interest (POI) were
expressed as HaloTag fusions following the following steps. Genes
were inserted into an appropriate expression vector that encodes
either an N-terminal or C-terminal HaloTag fusion partner. For E.
coli expression, the expression vector pCPD_nHalo containing the
gene was transformed into E. coli strain BL21(DE3). A 2 mL culture
can be grown in LB media with 50 ug/mL ampicilin at 37.degree. C.
until OD600=0.6, and induced with 1 mM IPTG. Expression for 20
hours at 18.degree. C. was sufficient to produce the HaloTag fusion
protein. The culture was pelleted by centrifugation at 5000.times.g
for 20 minutes, and resuspended in a standard lysis buffer
containing lysozyme and DNase. After 30 minutes of lysis, the
lysate can be analyzed for expression of the fusion protein.
[0072] For cell-free expression systems, the expression vector DNA
was added to the lysate, and expression can be performed following
the kit manufacturer's instructions.
[0073] Lysate from either in vivo or in vitro expression systems
can be assayed by incubating the lysate with a small concentration
of fluorescent HaloTag ligand. A 4 uM stock solution can be added
to each sample to make the final concentration 1 uM. Samples can be
incubated for 20 minutes and room temperature, and then denatured
with SDS and boiling. For testing on a LabChip system, samples can
be prepared following manufacturer's instructions.
Example 2
High-Throughput Detection of HaloTad Fusion Proteins in Lysate
Using LabChip
[0074] The HaloTag fusion proteins were produced following the same
procedure as described in Example 1. LabChip system was purchased
from Caliper, and no modifications were performed on the system.
The experiment follows an automated protocol according to the
technical manual of LabChip.
Example 3
[0075] FIG. 11 depicts 32 proteins expressed as N-terminal HaloTag
fusion proteins in HeLa cell lysate and incubated with
HaloTag-Alexa660 ligand. Expression reactions were denatured and
separated by PAGE. (A) Coomassie stain of 1 uL expression
reactions; (B) In-gel fluorescence of the same gels prior to
Coomassie staining; (C) LabChip virtual gel of collection.
References
[0076] 1. HaloTag Technology: Focus on Imaging Protocol TM260.
Promega Corporation. Accessed Oct. 22, 2012.
http://www.promega.com/resources/protocols/technical-manuals/0/halotag-te-
chnology-focus-on-imaging-protocol/2. [0077] 2. HaloTag Technology.
Promega Corporation. Accessed Oct. 22, 2012.
http://www.promega.com/.about./media/images/product%20marketing%20pages/p-
roduct%20
marketing%20images/halotag%20technology/halotag-web-banner.jpg?I-
a=en&w=600&h=150&as=1 [0078] 3. Gene Expression and
Cellular Analysis. New England Biolabs. Accessed Oct. 22, 2012.
http://www.neb.com/nebecomm/products/categoryl40.asp?#141 [0079] 4.
Lumio In-Cell Labeling Kits. Life Technologies. Accessed Oct. 22,
2012.
http://tools.invitrogen.com/content/sfs/manuals/lumioincelllabeling_man.p-
df [0080] 5. SWAP: A High Throughput Automated Microfluidic
Alternative to Western Blotting. PerkinElmer Inc. Accessed Oct. 23,
2012.
http://www.perkinelmer.com/CMSResources/Images/44-133015APP_SWAP.%20A%20H-
igh%20Throughput%20Automated%20Microfluidic%20
Alternative%20to%20Western%20Blotting. pdf [0081] 6. Technical
manual, Promega, "HaloTag Interchangeable Labeling Technology";
http://www.promega.com, 2006
* * * * *
References