JP3047020B1 - Method for producing immobilized protein - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To improve the immobilization yield in a method of immobilizing a protein via a carboxyl terminal carboxyl of a protein by using an amide bond forming reaction via a cyanocysteine residue. SOLUTION: General formula (1) NH ₂ —R ₁ —COOH (wherein R ₁
Represents a chain of arbitrary amino acid residues. In immobilization of the protein represented by the formula (2), NH ₂ —R ₁ —CO—NH—CH (CH
₂ -SCN) -CO-NH-R ₂ -COOH (wherein R ₁ has the above meaning, R ₂ is strongly negatively charged near neutral, and the isoelectricity of the compound of formula (2) A protein having a cyano group represented by the general formula (3) NH ₂ —Y (where Y represents a primary amine) Represents an immobilized carrier having a base.)
To produce an immobilized protein represented by the general formula (4) NH ₂ —R ₁ —CO—NH—Y (wherein R ₁ and Y have the above-mentioned meanings). how to.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an efficient method for producing an immobilized protein.

[0002]

2. Description of the Related Art For the purpose of widespread use of proteins, particularly for repeated use, attempts have been made to combine soluble proteins with insoluble carriers such as agarose gels and use them as immobilized proteins. For example, an apparatus for immobilizing a receptor protein and detecting a ligand recognized by the receptor by a surface plasmon resonance phenomenon (trade name: Biacore), or an immobilized enzyme in which an enzyme protein is bound to an insoluble carrier and the use thereof were used. Enzyme reactors are being manufactured.

[0003] For immobilization of an enzyme, chemical bonding to an insoluble carrier is mainly performed by utilizing the reactivity of the side chain of an amino acid constituting a protein. For example, a cysteine residue has an SH group as a functional group. As the reaction of the SH group, disulfide formation, alkylation, acylation and the like are known, and by utilizing this reactivity, the protein can be immobilized via the side chain of the cysteine residue. Further, the lysine residue has an amino group in a side chain. This amino group can form an amide bond with a carboxyl group using carbodiimide. Similarly, since aspartic acid and glutamic acid have a carboxyl group, an amide bond can be formed with a primary amine using carbodiimide. However, such an immobilization reaction utilizing a functional group of a side chain depends on the amino acid sequence of the protein, and since a protein contains a plurality of amino acids of the same type, an immobilization site cannot be specified. There is a problem that it is not possible to exclude the possibility of immobilization at the location.

In order to solve these problems, an amide bond forming reaction via a cyanocysteine residue (Japanese Patent Application Laid-Open No. 10-4)
A reaction for immobilizing the protein via the carboxy group at the carboxy terminus of the protein has been developed, and means for binding the protein at one position of the carboxy terminus and via the main chain has been developed (see 5798). See Japanese Patent Application No. 10-283669).

[0005] By binding the protein at one position of the carboxy terminus and via the main chain, the reversibility of denaturation can be enhanced, and an immobilized enzyme capable of heat-sterilizing the immobilized protein can be produced. The benefits were obtained.

[0006]

However, the amide bond formation reaction via a cyanocysteine residue is represented by the reaction formula (1) NH ₂ -R-CO-NH-CH (CH ₂ -SCN) -CO-X + NH ₂ -B → NH ₂ -R-CO-NH-B (where R is a chain of any amino acid residues, X is OH or a chain of any amino acid residues or any amino acid residue, NH ₂ -B represents any primary amine compound.)

The amide bond formation reaction is carried out by the reaction formula (2) NH ₂ —R—CO—NH—CH (CH ₂ —SCN) —CO—X + H ₂ O → NH ₂ —R—COOH + Z (formula Wherein, R is a chain of any amino acid residues, X is a chain of OH or any amino acid residue or any amino acid residue, and Z is a 2-iminothiazoline-4-carboxylyl derivative of X.)

[0008] The peptide chain cleavage reaction (GRJaco
bson, MHSchaffer, GRStark, TCVanaman, J. Biol
ogical Chemistry, 248, 6583-6591 (1973 ) refer), and _{(3) NH 2 -R-CO} -NH-CH (CH 2 -SCN) -CO-X → NH 2 -R-CO-NH-C ( CH ₂ ) -CO-X (wherein, R represents a chain of any amino acid residues, and X represents OH or a chain of any amino acid residues or any amino acid residue.)

A thiocyano group represented by the formula
Conversion of cyanocysteine residue to dehydroalanine by elimination reaction (Y.Degani, A.Patchornik, Biochemistr
y, 13, 1-11 (1974)), which caused problems with the reaction yield. That is, the immobilization of the protein using the reaction formula (1) has an advantage that it can be bonded at one position of the carboxy terminus and via the main chain, but has a difficulty in the immobilization yield. Have been.

