JPH03164178A - Protein containing human iga binding region and production thereof - Google Patents

Abstract

NEW MATERIAL:A protein containing amino acid sequence expressed by the formula and having immunoglobulin-A(IgA) binding activity. USE:Used as IgA detecting reagent and remedy of autoimmune disease owing to human IgA. PREPARATION:For instance, plasmid containing gene coding binding region of protein Arp4 having IgA binding activity expressed by the formula is cut with suitable restricting enzyme and each cut fragment is bonded to suitable vector to prepare recombinant plasmid. Next, said plasmid is inserted into host such as Escherichia coli and transformed, then the resultant transformant is cultured, thus gene is manifested. Next, a solution containing produced material is purified by an anion-exchange column chromatography and gel column chromatography to afford the aimed protein having IgA binding activity containing amino acid sequence expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a protein having an affinity for human immunoglobulin A (hereinafter abbreviated as [gA)]. A r p 4
The present invention relates to a protein containing one or more IgA binding regions of a protein called Arp4 (hereinafter abbreviated as Arp4), a gene for the protein, and the production of the protein using a genetically recombinant protein.

[Conventional technology and issues to be solved]

Protein Arp4 is a membrane protein present on the membrane surface of group A hemolytic streptococcus AP4 strain, and specifically binds to human IgA. The Arp4 gene has been cloned and its entire nucleotide sequence has been determined (Mo1, Vic
robiol. 3 1111-1119 (1989
))． According to this, Arp4 is a protein consisting of 386 amino acids, and the N-terminal 41 amino acids are a signal sequence necessary for protein secretion. excluding this signal sequence,
Mature Arp4 has a molecular weight of 395 and consists of 345 amino acids.
There are 44 proteins. The amino acid sequence, especially the latter half of the C-terminus, shows similarity to the M protein present on the membrane of group A hemolytic streptococci. Furthermore, in the central region, there are three repeating amino acid sequences each consisting of 54 amino acids. This Arp4 gene can also be expressed in E. coli, and continuous production in E. coli is also possible, for example, by linking it to an appropriate plasmid. In that case, the resulting Arp4 protein accumulates in large quantities in the periplasm, which is the space between the inner and outer membranes (Mo I. Microbiol. 3
1111-1119 (1989)).

Therefore, large amounts of Arp4 can be obtained relatively easily in E. coli. Fields of use for A'p4 obtained in this way include its use as a research reagent for binding, separation, and identification of human IgA, as well as the use of IgA from the blood of patients with autoimmune diseases whose main cause is human IgA. Possible uses include absorbing and removing water.

When used in such applications, one of the important factors is the binding capacity of Arp4 to IgA. That is, the binding molar ratio expressed as the ratio of the number of molecules of IgA binding to one molecule of Arp4, or the unit of it. A
The binding weight ratio, expressed as the weight of IgA bound to r p 4, greatly influences the removal efficiency of IgA from, for example, blood. Although it is expected that the removal efficiency will improve as the coupling ratio increases, no attempt has been made to date to improve the coupling capacity by using Arp4 and improving it.

In addition, protein engineering efforts have not yet been undertaken, such as extracting the [gA-binding region of Arp4 and fusing it with other proteins to impart new functions. It goes without saying that the identification of the IgA binding region on the Arp4 protein is essential as a prerequisite for starting such an attempt.

[Means for Solving the Problem] Under these circumstances, the present inventors used genetic engineering techniques to create various derivatives of Arp4, and their [g. l
In the process of examining I-binding activity, we succeeded in improving Arp4. That is, as a result of producing various derivatives of the Arp4 gene and examining their IgA binding activity, it was found that Arp4
The region to which IgA binds on the protein was identified. Based on this result, a mutant Arp with two IgA binding regions was found.
We created 4S and found that the binding molar ratio of this protein was improved.

This also indicates that the isolated IgA-binding region has a function as it is, and provides a means to create a new protein with a new function by fusing it with another protein7. The present invention provides polypeptides with IgA binding activity. As an example,
A polypeptide consisting of 70 amino acids derived from A r p 4 and having the amino acid sequence shown below and a gene encoding the same are provided.

