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Title:
PROTEIN G-OLIGONUCLEOTIDE CONJUGATE
Document Type and Number:
WIPO Patent Application WO/2008/156249
Kind Code:
A1
Abstract:
The present invention relates to a protein G conjugate, which is prepared by linking an N-terminal cysteine-tagged protein G variant with an oligonucleotide via a linker. The conjugate binds in a directional manner on the surface of a biochip and biosensor, thereby providing a biochip and biosensor having improved antibody immobilization ability.

Inventors:
CHUNG BONG HYUN (KR)
JUNG YONG WON (KR)
LEE JEONG MIN (KR)
Application Number:
KR2008/002739
Publication Date:
December 24, 2008
Filing Date:
May 16, 2008
Export Citation:
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Assignee:
KOREA RES INST OF BIOSCIENCE (KR)
CHUNG BONG HYUN (KR)
JUNG YONG WON (KR)
LEE JEONG MIN (KR)
International Classes:
C07K19/00
Foreign References:
US6365418B12002-04-02
Other References:
CHOI J.-W. ET AL.: "Fabrication of DNA-protein conjugate layer on gold-substrate and its application to immunosensor", COLLOIDS SURF. B BIOINTERFACES, vol. 25, February 2003 (2003-02-01), pages 173 - 177, XP008125423
LEE J.M. ET AL.: "Direct immobilization of protein g variants with various numbers of cysteine residues on a gold surface", ANAL. CHEM., vol. 79, April 2007 (2007-04-01), pages 2680 - 2687, XP009132789
BAE Y.M. ET AL.: "Study on orientation of immunoglobin G on protein G layer", BIOSENS. BIOELECTRON., vol. 21, July 2005 (2005-07-01), pages 103 - 110, XP004943509
See also references of EP 2155792A4
Attorney, Agent or Firm:
SON, Min (City Air Tower 159-9Samseong-dong, Gangnam-gu, Seoul 135-973, KR)
Download PDF:
Claims:

[CLAIMS]

[Claim l]

A protein G conjugate (gA-G conjugate) which is prepared

by linking an N-terminal cysteine-tagged protein G variant with

an oligonucleotide (gA) comprising an amine group using a linker

capable of selectively reacting with both amine and thiol groups,

represented by the following Formula: A x -Cys-L y -Protein G-Q 2

(wherein A is an amino acid linker, L is a linker linking

aprotein Gwith a cysteine tag, Q is a tag for protein purification, x is 0 to 2, and y or z is 0 or 1, respectively)

[Claim 2]

The protein G conjugate according to claim 1, wherein the

oligonucleotide (gA) is selected from the group consisting of

oligo DNA, RNA, PNA (peptide nucleic acid) and LNA (locked nucleic acid) .

[Claim 3] The protein G conjugate according to claim 2, wherein the

oligonucleotide (gA) is an oligo DNA.

[Claim 4 ]

The protein G conjugate according to claim 1, wherein the

oligonucleotide (gA) comprising an amine group is modified with

an amine group at its 5' -end.

[Claim 5]

The protein G conjugate according to claim 1, wherein the

linker capable of reacting with both amine and thiol groups is

selected from the group consisting of Sulfo-SMCC (SuIfosuccinimidyl

4- (N-maleimidomethyl) cyclohexane-1-carboxylate) , BMPS (N- [Maleimidopropyloxy] succinimide ester) ,

GMBS (N- [Malwimidobutyryloxy] succinimide ester), and SMPB (Succinimidyl 4- [p-maleimidophenyl] butyrate) .

[Claim β]

The protein G conjugate according to claim 1, wherein the

oligonucleotide (gA) has a length of 18 to 30 nt .

[Claim 7]

The protein G conjugate according to claim 1, wherein the

protein G variant and oligonucleotide (gA) are linked to each other one by one.

[Claim 8] The protein G conjugate according to claim 1, wherein the

linker (L) linking a protein G with a cysteine tag is a peptide

consisting of 2 to 10 amino acids.

[Claim 9] The protein G conjugate according to claim 8, wherein the

linker (L) linking a protein G with a cysteine tag has an amino

acid sequence of DDDDK (Asp-Asp-Asp-Asp-Lys) .

[Claim 10] A method for preparing the protein G conjugate of any one

of claims 1 to 9 comprising the step of linking an N-terminal

cysteine-tagged protein G variant and an oligonucleotide (gA)

comprising an amine group with a linker capable of reacting with both amine and thiol groups by a covalent bond, represented by

the following Formula:

A x -Cys-L y -Protein G-Q 2

(wherein A is an amino acid linker, L is a linker linking

a protein G with a cysteine tag, Qisatag for protein purification,

x is 0 to 2, and y or z is 0 or 1, respectively)

[Claim 11]

The method for preparing the protein G conjugate according

to claim 10, further comprising the step of isolating andpurifying

the protein G conjugate after the conjugate formation.

[Claim 12]

A biochip or biosensor which is fabricated by linking the protein G conjugate of any one of claims 1 to 9 onto the surface of a solid support.