[0010]

According to the present invention, there is provided a reaction scheme (1)
As a result of intensive studies to improve the reaction efficiency of the amide bond formation reaction represented by the formula, the immobilized carrier represented by the general formula NH ₂ -Y is positively charged at the commonly used reaction pH. By making the isoelectric point of the compound acidic, it is assumed that the adsorption reaction is efficiently performed by ionic interaction as a capture reaction, and the reaction of the amide bond formation reaction represented by the reaction formula (1) is performed by adsorbing the compound on an immobilized carrier. I got the idea that efficiency would increase.

That is, the present invention provides a method for immobilizing a protein sequence represented by the following general formula (1):
A peptide containing a cyanocysteine residue represented by the following general formula (6) and having a sequence obtained by bonding to a peptide having a peptide sequence on the carboxy terminal side that is negatively charged near neutrality: By reacting a protein having a cyano group represented by the formula (2) with an immobilized carrier having a primary amine represented by the following general formula (3),
Provided is a method for efficiently producing an immobilized protein represented by the following general formula (4).

General formula (1) NH ₂ —R ₁ —COOH General formula (2) NH ₂ —R ₁ —CO—NH—CH (CH ₂ —SCN) —CO—NH—R ₂ —COOH General formula (3) ) NH ₂ -Y general formula _{(4) NH 2 -R 1 -CO} -NH-Y general formula _{(6) NH 2 -CH (CH} 2 -SCN) -CO-NH-R 2 -COOH [ wherein, R ₁ is a chain of arbitrary amino acid residues, and R ₂ is any amino acid residue rich in acidic amino acids, which is strongly negatively charged in the vicinity of neutrality and acidicly brings the isoelectric point of the compound of the general formula (2). And Y represents an immobilized carrier having a primary amine as a functional group. ]

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In practicing the present invention, it is necessary to prepare a fusion protein represented by the general formula (2) for immobilization of the protein represented by the general formula (1). The production can be performed using a so-called recombinant DNA technique and a chemical modification method.

That is, a gene encoding the protein represented by the general formula (1) and a general formula (7) NH ₂ —CH (CH ₂ —SH) —CO—NH—R ₂ —COOH (7) R ₂ has the above meaning.) By binding to a gene encoding a peptide sequence represented by the following formula:
Formula _{(8) NH 2 -R 1 -CO} -NH-CH (CH 2 -SH) -CO-NH-R 2 -COOH (8) [ In the formula, R ₁ has the abovementioned meaning, R ₂ Is strongly negatively charged near neutral and is NH ₂ —R ₁ —CO—NH—CH (CH ₂ —SH) —CO—NH—R ₂
-A chain of arbitrary amino acid residues capable of making the isoelectric point of COOH acidic. ]

A gene encoding the fusion protein represented by (1) is prepared, expressed in a host organism such as Escherichia coli, and then the expressed protein is separated and purified. Such a fusion protein can be obtained by a known technique (for example, M. Iwakura et al., J. Bio. Chem. 111: 37-45 (199
Since any person skilled in the art can produce the fusion protein by using (2)), it is apparent that the present invention is not limited by the method for producing the fusion protein represented by the general formula (8). Alternatively, the fusion protein can be produced by a combination of a genetic engineering technique and a conventional protein synthesis technique, or by only a protein synthesis technique.

As R ₂ in the general formula (2) or (8), a sequence containing a large amount of aspartic acid or glutamic acid is preferable. The isoelectric point of a protein depends on the type and number of constituent amino acids. For example, when a large amount of basic amino acids such as lysine and arginine are contained, the number of aspartic acids and glutamic acids exceeding the total number of basic amino acids is required. A person skilled in the art can easily estimate the isoelectric point of a protein by calculation. Preferably, the isoelectric point of the general formula (2) or (8) is a value between 4 and 5,
Design a sequence rich in aspartic acid and glutamic acid.

Among such sequences, alanyl-polyaspartic acid is a preferred sequence. This is because, by making alanine the next amino acid after cyanocysteine, the amide bond formation reaction via the cyanocysteine residue is convenient, and the carboxyl group of aspartic acid is the most acidic in the amino acid side chain. It is. That is, in the embodiment of the present invention, a preferred example of the protein represented by the general formula (8) is represented by the general formula (9): NH ₂ —R ₁ —CO—NH—CH (CH ₂ —SH) —CO—NH—CH _{2 (CH 3) -CO- [NH-} CH (CH 2 -COOH) -CO] n -OH (9)

(Wherein, R ₁ is a chain of arbitrary amino acid residues, and n is a natural number). A preferred example of the protein represented by the general formula (2) is a compound represented by the general formula (5): NH ₂ -R ₁ -CO-NH-CH (CH ₂ -SCN) -CO-NH-CH ₂ (CH ₃ ) -CO- [ NH—CH (CH ₂ —COOH) —CO] _n —OH (5) (wherein, R ₁ and n have the above-mentioned meanings).