G 1 uLysAspProG 1 nTyrArg
A 1 aLeuMetG 1yG1 uAsnG 1
nAspLeuArgLygArgG 1 uG 1
yG 1 nTyrG I nAspLys I le
G l uG 1 uLauCl ul,ysG1 u
ArgLysG 1 uLysG 1nG 1 uAr
gG 1nG l uG 1nLeuG IuArgG
1nTyrG In [1eG1uA1aAspLy+
tH i sTyrG1 nG 1 uG1 nG I
nLy+tLys! IjsG1nG1nGluG1nG
In addition, the present invention provides a protein containing the amino acid sequence, a gene corresponding to the amino acid sequence, a recombinant plasmid or virus containing the gene, and a recombinant carrying such a recombinant plasmid. For example, it provides a recombinant that uses E. coli as a host. The present invention further provides a method for producing a protein having IgA binding activity, which comprises expressing a gene for a protein containing an amino acid sequence having gene binding activity in a host cell, and recovering the produced protein. It is something. More preferably, a method for producing a protein having IgA binding activity using E. coli DHI or JM109 as the host cell is provided. According to the invention. Proteins with IgA binding activity are
It can be manufactured as follows.

■ Create IgA containing one or more nucleotide sequences encoding the protein Arp4 binding region; ■ Incorporate this [gA into a plasmid or viral vector that can stably exist in the intended host cell; ■ This recombinant plasmid or The phage DNA is introduced into host cells, the recombinant obtained in
Collect proteins with binding activity.

The present invention will be explained step by step below.

l. Creation of a repeat mutant + repeat mutant with two gA binding regions 1 Cut the DNAW encoding the gA binding region with an appropriate restriction enzyme, connect multiple pieces of this, and then insert into IgA encoding an appropriate protein. This is possible. A specific example in which Arp4 itself is used as the protein to be inserted is shown in Examples.

2. Obtaining repeating mutant protein 1. E. coli can be transformed with the plasmid containing the duplicate mutant obtained in E. coli, and the duplicate mutant can be produced in E. coli. In this case, various known plasmids and viral DNAs can be used as vectors for the duplication mutant 1gA. Furthermore, various subspecies of the Kl2 strain can be used as hosts for E. coli. After culturing such a transformed strain, the produced repeat mutant can be isolated and purified by conventional column chromatography. An example of the purification of duplicate mutants by anion exchange and gel filtration is provided in the Examples.

3. Evaluation of IgA binding activity of duplicate mutant proteins The IgA binding activity of duplicate mutant proteins can be evaluated using various immunobiochemical parameters. For example, binding constant (affinity constant), binding specificity, binding capacity, etc.
These can be determined by known methods. (E. h
ae low et al., “Ant'ibodies” (19
88), Cold Spiring Harbor, or Experimental Procedures Volumes 1-13, Japanese Society of Immunology).

Example 5 shows a specific example in which the binding capacities of a protein with one IgA binding region and a protein with two IgA binding regions were determined and compared.

[Effect of the invention 1] By identifying the IgA-binding region, a new protein with added IgA-binding activity can be created by cutting out this region alone and fusing it with another protein using IgA engineering. For example, if a certain type of enzymatic activity is imparted, lgA can be easily detected using this enzymatic activity.

(2) IgA binding activity can be improved by having multiple identified IgA binding regions present in the same protein. For example, by increasing the binding capacity, it becomes possible to more effectively remove 1 gA from the blood, which is considered to be the intended use of the protein, and this has great economic benefits.

■ By creating a protein containing only the gA-binding region, it is also possible to impart high 1gA-binding activity.

It has the same effect as ■.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
It goes without saying that the present invention is not limited to this.

Example A "4 Deletion...Bam containing Arp4 gene"11-HindlII
The 2.2 kb fragment was inserted into plasmid pUcl8 (C. Ya
Nisch-Perron et al., Gene. Volume 33 (1
The structure of plasmid pARP401 prepared by inserting it into the BamHl/HindIII site of 985), 103) is shown in Figure 1 (E. Frijhz et al., Mol. Mi
crobiol. ,3. (1989). 1111
1119). To 2 μg of this Arp401 DNA, 10 units of restriction enzymes Kpnl and Bam were added, and TA buffer y-(33mM Tris acetate (pH 7.9)/66mM
M potassium acetate/lomM magnesium acetate/0.5m
The reaction was carried out at 37° C. for 2 hours in 30 ul (MDTT). After adding and mixing an equal amount of phenol and chloroform (1:1 mixture), the mixture was centrifuged and the upper layer was collected.