[Claim 13]

The biochip or biosensor according to claim 12, wherein an oligonucleotide (cA) having a base sequence complementary

to the oligonucleotide (gA) of the protein G conjugate is linked onto the surface of the solid support.

[Claim 14]

The biochip or biosensor according to claim 12, wherein the solid support is selected from the group consisting of ceramic, glass,

polymer, silicone and metal.

[Claim 15]

The biochip or biosensor according to claim 14, wherein

the biochip or biosensor is a gold thin film or gold nano-particle .

[Claim lβ] The biochip or biosensor according to claim 12, wherein an antibody is linked to the protein G conjugate.

[Claim 17]

Amethod for fabricating a biochip or a biosensor, comprising

the steps of

a) linking an oligonucleotide (cA) , which has a base sequence

being complementary to an oligonucleotide (gA) of the protein

G conjugate of any one of claims 1 to 9, onto the surface of a solid support;

b) linking the oligonucleotide (cA) on the surface of the

solid support with the oligonucleotide (gA) of the protein G

conjugate; and c) linking an antibody with the protein G conjugate

immobilized on the solid support.

[Claim 18]

The method according to claim 17, wherein the solid support

is selected from the group consisting of ceramic, glass, polymer,

silicone and metal.

[Claim 19]

The biochip or biosensor according to claim 18, wherein

thebiochiporbiosensorisagoldthinfilmorgold nano-particle .

[Claim 20] A method for analyzing an antigen using the biochip or biosensor of claim 12.

Description:

[DESCRIPTION]

[invention Title]

PROTEIN G-OLIGONUCLEOTIDE CONJUGATE

[Technical Field]

The present invention relates to a protein G conjugate (gA-G)

which is prepared by linking an N-terminal cysteine-tagged

protein G variant with an oligonucleotide using a linker, a method

for preparing the same, and a biochip and a biosensor fabricated

by using the conjugate.

[Background Art]

Antibodies have been widely used in medical studies

concerning diagnosis and treatment of diseases as well as in

biological analyses, because of their property of specifically binding to an antigen (Curr. Opin. Biotechnol. 12 (2001) 65-69,

Curr. Opin. Chem. Biol. 5(2001) 40-45). Recently, as an

immunoassay, immunosensors have been developed, which require the immobilization of an antibody on a solid support to measure

changes in current, resistance and mass, optical properties or

the like (Affinity Biosensors . vol.7: Techniques and protocols) .

Among them, a surface plasmon resonance-based immunosensormaking

use of optical properties has been commercialized. The surface plasmon resonance-based biosensor provides qualitative

information (whether two molecules specifically bind to each

other) and quantitative information (reaction kinetics and

equilibrium constants) , and also performs sensing in real time

without the use of labeling, thus being particularly useful for

measuring antigen-antibody binding (J. MoI. Recognit. 1999, 12,

390-408) .

In the immunosensor, it is very important that antibodies are selectively and stably immobilized on a solid support. The

techniques for immobilizing antibodies are classified into two

categories, physical immobilization and chemical immobilization.

The physical immobilization techniques (Trends Anal. Chem.2000 19, 530-540) have been minimally used because they cause

denaturation of the protein, and the results are less reproducible.

In contrast, the chemical immobilization techniques (Langumur, 1997, 13, 6485-6490) have been widely used because they show

good reproducibility and a wide range of applications, due to

their feature of allowing secure binding of proteins through

covalent bonding. However, when immobilization of antibodies

is performed using a chemical immobilization technique, the

antibodies, being asymmetric macromolecules, often lose their

orientation and activity to bind to antigens (Analyst 121,

29R-32R) .

In an attempt to enhance the ability of antibodies to bind

to antigens, a support may be used before the antibodies are

linked to a solid substrate, and a technology of using protein

G as the support is known. However, there is a problem that this

protein G itself also loses orientation and its ability to bind

to an antibody when bound to the support.

Accordingly, in order to solve such problem, a variety of

methods have been suggested. For example, Streptococcal protein

G is treated with 2-iminothiolane to thiolate the amino acid

group of a protein, and then the thiolated Streptococcal protein

G is immobilized on the surface. However, this method is directed to thiolating the amino groups of amino acids having an amino

group (Arg, Asn, GIn, Lys), instead of thiolating any specific

site, and thus the method results in low specificity and requires

additional purification processes after chemical treatments

(Biosensors and Bioelectronics, 2005, 21, 103-110) .

A DNA-directed immobilization method has been used for

immobilization of protein. The DNA surface is known to be stable,

and known to be easily prepared, as compared to a protein chip.

For the protein immobilization, the following factors have to be considered, such as storage for a long period of time,

immobilization of unstable protein, or protein storage under

unstable conditions. The DNA-directed antibody immobilization methods have also been reported, for example, an immobilization

method of biotinylated antibody using a streptavidin-DNA

conjugate, or directly linking DNA to antibodies . However, both

methods have a drawback in that a small molecule or DNA has to

be linked to the antibody, so as to cause loss of its orientation or chemical modification of antigen-binding site.