The general formula (8) [that is, the above formula NH ₂ -R ₁ -CO-NH-
From CH (CH ₂ —SH) —CO—NH—R ₂ —COOH], the general formula (2) [ie,
The conversion to the above formula NH ₂ —R ₁ —CO—NH—CH (CH ₂ —SCN) —CO—NH—R ₂ —COOH], a so-called cyanation reaction, can be carried out using a cyanation reagent.

As the cyanating reagent, 2-nitro-
5-Thiocyanobenzoic acid (2-nitro-5-thiocyanobennzoic
acid (NTCB)) (Y.Degani, A.Ptchornik, Biochemistry,
13,1-11 (1974)) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (1-cyano-4di
A method using methylaminopyridinium tetrafluoroborate (CDAP)) or the like is simple. Commercially available NTCB and CDAP can be used as they are. Cyanation using NTCB can be efficiently performed between pH 7 and 9, and the reaction efficiency is increased by increasing the absorbance of the released thionitrobenzoic acid at 412 nm (molecular extinction coefficient = 13,600 M ^-1 cm ^-1 ). You can find out. In addition, cyanation of the SH group is described in the literature (J. Wood & Catsipoo
las, J. Biol. Chem. 233, 2887 (1963)).

As the immobilized carrier represented by the general formula (3) "NH ₂ -Y" used in the present invention, any insoluble carrier having a primary amino group can be used. Commercially available carriers having a primary amino group include amino-cellulofine (sold by Seikagaku Corporation) and AF-aminotoyopearl (TOS
OH), EAH-Sepharose 4B and Lysine-Sepharose 4B (sold by Amersham Pharmacia), Porus 20NH (sold by Boehringer Mannheim) and the like are available. Further, a primary amine is introduced into glass beads or the like using a compound having a primary amine as a silane compound (for example, 3-aminopropylmethoxysilane or the like),
It is also possible to use it.

The formula (2) [that is, the above formula NH ₂ -R ₁ -CO-NH-
Reaction of a cyanated fusion protein represented by CH (CH ₂ -SCN) -CO-NH-R ₂ -COOH] with an immobilized carrier represented by the general formula (3) [ie, the above formula NH ₂ -Y] Is under weak alkaline conditions (pH 8-10)
And at room temperature. Under weak alkaline reaction conditions, the cyanated fusion protein represented by the general formula (2) is negatively charged, while the immobilized carrier represented by the general formula (3) is positively charged, and Since they bond to each other, they can be used as a capture reaction of the amide bond formation reaction represented by the reaction formula (1). Since this electrostatic interaction depends on the salt concentration in the solvent, it is preferable that the solvent used has as low a salt concentration as possible. In the embodiment of the present invention, a 10 mM borate buffer, pH 9, is used. However, any condition can be used as long as the salt concentration allows the electrostatic interaction to be properly performed.

Therefore, the solution for performing the immobilization reaction is
Any solvent can be used as long as it is a solvent that guarantees the above electrostatic interaction, dissolves the cyanated fusion protein represented by the general formula (2), and can adjust the pH. Various buffers such as a phosphate buffer and a borate buffer, alcohols such as methanol and ethanol, dimethylformamide, dimethyl sulfoxide and the like can be used.
As for the reaction temperature, a high reaction efficiency can be obtained at room temperature, but the solvent used does not freeze or boil, and the general formula (2)
Can be used without any problem within a temperature range in which the cyanated fusion protein represented by the above does not aggregate as a result of denaturation.

In the reaction involving cyanocysteine used in the present invention, a hydrolysis reaction, that is, the reaction of the above-mentioned reaction formulas (2) and (3) may occur as a side reaction. Since all the reactants generated from the side reaction are dissolved in the solvent, after the reaction, the side reaction product can be removed by washing the immobilized carrier with an appropriate solvent.
Therefore, all the immobilized enzymes produced by the immobilization reaction used in the present invention have the general formula (4) NH ₂ —R ₁ —CO—NH—Y (wherein R ₁ and Y have the above-mentioned meanings) )),
It binds to the immobilization carrier at one position of the carboxy terminal of the target protein.

The characteristic of the immobilized protein thus obtained is that the carboxy terminus is bonded to the carrier at only one place, and the protein functions well. For example, when using an enzyme protein having a catalytic function as the immobilized protein, once denatured by raising the temperature or adding a denaturing agent,
Although the catalytic function is lost, the immobilized enzyme prepared by immobilization according to the present invention can completely regenerate its function by removing the denaturing conditions (Japanese Patent Application No. 10-28366).
No. 9).

In order to show that the protein immobilization method of the present invention is actually effective, the following Examples
It has been demonstrated using 9 mutant E. coli-derived dihydrofolate reductase enzymes (abbreviated as AS-DHFR-G4).