Add twice the amount of cold ethanol to this and -20°0130
After a few minutes, the mixture was centrifuged and the DNA was collected as a precipitate.

By performing various enzyme treatments on this DNA, Bam
H! Kilo sequencing deletion kit (
(Takara Shuzo) was used to introduce the deletion. Then these D
NA was transformed into E. coli JM109.

After selecting ampicillin-resistant colonies and culturing, plasmid DNA was extracted by alkaline elution method, and restriction enzyme Ec
oRI. After digestion with HindI, it was subjected to agarose gel electrophoresis. (Maniatis et al. (mentioned above)
). DN after ethidium bromide staining
DNA having a deletion mutation was selected from the pattern of the A fragment. These DNAs were denatured with 0.2N sodium hydroxide, recovered by ethanol filtration, and then their base sequences were decoded by the tune terminator method using dideoxynucleotides using a Sequence kit (Toyobo). The obtained nucleotide sequence was compared with the entire nucleotide sequence of the Arp4 gene (E. Frithz et al. (mentioned above)), and the deleted nucleotide sequence was identified.

All of these deletion mutants are N-terminal deletions. Among them, those fused with the β-galactonase gene on the vector pucta were selected after examining their base sequences. Their expression in E. coli is controlled by the promoter and SD sequence of the β-galactosidase gene (C. Yanisch-Perronn et al. (mentioned above)). Five such mutants were selected and pLAC/. A
It was named RPi5. Furthermore, a mutant in which the central region of the Arp4 protein was deleted was created as follows.

Add 2 μg of pARp401 DNA to the restriction enzyme Pstl.
The reaction was carried out at 37°C for 2 hours at 10 units in 20 μITA buffer, and the decomposition products were electrophoresed on a low melting point agarose gel (Bethesda Research). 5
The gel containing the kb fragment was taken out, kept at 65°C for 30 minutes to melt the gel, and an equal amount of water-saturated phenol was added and mixed. The upper layer was collected, and further ethanol precipitated.
NA was collected. pUc18 2 μg pARP40
As in 1, Pstl digestion was performed, phenol/chloroform extraction, and ethanol/l were collected on the lees. This pUc1
8 Pstl decomposition product and the Pstl-Pstl l.
0.1 μg of each 5 kb fragment was mixed and religated using a T4 DNA ligase kit (Takara Shuzo). This DNA was transformed into Escherichia coli JMl09, white colonies resistant to ampicillin were selected in the presence of isoprobyl thiogalactoside (IPTG) and Xgal, the plasmid DNA was collected, digested with Pstl, and analyzed by agarose gel electrophoresis. A 'p4 gene-derived Pstl
-Pst[l. Plasmid p with 5kb fragment inserted
pARP420R in which the ARP420 or Pstl fragment was inserted in the opposite direction was obtained. After pARP420 was digested with restriction enzymes Sph[, Stul, deletions were introduced by the method described above. As a result, a mutant in which the C-terminal side of the Arp4 gene is deleted is obtained. The C-terminal side of this deletion contains Hin
dIII sites are present. In addition, separately pARP401
is cut at the center of the Arp4 gene with the restriction enzyme Nru[, and religated using a T4 DNA ligator kit (Takara Shuzo) in the presence of a Hind linker (Takara Shuzo). After converting the Nrul site into the old ndl[[[ site, the Nrul site was converted into the old ndlI[-H
indllI, 1. Okb is isolated and recovered, and introduced into the old nd■ site of the aforementioned deletion mutant. By decoding the nucleotide sequence from among the deletion mutants that had been humiliated in this way, we selected a mutant in which the nucleotide sequence corresponding to amino acids 113-218 of the Arp4 protein was deleted, and pARP- It was named 105. In addition, the pARP42
After digesting 0R with the restriction enzymes Kpnl and Bam, by introducing a deletion from the C-terminal side of the Arp4 gene and recombining it using the deletion kit, a joint stop codon (universal terminator, Pharmacia) is present. A plasmid pARP420D was obtained in which protein synthesis was stopped at the deleted end. This plasmid encodes only the first half of the N-terminal side of the Arp4 protein.