[Disclosure] [Technical Problem] It is an object of the present invention to solve the problem that antibodies lose their orientation upon binding to an

immunosensor, and to provide techniques for easily immobilizing

antibodies on a variety of solid supports in a consistent

orientation using well-defined DNA surfaces.

[Technical Solution]

Previously, the present inventors have prepared an

N-terminal cysteine-tagged protein G variant (Korean Patent

Application No. 10-2007-0052560) , and confirmed its usefulness

through experiments, in order to solve the problem that antibodies

lose their orientation upon binding to an immunosensor. Also, based on the invention, the present inventors have prepared a protein G conjugate (gA-G) by chemically linking an

oligonucleotide (gA) having an amine group with the cysteine-tagged protein G variant using a linker capable of

selectively reacting withboth amine and thiol groups . Theyfound

that antibodies can be easily immobilized on a variety of solid

supports in a consistent orientation and on intended areas of the surfaces by using the protein G conjugate, thereby completing the present invention.

[Description of Drawings]

Fig. 1 shows binding domains (Bl and B2) of Streptococcal

protein G that binds with antibodies,

Fig. 2 shows the structure of protein G variant used in

the present invention and an amino acid sequence of B2, which

is one of domains binding with antibodies,

Fig.3 is a photograph of protein electrophoresis (SDS-PAGE)

showing the expression patterns of the cysteine-tagged protein

G variants in E . coli transformed with the expression vector shown in Fig. 2 r Fig. 4 is a diagram showing a biosensor or biochip, prepared

by immobilizing the protein G conjugate (gA-G) having an

oligonucleotide (gA) on the surface of gold thin film having

the complementary oligonucleotide (cA) , and then immobilizing an antibody,

Fig.5 is a photograph of protein electrophoresis (SDS-PAGE)

to analyze the protein G conjugate (gA-G) ,

Fig. 6 is a graph showing the changes in the surface plasmon

resonance signal tomeasure the reaction of the protein G conjugate

(gA-G) , complementary oligonucleotide (gA) , and

noncomplementary control oligonucleotide (gB) with the

oligonucleotide (cA) on the surface of gold thin film,

Fig. 7 is a graph showing the changes in the surface plasmon

resonance signal to detect the reaction of 100 nM PSA and its

antibody in theproteinGconjugate (gA-G) -immobilizedbiosensor,

Fig. 8 is a photograph obtained by a fluorescent scanner,

in which after linking the oligonucleotide (cA) to the epoxy

group on the glass surface, an array was fabricated to immobilize

the oligonucleotides (cA or cB) using a DNA arrayer, and then

the surface was treated with the protein G conjugate (gA-G) and

Cy3-mouse IgGl (1 nM) labeled with a fluorescent marker Cy3,

and

Fig. 9 is a photograph of agarose gel electrophoresis to analyze the formation of antibody-immobilized gold nano-particle, in which (A) is a photograph of agarose gel after reacting the

gold nano-particle linked with oligonucleotide (cA) (AuNP-cA)

with the complementary oligonucleotide (gA) , protein G conjugate

(gA-G) , noncomplementary control oligonucleotide (gB) , and

noncomplementary oligonucleotide (gB) -protein G variant (gB-G) ,

(B) is a photograph of agarose gel for analysis of antibody immobilization, after reacting the protein G conjugate (gA-G)

with the gold nano-particles having two different numbers of

oligonucleotide (cA) (AuNP-cA-I, AuNP-cA-II) and removing the

unreacted protein G conjugate (gA-G) , and (C) is a schematic

diagram showing the IgG labeled AuNP-cA-I and AuNP-cA-II.

[Best Mode]

It is an object of the invention to provide a protein G

conjugate (gA-G) , which is prepared by linking an N-terminal

cysteine-tagged protein G variant with an oligonucleotide (gA)

comprising an amine group using a linker capable of selectively

reacting with both amine and thiol groups.

It is another object of the invention to provide a method for preparing the protein G conjugate (gA-G conjugate),

comprising the step of chemically linking the protein G variant with an oligonucleotide (gA) comprising an amine group using

a linker capable of selectively reacting with both amine and

thiol groups.

It is still another object of the invention to provide a biosensor fabricated by adhering the protein G conjugate (gA-G conjugate) onto the surface of a solid support, and a method for fabricating a biochip and a biosensor, characterized in that

the protein G conjugate is linked to the solid support, the surface

of which is linked with an oligonucleotide (cA) having a DNA

sequence complementary to the oligonucleotide (gA) comprising

an amine group.

It is still another object of the invention to provide a

method for analyzing an antigen using the biochip or biosensor.

In one embodiment to achieve the object of the present invention, the present invention relates to a protein G conjugate (gA-G conjugate) , which is prepared by linking an N-terminal cysteine-tagged protein G variant with an oligonucleotide (gA) comprising an amine group using a linker capable of selectively

reacting with both amine and thiol groups.

The N-terminal cysteine-tagged protein G variant used in

the present invention has the following structure.