[0027]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it should be understood that the present invention is not limited by these Examples. <Example> For preparation of dihydrofolate reductase for immobilization and immobilization reaction
Effect of ionic interaction on

In this embodiment, AS-DHFR-G4 is used as a protein corresponding to the general formula (1). AS-DHFR-
The amino acid sequence of G4 is shown in SEQ ID NO: 1. In this example, at the carboxy terminus of AS-DHFR-G4, Cys-Ala (abbreviated as CA), Cys-Ala-Asp-Asp (abbreviated as CAD2), Cys-Ala-Asp-A
sp-Asp-Asp (abbreviated as CAD4), Cys-Ala-Asp-Asp-Asp-Asp-As
p-Asp (abbreviated as CAD6), Cys-Ala-Asp-Asp-Asp-Asp-Asp-As
A protein was synthesized by fusing five types of polypeptides, p-Asp-Asp (abbreviated as CAD8), and the relationship between the calculated isoelectric point and the binding to the immobilized carrier having a primary amine was investigated. . Each fusion protein of AS-DHFR-G4 and CA, CAD2, CAD4, CAD6, and CAD8 was converted to AS-DHFR-G4-CA, AS-DHFR-G4-
CAD2, AS-DHFR-G4-CAD4, AS-DHFR-G4-CAD6, and AS-DHF
Abbreviated as R-G4-CAD8.

AS-DHFR-G4-CA, AS-DHFR-G4-CAD2, AS-DHF
The amino acid sequences of R-G4-CAD4, AS-DHFR-G4-CAD6, and AS-DHFR-G4-CAD8 are shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: : 6.
AS-DHFR-G4-CA, AS-DHFR-G4-CAD2, AS-DHFR-G4-CAD4, A
The production of S-DHFR-G4-CAD6 and AS-DHFR-G4-CAD8 was performed using AS-DHF
Genetic engineering was performed using the R gene. Already AS-
DHFR gene is known (M. Iwakura, BE Jones,
J. Luo, & CRMatthews, J. Biochemistry, 117: 480
-488 (1995)).

The nucleotide sequence of the AS-DHFR gene including the promoter sequence and ribosome binding sequence necessary for gene expression is shown in SEQ ID NO: 7. AS-DHFR represented by SEQ ID NO: 7
Is integrated into a plasmid named "pTZDHFR20" (M. Iwakura, BE Jones, J. Luo, &
CR Matthews, J. Biochemistry, 117: 480-488 (19
95)).

Based on this gene sequence, two primers
DNA, 5'-GGGGATCCTC TTGACAATTA GTTAACTATT TGTTATAAT
G TATTC -3 '(SEQ ID NO: 8) and 5'-GGGGATCCCT TATGC
Using ACAGC CACCGCCACC ACGACGCTCG AGGATTTCG-3 ′ (SEQ ID NO: 9) and pTZDHFR20 as a template, amplification was performed by PCR to prepare a gene sequence capable of expressing AS-DHFR-G4-CA. Next, the AS-D produced in this way
Using the HFR-G4-CA gene as a template, SEQ ID NO: 8 and SEQ ID NO:
A gene sequence capable of expressing AS-DHFR-G4-CAD2 was prepared by amplifying by PCR using 10 primer DNAs. Similarly, AS-DHFR-G4-CAD
Using two genes as templates and two primer DNAs of SEQ ID NO: 8 and SEQ ID NO: 11, the AS-DHFR-G4-CAD4 gene was
SEQ ID NO: 8 and SEQ ID NO: 12 using the R-G4-CAD4 gene as a template
AS-DHFR-G4-CAD6 gene using the two primer DNAs, and AS-DHFR-G2 using the two primer DNAs of SEQ ID NO: 8 and SEQ ID NO: 13 using the AS-DHFR-G4-CAD6 gene as a template. G4-CA
The D8 gene was created.

AS-DHFR-G4-CA, AS-DHFR-G4-CAD2, AS-DHF
The nucleotide sequences of the genes of R-G4-CAD4, AS-DHFR-G4-CAD6, and AS-DHFR-G4-CAD8 are shown in SEQ ID NO: 14 and SEQ ID NO: 1
5, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18. After digesting each DNA obtained by amplification with the restriction enzyme BamHI, ligating with the cloning vector pUC19 cut with BamHI, and introducing the obtained recombinant plasmid into Escherichia coli, AS-DHFR-G4-CA, AS -DHFR-G4-CAD2, AS-D
HFR-G4-CAD4, AS-DHFR-G4-CAD6, and AS-DHFR-G4-CAD8
Each could be expressed in E. coli cells and E. coli was transformed to trimethoprim resistance and ampicillin resistance.