An E. coli JMl09 transformant carrying a deletion mutant of the Arp4 gene obtained by further manipulation of the genetic binding region of the deletion mutation was treated with 100 μg/d of ampicillin (pLAC/, 1 mM in the case of ARPI-5).
(Also includes IPTG) L medium (10g/l Bactotryptone, 5g#' yeast extract, 5g/l NaCl
(pH 7.2)) and was cultured with shaking at 37°C and grown to the stationary phase. Collect the bacterial cells in culture solution 1-1 and add them to 0.3-sample buffer 1 (62.5mMT Tris-HCl (pH 6).
．． 8). After suspending in 2X SDS, 5X 2-merfy ethanol), dissolve by treating at 100'C for 2 minutes, and dissolve 20Itl in laemmli? Natu
re, 227, 680 (1970)) +: Jutte, SOS presence, 10-20X nopolyacrylic electrophoresis (hereinafter abbreviated as SOS-PAGE)-containing protein, and Coomassie Brilliant. After staining with blue, the contained proteins were detected. In addition, the gel obtained as described above was separately layered on a nitrocellulose filter, and electricity was applied (IOOV, 1 hour) in a methanol solution containing 25 mM Tris/192 mM glycine (8.3)/20 to transfer the protein to the filter. did. The gene-binding activity of the protein on this filter was detected by enzyme-antibody staining using human gene (Kappel) and alkaline phosphatase-labeled anti-1gA antibody (KPL).

In this way, the Ig of the Arp4 protein deletion mutant
A was investigated and the results are shown in Figure 2.

As shown in FIG. 2, the IgA binding region of the Arp4 protein was found to exist in an amino acid sequence consisting of 70 amino acids at positions 43-1.12.

In addition, among the Arp4 protein deletion mutants that were created, [g
The one with the smallest molecular weight that has A-binding activity is pAR
This is a protein produced by P420D. This protein is derived from ArgLeu1, which is derived from the amino acid sequence 1-165 of protein Arp4 and a synthetic stop codon.
1eAsn is attached to the C-terminus.

Duplication mutant Arp4S, a duplication mutant with two units of Ar4' l g A-binding region
was prepared as follows. pARP40 1D
After restriction of NA with restriction enzyme Stu I, Sac I
A linker (8mer, Takara Shuzo Co., Ltd.) was present, and a Sac I linker was attached using a T4 DNA ligase kit (Takara Shuzo Co., Ltd.) to convert the Stu ■ site into a Sac I site. This DNA was digested with Sac T, and the resulting Sac r
-0 of Sac I. The 3 Kb fragment was separated and recovered by low melting point agarose gel electrophoresis. This fragment encodes amino acids 28-132, which include the IgA-binding region of the Arp4 protein. On the other hand, pARP401 was digested with restriction enzymes EcoR I and BamH I, and sticky ends were changed to blunt ends using Klenow fragment (Takara Shuzo).
pARP4 by religating with T4 DNA ligase
Sac I present in the vector pucl8 part of 01
After cutting at the above Sac I-Sac I site and removing the 5° phosphate group with E. coli alkaline phosphatase (Takara Shuzo), T in the presence of a 3 Kb fragment
4 DNA ligase, and 0.3 of Sac I-Sac I was added to the Sac I site on the Arp4 gene.
A plasmid pARP401s into which the Kb fragment was inserted was constructed. pARP401s contains the 27-
A mutant Arp4S in which the 132nd amino acid is duplicated is encoded. Arp4S protein is SOS-PAG
Molecular weight s. Indicates sooo.

A'4, Arp4 manufactured by Ar4S, plasmid pAR containing Arp4S IgA
P401, pARP401s was infected with E. coli D}II (D.
Hanahann. J. Mol. Biol. 1
66, 557 (1983)), and the transformant was cultured with shaking in 3f, LB medium at 37°C until reaching the stationary growth phase, and then the bacterial cells were collected. 12-14 g of wet bacterial cells were obtained from 3 liters of culture solution. Osmotic shock (Nossal & Heppel)
, J. B. C. ．． Shadow 11. 3055 (1966)
), the periplasmic fraction was collected. Ar in this fraction
p4, Arp4S protein is included.
Confirmed with PAGE. Next, this fraction was added to buffer 7-A (1
Anion exchange column (Q Sepharose (Pharmacia)), diluted 5 times with 0mM Tris-HCl (pH 7.6) and equilibrated with buffer A, diameter 1.0cm x 8. Ocm)
was adsorbed to. 0-20X Buffer B (10mM
T Tris-HCl (pH 7.6)/0.5M NaCI)
Elution was performed using a concentration gradient of 30 minutes. SOS-PA
The eluted fractions were confirmed by GE, and the fractions containing Arp4 and Arp4S proteins were concentrated by ultrafiltration using YM-30 (Amicon), followed by gel filtration ram (TSK-3000SW,
φ2.8 x 60cm, Tosoh) and PBS(-)(
NaCI. 8g/l, KCI, 0.2g/l
, NatHPO. - 12H. 0 2.9g/
1.KH2PO4. 0.2 g/l). SOS-PAGEi. : From the analysis, Arp4, A
The rp4S protein was confirmed to be present in the first peak. These were made into refined products. The recovered amount is 11
The amount of Arp4 protein and Arp4S protein was 5 and 4, respectively. Protein concentration was quantified using purified bovine IgG (Bio
Rad) was used as a standard for the BCAl removal kit (Pierce).

The purified Arp4 and Arp4S proteins obtained in Example 4 were purified by CN
It was immobilized on Br-activated Sepharose (Pharmacia). Fixation was performed according to the attached instructions. 5mg of both proteins
/- The gel was immobilized. This fixed gel 20
Various concentrations of human serum 1 dissolved in PBS(-) in μl
500 μl of gA was added and allowed to bind overnight at room temperature. Unbound IgA was centrifuged and quantified by the BCA method, and the added IgA
The amount of bound IgA was determined from the difference from the amount of gA. From this value, the molar ratio of 1 gA bound to the immobilized Arp4 and Arp4S proteins at the time of maximum binding was determined. The results are shown in the table below. At that time, Arp4, Arp4S, Ig. The molecular weight of the protein was calculated as 40,000, 55,000, and 150,000.

Binding capacity for human 1gA Arp4 0.4l Arp4S 0.50 As shown in the table above, immobilized Arp4S is
The binding capacity for 20X IgA was increased. This was considered to be an effect due to the presence of two rgA binding proteins in the Arp4S protein.

mold

[Brief explanation of the drawing]

? Figure 1 shows the structure of plasmid pARP401. 1 indicates the position of the Arp44 gene, and ■ indicates pUc18. E, B, H, Sc, St, and N are restriction enzymes EcoRI, BamHI, and HindI, respectively.
The cleavage sites of IT, Sac I, StuI%NruI are shown. FIG. 2 shows the structure and gene binding properties of various deletion mutants of Arp4. The top row shows IgA protein. C.I.
, 2.3 shows a repetitive sequence characteristic of Arp4 protein. Sono shows the Arp4 sequence derived from β-galactosidase, and the others show the amino acid sequences derived from Arp4. The numbers are the numbers of the terminal amino acids. The 28th row represents the deletion mutant, and the bottom row represents the IgA binding region obtained as a result. Figure 1 To →

Claims (1)

[Claims] (1) A protein containing the following amino acid sequence and having immunoglobulin A binding activity [there is a gene sequence]. (2) A protein according to claim 1 which contains two or more amino acid sequences according to claim 1. (3) A DNA encoding the protein according to claim 1 or 2. (4) A DNA characterized by encoding the protein according to claim 1 or 2. An expression vector capable of expressing the DNA of claim 3 in a host (5) A host cell containing the expression vector of claim 4 (6
) The method according to claims 1 and 2 by expressing the gene using a plasmid or virus that contains the DNA according to claim 3 and which can be expressed in a host, and recovering the product. (7) The method for producing the protein according to claim 6, wherein the host is Escherichia coli (8) The amino acid sequence from 1 to 165 of protein Arp4, and the four amino acids ArgLeuIleAsn are added to the C-terminus. The protein according to claim 1, characterized in that it has a structure that