A x -Cys-L y -Protein G-Q 2

(wherein A is an amino acid linker, L is a linker linking

a protein G with a cysteine tag, Q is a tag for protein purification,

x is 0 to 2, and y or z is 0 or 1, respectively)

Protein G is a bacterial cell wall protein isolated from

the group G streptococci, and has been known to bind to Fc and

Fab regions of a mammalian antibody (J. Immuunol. Methods 1988,

112, 113-120) . However, the protein G has been known to bind

to the Fc region with an affinity about 10 times greater than

the Fab region. A DNA sequence of native protein G was analyzed and has been disclosed. A Streptococcal protein G and

Staphylococcal protein A are among various proteins related to cell surface interactions, which are found in Gram-positive bacteria, and have the property of binding to an immunoglobulin antibody. The Streptococcal protein G variant, inter alia, is more useful than the Staphylococcal protein A, since the

Streptococcal protein G variant can bind to a wider range of

mammalian antibodies, so as to be used as a suitable receptor

for the antibodies.

The origin of the protein G used in the present invention

is not particularly limited, and the native protein G, an amino acid sequence of which is modified by deletion, addition,

substitution or the like, may be suitably used for the purpose

of the present invention, as long as it holds the ability to

bind to an antibody. In one embodiment of the present invention,

only the antibody-binding domains (Bl, B2) of the Streptococcal

protein G were used.

The protein G-Bl domain consists of three β-sheets and one

α-helix, and the third β-sheet and α-helix in its C-terminal

part are involved in binding to the antibody Fc region. The Bl domain is represented by SEQ ID NO. 1, and the B2 domain is

represented by SEQ ID NO. 2. As the amino acid sequences of Bl and B2 domains are compared to each other, there are differences in the four sequences, but little difference in their structures .

In one embodiment of the present invention, a Bl domain, in which

ten amino acids were deleted at its N-terminus, was used (Fig. 1) . It was reported that even though a form of the Bl domain

having a deletion of ten amino acid residues from its N-terminus

was used, there was no impact on the function of binding with an antibody (Biochem. J. (1990) 267, 171-177, J. MoI. Biol (1994)

243, 906-918, Biochemistry (2000) 39, 6564-6571).

As used herein, the term "cysteine tag (Cys)" refers to

a cysteine, which is fused at the N-terminus of protein G. A

preferred cysteine tag consists of one cysteine.

In the protein G variant of the present invention, the

cysteine tag may be directly linked to the protein G by a covalent

bond, or may be linked through a linker (L) . The linker is a

peptide having any sequence, which is insertedbetween the protein

G and cysteine. The linker may be a peptide consisting of 2 to 10 amino acids. In embodiments of the present invention, the linker consisting of 5 amino acids was used. The cysteine tag

of the present invention is not inserted inside the amino acid sequence of the protein G, and it provides the protein G with

orientation upon attaching to a solid support. If the linker

is attached, a thiol group is readily exposed to the outside.

Thus, the protein G can be more efficiently bound to a biosensor

with directionality.

In addition, 0 to 2 amino acid(s) maybe linked to the cysteine

tag of the protein G variant used in the present invention. A

preferred amino acid is methionine.

In order to easily isolate the protein G variant of the

present invention, a tag (Q) for protein purification may be

further included at its C-terminus . In embodiments of the present

invention, hexahistidine was tagged at its C-terminus, but as

the tag for protein purification, any known tag can be used for the purpose of the invention without limitation. The variant

of the present invention may contain methionine, which serves

as an initiation codon in prokaryotes, or not. In one embodiment

of the present invention, the present inventors prepared a one

cysteine-tagged variant.

The protein G variants of the present invention can be

prepared by a known method such as a peptide synthesis method,

in particular, efficiently prepared by a genetic engineering

method. The genetic engineering method is a method for expressing

large amounts of the desired protein in a host cell such as E . coli

by gene manipulation, and the related techniques are described in detail in disclosed documents (Molecular Biotechnology:

Principle and Application of Recombinant DNA ; ASM Press: 1994,

J. chem. Technol. Biotechnol. 1993, 56, 3-13). Using the known

techniques, a nucleic acid sequence encoding the protein G variant

used in the present invention is contained in a suitable expression

vector, and a suitable host cell is transformedwith the expression

vector, and cultured to prepare the protein G variants. The

preparation method of the protein G variant used in the present

invention is described in detail in Korean Patent Application

No.10-2007-0052560, applied by the present inventors, the entire

contents of which are fully incorporated herein by reference.

In one embodiment of the present invention, an expression

vector (pET-cysl-L-proteinG) comprising a base sequence that encodes the N-terminal cysteine-tagged Streptococcal protein

G variant was prepared as shown in Fig. 2.

Cysteine is an amino acid having a thiol group, and has

been known to specifically immobilize a protein by its insertion

into the protein (FEBS Lett. 1990, 270, 41-44, Biotechnol. Lett.

1993, 15, 29-34) . Disclosed is a method for binding cysteine at the C-terminus of Streptococcal protein G. However, in the

present invention, cysteine having a thiol group was used to tag the N-terminus, which is remote from the active domain of

the Streptococcal protein G variant. The active domain of the

Streptococcal protein G that binds with an antibody is located

in its C-terminus (the third β-sheet and α-helix) . Accordingly,

cysteine was not used to tag the inside of the protein G variant

but at the N-terminus thereof, thereby minimizing the loss of antibody-binding ability, in which the loss can occur by tagging the C-terminus with cysteine residues.