The thus obtained Escherichia coli was cultured overnight at 37 ° C. in 3 liters of a medium (containing 15 g of salt, 15 g of yeast extract, 24 g of tryptone, and 30 mg of ampicillin sodium). Then, cells having a wet weight of about 10 g were obtained. The cell-free extract of the cells was treated with streptomycin sulfate, ammonium sulfate fractionation, methotrexate affinity chromatography and DEAE Toyopearl chromatography to purify the protein to homogeneity, and about 100 mg of uniform protein was obtained. AS-DHFR-G4-CA, AS-DHFR-G4-CA
D2, AS-DHFR-G4-CAD4, AS-DHFR-G4-CAD6, and AS-DHFR-
G4-CAD8 was obtained respectively. Each protein concentration was determined from the absorbance at 280 nm using the molecular extinction coefficient of AS-DHFR at 280 nm of 31100 M ^-1 cm ^-1 .

The obtained AS-DHFR-G4-CA, AS-DHFR-G4-CAD
2, AS-DHFR-G4-CAD4, AS-DHFR-G4-CAD6, and AS-DHFR-G
4-CAD8 The cyanation of the cysteine residue at the 164th amino acid in each sequence was performed in 0.1 M Tris-HCl buffer, pH 7.4, containing 5 mM ethylenediaminetetraacetic acid (EDTA).
AS-G4CA (about 0.032 mM) 5-fold (0.16 mM) 2-nitro
The reaction was carried out by adding -5-thiocyanobenzoic acid (NTCB) and reacting at room temperature for 4 hours. Increase in the absorbance at 412 nm of the released thionitrobenzoic acid (molecular extinction coefficient = 13,600 M ^-1 cm
^-1 ), it was confirmed that cysteine residues were almost cyanated.

Unreacted NTCB and thionitrobenzoic acid were removed from the cyanation reaction solution by gel filtration using a Sephadex G50 column (column size: φ25 × 150 mm). As an eluent, 10 mM borate buffer, pH 9.5, containing 5 mM ethylenediaminetetraacetic acid (EDTA) was used. The cyanated protein thus obtained was used immediately for the immobilization reaction.

AS-D in which the obtained cysteine is cyanated
HFR-G4-CA, AS-DHFR-G4-CAD2, AS-DHFR-G4-CAD4, AS-DH
About 20 mg each of FR-G4-CAD6 and AS-DHFR-G4-CAD8 (about
10 ^-8 moles) using, sold amino-Cellulofine (Seikagaku of 5 ml; amine content, mixed with about 10 ^-5 moles NH ₂ / ml gel), the free protein content immediately after mixing was measured. To measure the amount of free protein, centrifuge the reaction solution at 1,000 rpm for 2 minutes,
The measurement was performed by measuring the absorbance at 280 nm of the supernatant. Immediately after the mixing, the immobilization reaction hardly proceeds, so it is considered that the amount of protein obtained by subtracting the amount of free protein from the amount of input protein was bound to aminocellulofine by ionic interaction. Thereafter, the immobilization reaction was performed at room temperature for 24 hours while gently mixing. To this was added KCl to a final concentration of 1 M, and the amount of free protein was measured.
By adding KCl to a final concentration of 1M, proteins bound only by ionic interaction will be released from aminocellulofine, and the amount of input protein minus the amount of free protein will be fixed. It was determined as the amount of protein converted.

The correctness of the estimation of the amount of the immobilized protein thus obtained was confirmed as follows. Each of the obtained immobilized enzymes was packed in a column, and once passed through a 4 M guanidine hydrochloride solution to completely wash away non-specifically bound proteins, and then equilibrated with 10 mM phosphate buffer, pH 7.0. The immobilized protein denatured by guanidine hydrochloride treatment was completely regenerated. This is DHFR
Is a potent inhibitor of methotrexate (MTX) (0.1 mM)
After binding to the immobilized protein through 10 mM phosphate buffer containing 1 M KCl, pH 7.0, non-specifically bound MT
X was washed out, and then the immobilized protein was denatured again by flowing a 4 M guanidine hydrochloride solution, and bound.
The MTX is eluted, the eluted MTX is recovered, the amount of the recovered MTX is measured, and the amount of the recovered MTX is determined (molecular extinction coefficient of MTX: 22100 M ^-1 cm ^-1 ). The amount was measured. The above results are summarized in Table 1.

[0038]

[Table 1]

The results show that the amount of free protein reduced immediately after mixing with aminocellulofine as the immobilization carrier almost coincides with the amount of immobilized protein. Suggest that the ionic bond of can be used as a capture reaction.
To confirm this, the concentration of the borate buffer used was increased to 50 mM, and the immobilization efficiency was measured as described above. The immobilization efficiency (%) is given by 100 × immobilized protein amount / input protein amount.

Table 2 compares the immobilization efficiencies when using 10 mM and 50 mM borate buffers. At the same time,
The isoelectric point of each protein determined from the amino acid composition, and the charge at pH 7 are also shown.

[0041]

[Table 2]

As is clear from Table 2, the immobilization efficiency is
Introducing neutral to alkaline, negatively charged polyasparagine at the carboxy terminal side has been shown to significantly improve. Also, increasing the concentration of the borate buffer (ie, the salt concentration) lowers the immobilization efficiency, but it is clear that longer polyasparagine is less affected by the salt concentration. This indicates that the decrease in free protein immediately after mixing is mainly due to ionic interaction, and that the "capture reaction due to ionic interaction" intended in the present invention certainly works.

[0043]

According to the present invention, the target protein can be immobilized via the carboxy group at the carboxy terminus in a high yield of about 70% of the input amount. Already Patent Application 10-283669
As described in the above item, the immobilized protein immobilized via the carboxy group at the carboxy terminus can exhibit all its functions. Then, the original function can be completely recovered, that is, the denaturation can be completely reversible, and it is of high practical value.

[0044]

[Sequence List] SEQUENCE LISTING <110> DIRECTOR-GENERAL OF AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY <120> PROCESS FOR PREPARING IMMOBILIZED PROTEINS <130> 11900258 <160> 18 <170> Windows 95 <210> 1 <211> 163 <212 > PRT <213> Escherichia coli <400> 1 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Ala Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Ser Phe Glu Ile Leu Glu Arg Arg Gly 145 150 155 160 Gly Gly Gly <210> 2 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Designated is an amino acid sequence of AS-DHFR-G4-CA. <400> 2 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Ala Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Ser Phe Glu Ile Leu Glu Arg Arg Gly 145 150 155 160 Gly Gly Gly Cys Ala 165 <210> 3 < 211> 167 <212> PRT <213> Artificial Sequence <220> <223> Designated is an amino acid sequence of AS-DHFR-G4-CAD2. <400> 3 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Ala Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Ser Phe Glu Ile Leu Glu Arg Arg Gly 145 150 155 160 Gly Gly Gly Cys Ala Asp Asp 165 <210> 4 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> Designated is an amino acid sequence of AS- DHFR-G4-CAD 4. <400> 4 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Ala Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Ser Phe Glu Ile Leu Glu Arg Arg Gly 145 150 155 160 Gly Gly Gly Cys Ala Asp Asp Asp Asp 165 <210> 5 <211 > 171 <212> PRT <213> Artificial Sequence <220> <223> Designated is an amino acid sequence of AS-DHFR-G4-CAD6. <400> 5 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala A sp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Ala Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Ser Phe Glu Ile Leu Glu Arg Arg Gly 145 150 155 160 Gly Gly Gly Cys Ala Asp Asp Asp Asp Asp Asp 165 170 <210> 6 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Designated is an amino acid sequence of AS-DHFR-G4-CAD8. <400> 6 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Ala Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Ser Phe Glu Ile Leu Glu Arg Arg Gly 145 150 155 160 Gly Gly Gly Cys Ala Asp Asp Asp Asp Asp Asp Asp Asp 165 170 <210> 7 <211> 554 <212> DNA <213> Escherichia coli <400> 7 ttgacaatta gttaactatt tgttataatg tattcatgag cttaactaac taatccggaa 60 aaggaggaac ttccatgatc agtctgattg cggcgctagc ggtagatcgc gttatcggca 120 tggaaaacgc catgccatgg aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct 180 taaataaacc cgtgattatg gggcgccata cctgggaatc aatcggtagg cctttgcccg 240 gccgcaaaaa tattatcctc agcagtcaac c cgggaccga tgatcgggtt acctgggtta 300 aatcggtcga cgaagccatc gcggccgcag gtgacgtacc agaaatcatg gtgattggcg 360 gcggacgcgt ttatgaacag ttcttgccaa aagcgcaaaa gctttatctg acgcatatcg 420 atgcagaagt ggaaggcgac acccattttc cggattacga gccggatgac tgggaatcgg 480 tattcagcga attccacgat gctgatgcgc agaactcgca tagctattcg ttcgaaatcc 540 tcgagcgtcg ttaa 554 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Designated is a primer for the amplification of AS-DHFR-G4-CA gene. <400> 8 ggggatcctc ttgacaatta gttaactatt tgttataatg tattc 45 <210> 9 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Designated is a primer for the amplification of AS-DHFR-G4-CA gene. <400> 9 ggggatccct tatgcacagc caccgccacc acgacgctcg aggatttcg 50 <210> 10 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Designated is a primer for the amplification of AS-DHFR-G4-CAD2 gene. <400> 10 ggggatccct taatcatctg cacagccacc gccaccacga cgctcgagga tttcg 55 <210> 11 <211> 55 <212> DNA < 213> Artificial