In embodiments of the present invention, the cysteine-tagged Streptococcal protein G variant was prepared (Example 1) . As mentioned above, after gene manipulation, the gene was inserted

into a protein expression vector to express the protein, and

then the protein G variant was separated by protein electrophoresis .

As usedherein, the oligonucleotide (guide oligonucleotide,

hereinafter, also referred to as gA) is an oligomer of 18 to

30 nt in length, and may include DNA, RNA, PNA and LNA, preferably oligo DNA. Any sequence, readily selected by those skilled in

the art, may be used depending on the purpose, and may be prepared

by a knownmethod or a commercially available sequence, for example,

a custom oligonucleotide (manufactured by Bioneer or IDT) may

be used. The method of oligomer preparation is well known in

the art. In addition, the oligonucleotide (gA) used in the

present invention comprises an amine group to bind with the protein

G via a linker, and the amine group may be located at the 5' -end,

3' -end or inside of the base sequence. To include the amine group

in the oligonucleotide (gA) , a specific region of the

oligonucleotide (gA) may be modified with the amine group by a known method in the art. A preferred oligonucleotide (gA) is

an oligonucleotide modified with the amine group at its 5' -end.

The amine group of the oligonucleotide is linked to the protein

G variant via a linker.

In addition, the oligonucleotide (gA) used in the present

invention has a base sequence being complementary to an

oligonucleotide (hereinafter, referred to as cA) , which is linked

onto the surface of the biosensor.

In the present invention, a linker (C) capable of reacting

with both amine and thiol groups is used to prepare the protein

G conjugate by linking the oligonucleotide (gA) with the protein

G variant. The linker of the present invention is used for the

purpose of linking the oligonucleotide (gA) comprising an amine

group with the protein G variant, and exemplified by

Sulfo-SMCC (Sulfosuccinimidyl

4- (N-maleimidomethyl) cyclohexane-1-carboxylate) ,

BMPS (N- [Maleimidopropyloxy] succinimide ester) ,

GMBS (N- [Malwimidobutyryloxy] succinimide ester) and

SMPB (Succinimidyl 4- [p-maleimidophenyl] butyrate) , but any

linker may be used without limitation, as long as it has a property of selectively reacting with both amine and thiol groups. A

preferred linker is Sulfo-SMCC.

The oligonucleotide (gA) modified with an amine group at

its end and the protein G variant are linked to each other via

the linker (C) to prepare the protein G conjugate (gA-G) . In this connection, the protein G variant and oligonucleotide (gA)

of the present invention have one thiol group and one amine group,

respectively. Thus, upon forming the conjugate, the oligonucleotide (gA) and protein G variant are linked to each

other one by one.

The protein G conjugate (gA-G) according to the present

invention binds in a directional manner with oligonucleotide

(cA) on the surface of the solid support of a biosensor by

complementary binding, thereby efficiently binding with

antibodies. Thus, the protein G conjugate can be satisfactorily

used in biochips and biosensors which utilize antigen-antibody

reactions .

In still another embodiment, the present invention relates to a method for preparing the protein G conjugate (gA-G) ,

comprising the step of linking the protein G variant and the

oligonucleotide (gA) modified with an amine group at its end to a linker capable of reacting with both amine and thiol groups

by a covalent bond.

The method for preparing the protein G conjugate (gA-G)

according to the present invention, as mentioned above, comprises

the step of linking the protein G variant and the oligonucleotide (gA) modified with an amine group at its end to a linker capable

of reacting with both amine and thiol groups by a covalent bond, in which any one of protein G variant and oligonucleotide (gA)

may be first linked to the linker, and then the other one may

be linked thereto.

In one preferred embodiment, the preparation method of the

present invention may further include the step of isolating and

purifying the protein G conjugate (gA-G) after the conjugate

formation. In the isolation/purification step, one or more known

methods for isolating/purifying a protein may be suitably

selected by those skilled in the art.

In a specific embodiment, the present inventors isolated the protein G conjugate (gA-G), which is prepared by linking the oligonucleotide (gA) modified with an amine group at its

5' -end and the Streptococcal protein G variant tagged with one cysteine to SuIfo-SMCC, by chromatography using both of the column

packed with anion exchange excellulose and the column packed with IDA excellulose.

In still another embodiment, the present invention relates

to a biochip or a biosensor fabricated by linking the protein

G conjugate (gA-G) onto the surface of a solid support, and to

a method for fabricating a biochip or a biosensor, comprising

the steps of

a) linking an oligonucleotide (cA) , which has abase sequence

being complementary to an oligonucleotide (gA) of protein G

conjugate (gA-G) , on the surface of a solid support,

b) linking the oligonucleotide (cA) on the surface of a

solid support with the oligonucleotide (gA) of the protein G

conjugate (gA-G) ; and c) linking an antibody with the protein G conjugate (gA-G)

immobilized on the solid support.