Sequence <220> <223> Designated is a primer for the amplification of AS-DHFR-G4-CAD4 gene. <400> 11 ggggatccct taatcatcat catctgcaca gccaccgcca ccacgacgct cgagg 55 <210> 12 <211> 55 <212> DNA <213> Artificial Sequence < 220> <223> Designated is a primer for the amplification of AS-DHFR-G4-CAD6 gene. <400> 12 ggggatccct taatcatcat catcatcatc tgcacagcca ccgccaccac gacgc 55 <210> 13 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Designated is a primer for the amplification of AS-DHFR-G4-CAD8 gene. <400> 13 ggggatccct taatcatcat catcatcatc atcatctgca cagccaccgc caccacgacg 60 <210> 14 <211> 651 <212> DNA <213 > Artificial Sequence <220> <223> Designated is a nucleotide sequence of AS-DHFR-G4-CA gene. <400> 14 ggggatcctc ttgacaatta gttaactatt tgttataatg tattcatgag cttaactaac 60 taatccggaa aaggaggaac ttccatgatc agtctgattg cggcgctagc ggtagatcgc 120 gttatcggca tggaaaacgc catgccatgg aacctgcctg ccgatctcgc ctggtttaaa 240 cgcaacacct taaataaacc cgtgattatg gggcgccata cctgggaatc aatcggtagg 300 cctttgcccg gccgcaaaaa tattatcctc agcagtcaac ccgggaccga tgatcgggtt 360 acctgggtta aatcggtcga cgaagccatc gcggccgcag gtgacgtacc agaaatcatg 420 gtgattggcg gcggacgcgt ttatgaacag ttcttgccaa aagcgcaaaa gctttatctg 480 acgcatatcg atgcagaagt ggaaggcgac acccattttc cggattacga gccggatgac 540 tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactcgca tagctattcg 600 ttcgaaatcc tcgagcgtcg tggtggcggt ggctgtgcat aagggatccc c 651 <210> 15 <211> 657 < 212> DNA <213> Artificial Sequence <220> <223> Designated is a nucleotide sequence of AS-DHFR-G4-CAD2 gene. <400> aacctgcctg ccgatctcgc ctggtttaaa 240 cgcaacacct taaataaacc cgtgattatg gggcgccata cctgggaatc aatcggtagg 300 cctttgcccg gccgcaaaaa tattatcctc agcagtcaac ccgggaccga tgatcgggtt 360 acctgggtta aatcggtcga cgaagccatc gcggccgcag gtgacgtacc agaaatcatg 420 gtgattggcg gcggacgcgt ttatgaacag ttcttgccaa aagcgcaaaa gctttatctg 480 acgcatatcg atgcagaagt ggaaggcgac acccattttc cggattacga gccggatgac 540 tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactcgca tagctattcg 600 ttcgaaatcc tcgagcgtcg tggtggcggt ggctgtgcag atgattaagg gatcccc 657 <210> 16 <211> 663 <212> DNA <213> Artificial Sequence <220> <223> Designated is a nucleotide sequence of AS-DHFR-G4-CAD4 gene. <400> 16 ggggatcctc ttgacaatta gttaactatt tgttataatg tattcatgag cttaactaac 60 taatccggaa aaggaggaac ttccatgatc agtctgattg cggcgctagc ggtagatcgc 120 gttatcggca tggaaaacgc catgccatgg aacctgcctg ccgatctcgc ctggtttaaa 240 cgcaacacct taaataaacc cgtgattatg gggcgccata cctgggaatc aatcggtagg 300 cctttgcccg gccgcaaaaa tattatcctc agcagtcaac ccgggaccga tgatcgggtt 360 acctgggtta aatcggtcga cgaagccatc gcggccgcag gtgacgtacc agaaatcatg 420 gtgattggcg gcggacgcgt ttatgaacag ttcttgccaa aagcgcaaaa gctttatctg 480 acgcatatcg atgcagaagt ggaaggcgac acccattttc cggattacga gccggatgac 540 tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactcgca tagctattcg 600 ttcgaaatcc tcgagcgtcg tggtggcggt ggctgtgcag atgatgatga ttaagggatc 660 ccc 663 <210> 17 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> Designated is a nucleotide sequence of AS-DHFR-G4-CAD6 gene. <400> 17 ggggatcctc ttgacaatta gttaactatt tgttataatg tattcatgag cttaactaac 60 taatccggaa aaggaggaac ttccatgatc agtctgattg cggcgctagc ggtagatcgc 120 gttatcggca tggaaaacgc catgccatgg aacctgcctg ccgatctcgc ctggtttaaa 240 cgcaacacct taaataaacc cgtgattatg gggcgccata cctgggaatc aatcggtagg 300 cctttgcccg gccgcaaaaa tattatcctc agcagtcaac ccgggaccga tgatcgggtt 360 acctgggtta aatcggtcga cgaagccatc gcggccgcag gtgacgtacc agaaatcatg 420 gtgattggcg gcggacgcgt ttatgaacag ttcttgccaa aagcgcaaaa gctttatctg 480 acgcatatcg atgcagaagt ggaaggcgac acccattttc cggattacga gccggatgac 540 tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactcgca tagctattcg 600 ttcgaaatcc tcgagcgtcg tggtggcggt ggctgtgcag atgatgatga tgatgattaa 660 gggatcccc 669 <210> 18 <211> 675 <212> DNA <213> Artificial Sequence <220> <223> Desig nated is a nucleotide sequence of AS-DHFR-G4-CAD8 gene. <400> 18 ggggatcctc ttgacaatta gttaactatt tgttataatg tattcatgag cttaactaac 60 taatccggaa aaggaggaac ttccatgatc agtctgattg cggcgctagc ggtagatcgc 120 gttatcggca tggaaaacgc catgccatgg aacctgcctg ccgatctcgc ctggtttaaa 240 cgcaacacct taaataaacc cgtgattatg gggcgccata cctgggaatc aatcggtagg 300 cctttgcccg gccgcaaaaa tattatcctc agcagtcaac ccgggaccga tgatcgggtt 360 acctgggtta aatcggtcga cgaagccatc gcggccgcag gtgacgtacc agaaatcatg 420 gtgattggcg gcggacgcgt ttatgaacag ttcttgccaa aagcgcaaaa gctttatctg 480 acgcatatcg atgcagaagt ggaaggcgac acccattttc cggattacga gccggatgac 540 tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactcgca tagctattcg 600 ttcgaaatcc tcgagcgtcg tggtggcggt ggctgtgcag atgatgatga tgatgatgat 660 gattaaggga tcccc 675