Examples of the solid support include metal or membrane, ceramic, glass, polymer surface or silicone, as described in the following Table 1. A preferred solid support is a gold thin film or gold nano-particle .

[Table 1]

Substrate for self-assembledmonolayer formation of protein having cysteine group

Presence or absence of Applications

Type of surface chemical Thin film Nano-particle or substrate pretreatment surface nano-structure

Absence Ag 0

Ags 0

Au 0 0

CdSe O

CdS 0

AuAg 0

AuCu 0

Cu 0 0

FePt O

GaAs 0

Ge 0

Hg 0

Pd 0 0

Pt 0 0

Stainless

O

Steel316L

Zn 0

ZnSe 0

In addition, on the surface of the solid support, the oligonucleotide (complementary oligonucleotide, hereinafter also referred to as cA) having a base sequence being complementary

to the oligonucleotide (gA) of the protein G conjugate (gA-G) of the present invention is linked. The oligonucleotide may be

linked onto the surface of the solid support by a known method which is selected by those skilled in the art, depending on the

structure of the solid support of a biochip and biosensor. For

example, in the case of a glass slide, the complementary oligonucleotide (cA) modified with an amine group may be linked onto the glass slide activated with an epoxy group, and in the case of a gold surface, the complementary oligo DNA (cA) modified

with a thiol group may be linked thereto, but is not limited

thereto .

In the biochip and biosensor of the present invention, the

oligonucleotide (gA) which constitutes the protein G conjugate

(gA-G) of the present invention is linked onto the solid support

by complementary binding with the oligonucleotide (cA) on the

surface of the solid support, and the protein G conjugate linked

to the solid support binds with an antibody. The biochip and biosensor of the present invention may be easily fabricated by contacting the protein G conjugate and antibody with the solid

support .

In still another embodiment, the present invention relates

to amethod for analyzing an antigen using the biochip orbiosensor.

The biochip or biosensor of the present invention is one type

of immunosensors, and thus antigen analysis may be performed

by any method using the immunosensor, which is widely known in the art. A surface plasmon resonance-based method may be

preferably used to analyze the antigen.

Hereinafter, the present invention will be described in

detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended

to be limited thereto.

[Mode for Invention]

Example 1 : Protein expression analysis of cysteine-tagged Streptococcal protein G variant

<1-1> Gene preparation of cysteine-tagged Streptococcal

protein G variant

Two primers were prepared in order to tag with cysteine at the N-terminus. In the base sequence of the 5' -primer, an

initiation codon (ATG) was followed by GAT (Asp codon) and TGC

(cysteine codon) , and in order to provide a link to protein G,

GGC GGC GGC GGC AGC (four GIy codons and one Ser codon) were

included. Furthermore, in order to insert the gene into an

expression vector pET21a (Novagen) , the Ndel restriction site was introduced into the N-terminal primer and the Xhol restriction site was introduced into the C-terminal primer. The

Streptococcal genomic gene was obtained, and a polymerase chain reaction (PCR) was performed with the primers. Thus, only the

amino acid regions (Bl [a formhaving 10 initial amino acid residues

cleaved], B2) , which are known as domains to which an antibody binds, were obtained. The obtained fragments were cleaved with

the restriction enzymes, which were the same enzymes as introduced

into each primer. Then, the cleaved fragment was inserted into

the pET21a vector cleaved with Ndel and Xhol restriction enzymes

to prepare a pET-cysl-L-protein G vector. The expression vector

expresses Met at the N-terminus.

5' Primer 1: Sense (SEQ ID NO. 1)

5-GGGAATTCCATATGGATTGCGGCGGCGGCGGCAGCAAAGGCGAAACAACTAC TGAAGCT-3

3' Primer 2: Antisense (SEQ ID NO. 2)

δ-GAGCTCGAGTTCAGTTACCGTAAAGGTCTTAGTC-S

<l-2> Protein electrophoresis of cysteine-tagged Streptococcal protein G variants

E. coli BL21 cells were transformed with the prepared pET-cysl-L-protein G, and cultured with shaking at 37 °C until

an O. D. (optical density, A600 nm) became 0.6. Then, IPTG

(isopropyl β-D-thiogalactopyranoside, total concentration of

1 mM) was added thereto, so as to induce protein expression at

25 ° C After 14 hrs, the E. coli cells were centrifuged, and the

obtained cell pellets were disrupted by sonication (Branson,

Sonifier450, 3 KHz, 3 W, 5 min) to give a total protein solution.

The total protein solution was separated by centrifugation into

a solution of soluble protein fraction and a solution of

non-soluble protein fraction. To purify the protein solution,

a solution of disrupted cells in which the recombinant gene

conjugated with hexahistidine were expressed, was loaded on a column packed with IDA excellulose. The recombinant protein

conjugatedwithhistidine was elutedwith aneluent (5OmM Tris-Cl,

0.5 M imidazole, 0.5 M NaCl, pH 8.0). To purify the obtained protein solution once more, the solution was loaded on a column packed with Q cellulose, and eluted with 1 M NaCl. Then, the

eluted protein solution was dialyzed in PBS (phosphate-buffered

saline, pH 7.4) buffer solution.

For protein electrophoresis, the protein solution obtained in the above was mixed with a buffer solution (12 mM Tris-Cl,

pH 6.8, 5% glycerol, 2.88 mM mercaptoethanol, 0.4% SDS, 0.02%

Bromophenol Blue) and heated at 100 ° C for 5 min, and then the

resultant was loaded on a poly-acrylamide gel, which consisted

of a 1 mm- thick 15% separating gel (pH 8.8, width 20 cm, length

10 cm) covered by a 5% stacking gel (pH 6.8, width 10 cm, length

12.0 cm). Subsequently, electrophoresis was performed at 200

to 100 V and 25 mA for 1 hr, and the gel was stained with a Coomassie

staining solution to confirm the recombinant protein.

The description of lanes in Fig. 3 is as follows;

Lane 1: protein size marker,

Lane 2: total protein of E. coli transformed with plasmid

pET-cysl-L-proteinG,

Lane 3: soluble protein fraction of E. coli transformed

with plasmid pET-cysl-L-protein G,

Lane 4: purified protein by IDA column,

Lane 5: purified protein by Q cellulose column.

Example 2 : Preparation of protein G conjugate (gA-G)

Using Sulfo-SMCC (SuIfosuccinimidyl

4- (N-maleimidomethyl) cyclohexane-1 carboxylate) , an

oligonucleotide (gA) modified with an amine group and a Streptococcal protein G variant tagged with one cysteine were

chemically linked to each other to prepare a protein G conjugate

(gA-G) .

In particular, 60 nmol of the oligo DNA (gA) modified with

an amine group at 5' -end was dissolved in 400 μl of 0.25 M phosphate buffer, and then reacted with 1.5 mg of Sulfo-SMCC (3400 nmol)

dissolved in 75 μl of DMF solution. The mixture was reacted at normal temperature for 1 hr, and then the activated oligo DNA

(gA) was separated from the excess unreacted Sulfo-SMCC using

a binding buffer (20 mM Tris, 50 mM NaCl, 1 mM EDTA pH7.0) by

Sephadex G25 gel filtration. While performing the activation

of oligo DNA, the cysteine tagged-protein G variant was reacted with 20 mM DTT for complete reduction, followed by gel filtration

to remove DTT. Consequently, the obtained cysteine

tagged-protein G variant was immediately reacted with the activated oligo DNA (gA) at normal temperature for 2 hrs.

The residual oligo DNA (gA) which was not linked to the

protein G was separated from the protein G variant and cysteine tagged-protein G conjugate (gA-G) using a His-tagged affinity column (IDA column) . Then, the protein G conjugate (gA-G) was purified using an ion exchange column to remove the unboundprotein

G variant.

The protein G conjugate (gA-G) was separated by

chromatography with two columns (column packed with IDA excellulose, and column packed with anion exchange Q cellulose) ,

and then the protein G conjugate (gA-G) was analyzed by protein

electrophoresis (Native gel, SDS-PAGE) . After protein

electrophoresis, the gels were stained with Gel Red and Coomassie,

which are DNA and protein-specific staining reagents,

respectively. As a result, it was found in a Native gel that the protein G variant-DNA conjugate (gA-G) was specifically linked to the oligomer (cA) having a complementary DNA sequence

to cause a difference in its migration (lane 2 vs lane 3) . Also, the band strength was found to be increased only in the DNA staining .

Therefore, it can be seen that the protein G conjugate (gA-G) was specifically reacted with the complementary oligomer (cA) .

The above results indicate that the protein G variant (G)

and the oligomer (gA) are linked to each other one-to-one in

the prepared protein G conjugate (gA-G) (Fig. 5) .

Example 3 : Fabrication of protein G conjugate (gA-G) -immobilized biosensor and biochip

The oligo DNA (gA) was chemically linked to the one

cysteine-tagged Streptococcal protein G variant , and then reacted

with the surface of gold thin film, on which the oligo DNA (cA)

complementary to oligo DNA (gA) was linked, to fabricate a protein

G conjugate (gA-G) -immobilized biosensor and biochip.

Inparticular, the oligo DNA (cA) was reacted with the surface ofgoldthinfilm, and then changes in the surfaceplasmon resonance signal were measured by means of a surface plasmon resonance

(SPR) -based biosensor to detect the immobilization reaction of

the complementary oligo DNA (gA) , protein G conjugate (gA-G) , and noncomplementary control oligo DNA (gB) in real-time.

As a result, when the noncomplementary control oligo DNA

(gB) was injected, there was little change in the surface plasmon

resonance signal. When the complementary oligo DNA (gA, 7.5 kDa)

was injected, the surface plasmon resonance signal was increased

by 231 RU. When the oligo DNA (gA) -protein G conjugate (gA-G,

21.5kDa) was injected, the surface plasmon resonance signal was increased by 564 RU, indicating that the oligo DNA (gA, 7.5 kDa)

and protein G conjugate (gA-G, 21.5 kDa) were specifically linked

onto the surface of oligo DNA (cA) -immobilized gold thin film.

In addition, the numbers of oligo DNA (gA, 7.5 kDa) and

protein G conjugate (gA-G, 21.5 kDa) linked on the surface (mm 2 )

were calculated. The number of oligo DNA (gA, 7.5 kDa) was 1.8

x 10 10 molecules/mm 2 . The number of protein G conjugate (gA-G,

21.5 kDa) was 1.6» 10 10 molecules/mm 2 , which had a slightly lower

density than the oligo DNA (gA, 7.5 kDa) . The result indicates

that the protein G variant slightly interfered with the complementary reaction of oligo DNA.

However, changes in the surface plasmon resonance signal

were measured by means of a surface plasmon resonance (SPR) -based

biosensor to detect the ability of the protein G conjugate (gA-G) to efficiently bind with an antibody, upon reacting the surface with various antibodies (Fig. 6) .

Example 4 : Detection of antigen using protein 6 conjugate

(gA-G) -immobilized biosensor

Antigen detection was performed using the biosensor which

binds with an antibody via the Streptococcal protein G conjugate

immobilized by complementary reaction of oligo DNA.

In particular, 50 nM protein G conjugate (gA-G) was

immobilized on the surface of complementary oligo DNA

(cA) -immobilized gold thin film for the immobilization time of

10 min and 7 min, and then changes in the surface plasmon resonance

signal weremeasured bymeans of a surface plasmon resonance-based

biosensor to detect the reaction between an antibody (anti-human

Kallikrein 3/PSA antibody, R&D systems, 100 nM) and its antigen

(Recombinant Human kallikrein 3/PSA, 100 nM) .

As a result, when the protein G conjugate (gA-G) was reacted

for 10 min, the surface plasmon resonance signal was increased

by 775 RU. When the protein G conjugate (gA-G) was reacted for 7 min, the surface plasmon resonance signal was increased by 297 RU. When the antibody was reacted with the gA-G immobilized

surface of 775 RU, the surface plasmon resonance signal was

increased by 2440 RU. When the antibody was reacted with the

gA-G immobilized surface of 297 RU, the surface plasmon resonance

signal was increased by 1296 RU. When the antigen was reacted with the antibody of 2440 RU on the gA-G immobilized surface

of 775 RU, the surface plasmon resonance signal was increased

by 435 RU. When the antigen was reacted with the antibody of

1296 RU on the gA-G immobilized surface of 297 RU, the surface

plasmon resonance signal was increased by 231 RU (Fig. 7).

Example 5 : Antibody immobilizationusingproteinGconjugate

linked onto DNA array

The DNA array was fabricated on other surfaces than the

surface of gold thin film, and then antibody was immobilized

using the protein G conjugate linked to DNA (gA, 21.5 kDa) .

In particular, the oligonucleotides (cA and cB) with amine

groups were linked to the epoxy groups on the glass surface, an array was fabricated using a DNA arrayer, and then non-specific

reaction was blocked with BSA. Then, a mixed solution of the

protein G conjugate (gA-G) and antibody labeled with a fluorescent marker (Monoclonal mouse IgG-Cy3 (15OnM) ) were reacted with the

surface, and fluorescent signals were measured using a fluorescent scanner.

As a result, since the protein G conjugate (gA-G) binding

with the antibody binds with the complementary oligonucleotide cA, fluorescence was observed not in the oligonucleotide cB but

in the complementary oligonucleotide cA, indicating that the antibody can be easily immobilized using a DNA array without

non-specific reaction (Fig. 8).

Example 6: Fabrication of antibody-immobilized gold

nano-particle via protein G conjugate (gA-G)

Antibody-immobilized gold nano-particles were fabricated using the protein G conjugate (gA-G) .

In particular, when the complementary oligonucleotide

cA-linked gold nano-particle was linked to gA-G (21.5 KDa) and

gA (7.5KDa) , the gA-G (21.5KDa) -linkedband, which is a relatively upper band, was less migrated than the gA (7.5 KDa) -linked band in the negative gel. In addition, to sufficiently link the

protein G conjugate (gA-G) to cA, two different numbers of

complementary oligonucleotide (cA) were linked to the gold nano-particles (allowed to link with the average number of 21

or 9.5 gA) , the protein G conjugate (gA-G) was linked thereto,

and then antibodies were linked (human IgGs).

As a result, it was found that more numbers of protein G

conjugate (gA-G) and antibody were linked onto the gold nano-particle capable of binding with the average number of 21

gA.

In the present experiment, the gold nano-particle-cA linked

with gA-G (21.5 KDa) was recovered using a centrifuge, and then

any unreacted antibody was removed using the hexahistidine tagged

to the protein variant. Based on the above results, the protein G conjugate (gA-G) is very useful for immobilizing antibodies

on the gold nano-particle (Fig. 9) .

[industrial Applicability]

The protein G conjugate (gA-G) according to the present invention, which is prepared by linking an N-terminal

cysteine-tagged protein G variant with an oligonucleotide via

a linker, binds in a directional manner with oligonucleotide

(cA) on the surface of the solid support of a biosensor, and thus efficiently binds with antibodies, thereby being

satisfactorily used in biochips and biosensors which utilize

antigen-antibody reactions.