[0045]

[Sequence List Free Text]

SEQ ID NO: 2 shows the amino acid sequence of AS-DHFR-G4-CA. SEQ ID NO: 3 shows the amino acid sequence of AS-DHFR-G4-CAD2. SEQ ID NO: 4: Shows the amino acid sequence of AS-DHFR-G4-CAD4. SEQ ID NO: 5: Shows the amino acid sequence of AS-DHFR-G4-CAD6. SEQ ID NO: 6: Shows the amino acid sequence of AS-DHFR-G4-CAD8. SEQ ID NO: 8: shows a primer for amplifying AS-DHFR-G4-CA gene. SEQ ID NO: 9 shows a primer for amplifying AS-DHFR-G4-CA gene. SEQ ID NO: 10 shows a primer for amplifying AS-DHFR-G4-CAD2 gene. SEQ ID NO: 11 shows a primer for amplifying AS-DHFR-G4-CAD4 gene. SEQ ID NO: 12: This shows a primer for amplifying AS-DHFR-G4-CAD6 gene. SEQ ID NO: 13 shows a primer for amplifying AS-DHFR-G4-CAD8 gene. SEQ ID NO: 14: shows the nucleotide sequence of AS-DHFR-G4-CA gene. SEQ ID NO: 15: shows the nucleotide sequence of AS-DHFR-G4-CAD2 gene. SEQ ID NO: 16: shows the nucleotide sequence of AS-DHFR-G4-CAD4 gene. SEQ ID NO: 17: shows the nucleotide sequence of AS-DHFR-G4-CAD6 gene. SEQ ID NO: 18: shows the nucleotide sequence of AS-DHFR-G4-CAD8 gene.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 17/00 C12N 11/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] _1. An immobilization of a protein represented by the general formula (1) NH ₂ —R ₁ —COOH (1) (wherein R ₁ represents a chain of arbitrary amino acid residues). 2) NH ₂ —R ₁ —CO—NH—CH (CH ₂ —SCN) —CO—NH—R ₂ —COOH (2) wherein R ₁ has the above meaning and R ₂ is neutral A protein having a cyano group which is strongly negatively charged in the vicinity and represents a chain of any amino acid residue rich in acidic amino acids, which acidifies the isoelectric point of the compound of formula (2). equation _{(3) NH 2 -Y (3} ) ( in the formula, Y represents. a carrier for immobilization having a primary amine as a functional group) by reaction with the immobilized carrier represented by the general formula (4) NH ₂ -R ₁ -CO-NH-Y (4) A method for producing an immobilized protein represented by the formula ( ₁ ) wherein R ₁ and Y both have the above-mentioned meanings. 2. The protein represented by the general formula (2) is represented by the general formula (5): NH ₂ —R ₁ —CO—NH—CH (CH ₂ —SCN) —CO—NH—CH ₂ (CH ₃ ) —CO— [NH-CH (CH ₂ -COOH) -CO] _n -OH (5) (wherein, R ₁ has the above-mentioned meaning and n represents a natural number.) The method of claim 1, wherein: