JP2008220317A - Penicillin-binding protein(pbp) 3-transpeptidase domain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein for penicillin-binding protein(PBP) 3-transpeptidase domain, usable in crystallization and in assaying compounds. <P>SOLUTION: The protein for Haemophilus influenzae and Escherichia coli PBP 3-transpeptidase domain is produced through identifying Haemophilus influenzae PBP 3-transpeptidase domain. A modified protein with a loop region in the above domain shortened is also produced. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to penicillin binding protein (PBP) 3 transpeptidase domains for use in crystallization and compound assays.

Penicillin-binding protein (PBP) is a series of enzymes that assemble cell wall peptidoglycan using murein monomer, which is a structural unit that constitutes the cell wall in bacteria, as a substrate. It is a membrane-bound protein that acts on the polymerization and crosslinking reaction of bacterial peptidoglycan. is there. Several types of PBP have been confirmed in Haemophilus influenzae, but in β-lactamase non-producing ampicillin resistant (BLNAR) Haemophilus, which tends to increase as resistant bacteria, mutations in PBP3 are sensitive to β-lactam drugs. It is greatly involved in the decrease (see Non-Patent Document 1). In addition, in the PBP3 derived from the Rd strain (ATCC 51907) shown in SEQ ID NO: 1, methionine at amino acid residue number 377, 385 serine, 389 leucine, 517 arginine and 526 asparagine are isoleucine, threonine, phenylalanine, histidine, respectively. It has been suggested that mutations substituted with lysine are important for drug resistance (see Non-Patent Document 2). Therefore, elucidation of the three-dimensional structure of PBP3 by crystal structure analysis is very important not only for explaining the mechanism of resistance but also for creating a new drug effective against resistant bacteria.

Haemophilus influenzae-derived PBP3 is a membrane-bound protein composed of an intracellular domain, a transmembrane region, an N-terminal domain, and a transpeptidase domain. The intracellular domain and the transmembrane region were removed, and the membrane-bound function was lost. Although the preparation method of PBP3 soluble part is clarified, since it is stored in a solution containing 0.5 M sodium chloride, it is unclear whether it can exist stably at a low salt concentration (see Non-Patent Document 3). ). In addition, a method for preparing a transpeptidase domain in which the N-terminal domain is further removed from the PBP3 soluble part has not yet been clarified. Regarding crystallization, although N-terminal and C-terminal sites were not specifically mentioned, there was a report that expressed, purified and crystallized PBP3 from which the membrane binding site was removed. In that report, a crystal photograph was presented, and when a crystal was irradiated with X-rays from a certain direction, a diffraction point with a resolution of about 3.5 mm was obtained, but depending on the direction of X-ray irradiation, It was orally announced that it was difficult to collect diffraction data necessary to determine a new structure because of its weak intensity (see Non-Patent Document 4). Furthermore, at present, no crystal structure has been reported for any of the membrane-bound PBP3, the PBP3 soluble portion and the PBP3 transpeptidase domain. Therefore, it is desired to create a high-quality PBP3 crystal that determines the three-dimensional structure of PBP3 by crystal structure analysis and enables efficient inhibitor design.

In general, the N-terminal, C-terminal, and loop regions of proteins may fluctuate in structure, and in that case, multiple conformations can be taken. This is an effective means for producing a crystal having high-resolution X-ray diffraction ability composed of conformation (see Non-Patent Document 5). As one of the methods, there is known a method for estimating a domain using limited degradation by a proteolytic enzyme. In general, a proteolytic enzyme cleaves a specific part of a protein according to substrate specificity, but a loop part not having a domain structure tends to be cleaved. Therefore, it is possible to estimate the domain by identifying a protein fragment digested with a proteolytic enzyme (see Non-Patent Document 6). However, it cannot be determined whether the estimated region is the optimal domain for crystallization with little fluctuation, and it is necessary to actually manufacture proteins and confirm their properties for various proteins with lengths before and after the estimated region. There is. In Haemophilus influenzae PBP3, reports of limited degradation by proteolytic enzymes are not known, and the region of the transpeptidase domain suitable for crystallization is not yet clear. If a new PBP3 transpeptidase domain suitable for crystallization can be found, it is thought that it can contribute greatly to the production of high-quality crystals and elucidation of the three-dimensional structure of PBP3.

Further, the structure of the loop portion existing in the internal region other than the N-terminal and C-terminal of the protein may be unfavorable for crystallization because the structure fluctuates. For example, in PBP2x derived from Streptococcus pneumoniae R6 strain having the amino acid sequence registered in SWISSPROT Accession No. P14677, each polypeptide corresponding to amino acid residues Nos. 74 to 93, 184 to 199, 218 to 229, and 257 to 750 is used. There is a report of crystallization of mini-PBP2x expressed by connecting with a linker of 1 to 3 amino acid residues (see Patent Document 1). However, in the transpeptidase domain of Haemophilus influenzae PBP3, there is no report as to which region is the loop portion where the structure is fluctuating. Therefore, in H. influenzae PBP3, the method for shortening the loop portion for the purpose of improving crystallization is not clear.

In addition, Escherichia coli PBP3 has a high homology with PBP3 derived from Haemophilus influenzae, about 80%, and is assumed to be similar in three-dimensional structure. Therefore, elucidation of the three-dimensional structure of E. coli-derived PBP3 is useful not only for designing an inhibitor exhibiting antibacterial activity against E. coli but also for designing an inhibitor exhibiting antibacterial activity against Haemophilus influenzae. E. coli PBP3 has a report on expression and purification of a soluble part consisting of a sequence starting from any one of amino acids 37 and 57 of SEQ ID NO: 25 and ending with amino acid of amino acid 577 (see Non-Patent Document 7). Regarding crystallization, the N-terminal and C-terminal sites are not specifically described, but there are reports of diffraction measurement of PBP3 crystals. In that report, only diffraction points with a resolution of about 6-7 mm have been obtained, and the crystal structure has not been determined (see Non-Patent Document 8). Accordingly, in E. coli PBP3, as in the case of Haemophilus influenzae PBP3, the region of the transpeptidase domain suitable for the production of high-quality crystals has not yet been clarified, and a new PBP3 transpeptidase domain suitable for crystallization can be found. If possible, it may be possible to make a high-quality crystal and contribute much to the elucidation of the three-dimensional structure of PBP3.
WO2004 / 007541 Antimicrob. Agents Chemother. 45 (6) 1693-1699 (2001) Antimicrob. Agents Chemother. 49 (7) 2834-2839 (2005) Antimicrob. Agents Chemother. 48 (5) 1630-1639 (2004) Park San-yo, “Development of New Antibiotics Based on the Structure of Penicillin-Binding Protein”, December 13, 2006, Yokohama City University 3rd Industry-Academia Collaboration Forum “Yokohama City University-Protein Structural Analysis Consortium Joint Symposium-Created from Protein Research To Drugs "Proceedings BIO Venture 3 (5), 36-39 (2003) METHODS IN ENZYMOLOGY. 368, 77-84 (2003) Biochem. J. et al. 298, 189-195 (1994) CHARLIER Paulette, 2 others, “Structure of Penicillin-Binding Protein 3 from Escherichia coli.” [Online], August 30, 1999, ESRF, [February 9, 2007 search], Internet </ http www. esrf. eu / smis / reports / gen / ls_1286_a. pdf>

Until now, the crystal structures of H. influenzae and E. coli PBP3 have not been reported. Therefore, there is no 3D structure information of PBP3 which is important in drug discovery, and it is difficult to design an efficient inhibitor. In addition, a method for producing a PBP3 transpeptidase domain suitable for crystallization is unknown.

The present invention solves the above problem by digesting a PBP3 soluble portion with a proteolytic enzyme, estimating a transpeptidase domain region, verifying various candidate proteins, and producing a transpeptidase domain suitable for crystallization. Resolve.

The present inventors have succeeded in producing a PBP3 transpeptidase domain that has improved stability by digestion with a proteolytic enzyme and is soluble even at a low salt concentration. In addition, by shortening the loop region present in the domain, we succeeded in producing a modified protein that is thought to have reduced conformational fluctuations. These proteins can be used for crystallization.

That is, the present invention includes the following inventions.
(1) a protein having transpeptidase activity selected from the group consisting of:
(A) Haemophilus influenzae penicillin binding protein (PBP) comprising a sequence beginning with any one of amino acids 225 to 262 of SEQ ID NO: 1 and ending with any one of amino acids 566 to 607 A protein consisting of three transpeptidase domains.
(B) any one of the amino acid numbers corresponding to amino acid numbers 566 to 607 starting from any one amino acid corresponding to amino acid numbers 225 to 262 of the amino acid sequence having at least 85% homology in SEQ ID NO: 1 A homologous protein consisting of a sequence ending with one amino acid.
(C) starting from any one of the amino acid numbers corresponding to amino acid numbers 225 to 262 of the amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted in SEQ ID NO: 1, and amino acid number 566 A modified protein consisting of a sequence ending with any one of the amino acid numbers corresponding to 607.
(2) 3 to 5 polypeptides starting from any one amino acid corresponding to amino acid numbers 301 to 308 in SEQ ID NO: 1 and ending with any one amino acid corresponding to amino acid numbers 315 to 322 The protein according to (1), wherein the protein is substituted with a polypeptide consisting of any amino acid as a residue.
(3) The polypeptide according to (1), wherein the polypeptide consisting of the sequence corresponding to amino acid numbers 306 to 317 in SEQ ID NO: 1 is substituted with a polypeptide consisting of any amino acid having 3 to 5 residues. protein.
(4) The polypeptide consisting of the sequence corresponding to amino acid numbers 306 to 317 in SEQ ID NO: 1 is substituted with a polypeptide consisting of 4-residue amino acids which are glycine, threonine, glycine and glycine, (1) The protein according to the description.
(5) Starting from any one amino acid of amino acid numbers 235, 242, 249, 252 or the amino acid corresponding thereto in SEQ ID NO: 1, and any one amino acid of amino acid numbers 576, 581, 586, 593, 597 or equivalent thereof The protein according to any one of (1) to (4), which ends with an amino acid.
(6) The protein according to any one of (1) to (5), comprising a sequence selected from the group consisting of SEQ ID NOs: 2 to 24.
(7) a protein having transpeptidase activity selected from the group consisting of:
(A) Escherichia coli penicillin binding protein (PBP) 3 consisting of a sequence starting from any one amino acid of amino acid numbers 205 to 242 of SEQ ID NO: 25 and ending with any one amino acid of amino acid numbers 559 to 579 A protein consisting of the transpeptidase domain.
(B) any one of the amino acid numbers corresponding to amino acid numbers 559 to 579, starting from any one amino acid corresponding to amino acid numbers 205 to 242 of the amino acid sequence having at least 85% homology in SEQ ID NO: 25 A homologous protein consisting of a sequence ending with one amino acid.
(C) starting from any one amino acid corresponding to amino acid numbers 205 to 242 of the amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted in SEQ ID NO: 25; A modified protein consisting of a sequence ending with any one of the amino acid numbers corresponding to 579.
(8) The protein according to (7), comprising a sequence that starts with any one amino acid of amino acid numbers 215 and 232 in SEQ ID NO: 25 or an amino acid corresponding thereto, and ends with the amino acid of amino acid number 569 or an amino acid corresponding thereto.
(9) The protein according to any one of (7) and (8), comprising a sequence selected from the group consisting of SEQ ID NOs: 26 and 27.
(10) A protein in which at least one methionine residue in the protein according to any one of (1) to (9) is substituted with selenomethionine.
(11) A protein in which at least one lysine residue in the protein according to any one of (1) to (10) is reductively dimethylated.
(12) A polynucleotide encoding the protein according to any one of (1) to (10).
(13) A recombinant vector containing the polynucleotide according to (12).
(14) A host cell transformed with the recombinant vector according to (13).
(15) a step of culturing the host cell according to (14), and a step of collecting the protein according to any one of (1) to (11) from the host cell obtained by the culture or a culture thereof. A method for producing a protein.
(16) In the method for assaying a compound that binds to Haemophilus influenzae or E. coli PBP3, the protein according to any one of (1) to (11) is contacted with the compound, and then whether or not the compound binds to PBP3 is determined. A method that consists of detecting.
(17) A method for producing a crystal of a transpeptidase domain of Haemophilus influenzae or Escherichia coli PBP3, the step of producing the protein according to any one of (1) to (11), and the produced protein and crystallization And a step of contacting with an agent to precipitate crystals of the transpeptidase domain of H. influenzae or E. coli PBP3.
(18) A primer having any one of SEQ ID NOs: 28 to 31.
(19) A method for producing a modified protein obtained by shortening a large fluctuation loop structure present in a protein of Haemophilus influenzae PBP3 transpeptidase domain, comprising a step of using the primer according to (18) in a gene amplification reaction method A method for producing the protein according to any one of (2) to (4).

The PBP3 transpeptidase domain of the present invention is suitable for the production of high-quality crystals because the region where the conformation is fluctuating is reduced as compared with the soluble portion from which only the membrane-bound region is removed. The present invention is extremely useful in drug discovery because it can be used to obtain crystal structure information useful for efficient design of compounds that inhibit PBP3.

Hereinafter, the present invention will be described in detail.
<< PBP3 soluble part and transpeptidase domain >>
The protein constituting the PBP3 transpeptidase of the present invention is a protein derived from Haemophilus influenzae or Escherichia coli. In the present invention, the term “transpeptidase” refers to a polypeptide chain that extends from N-acetylmuramic acid, which is involved in the final stage of biosynthesis of peptidoglycan, which is one component of the bacterial cell wall, and constitutes the peptidoglycan chain. An enzyme that catalyzes the crosslinking of Furthermore, the “transpeptidase domain” is a structural unit of a protein having the ability to crosslink the polypeptide chain and capable of binding to a β-lactam drug. The “homologous protein” in the present invention is an amino acid having at least 85%, preferably 90%, more preferably 95%, and still more preferably 98% homology with a protein having the amino acid sequence shown in SEQ ID NO: 1 or 25. A protein containing a sequence and having transpeptidase activity. The homology values shown here are FASTA (Science, 227, 1434-1441 (1985); Proc. Natl. Acad. Sci. USA, 85, 2444-2448 (homology search program known to those skilled in the art). 1988); http://www.ddbj.nig.ac.jp/search/fasta-j.html), and numerical values calculated using default (initial setting) parameters. The “modified protein” in the present invention includes an amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 1 or 25, and transpeptidase It is a protein having activity. Here, the number of amino acids related to the modification such as “substitution, deletion, addition or insertion” is preferably 1 to 40, more preferably 1 to 20, and still more preferably 1 to 12. Specific examples of the modified protein include a point mutant protein selected from the group consisting of SEQ ID NOs: 14 to 21 and a loop region described in Examples 7 and 8 described later, selected from the group consisting of SEQ ID NOs: 22-24. The modified protein which shortened is mentioned.

PBP3 is a membrane-bound protein composed of an intracellular domain, a transmembrane region, an N-terminal domain, and a transpeptidase domain. The PBP3 soluble part is PBP3 in which the intracellular domain and the transmembrane region are removed and the membrane-bound function is lost. The PBP3 soluble part can be derived from various strains with different origins and N-terminal and C-terminals. However, in H. influenzae, amino acid residues 67 to 610 of the amino acid sequence shown in SEQ ID NO: 1 are preferred. In E. coli, it is preferably composed of a protein starting with an amino acid corresponding to amino acid residue 37 or 57 of the amino acid sequence shown in SEQ ID NO: 25 and ending with an amino acid corresponding to 610. The PBP3 transpeptidase domain is obtained by further removing the N-terminal domain from the soluble portion of PBP3, and various types can exist depending on the region to be removed. As a method for estimating the transpeptidase domain, a candidate site can be determined by mass analysis of a sample obtained by restricting a purified PBP3 soluble portion in the presence of various concentrations of proteolytic enzymes and N-terminal amino acid sequence analysis of each resulting fragment. it can. However, it cannot be determined whether the estimated region is the optimal domain for crystallization with little fluctuation, and various proteins consisting of the lengths before and after the estimated region are actually manufactured and properties such as solubility. It is necessary to confirm.

The H. influenzae PBP3 transpeptidase domain starts with any one amino acid of amino acid numbers 225 to 262 or its corresponding amino acid in SEQ ID NO: 1 and ends with any one amino acid of amino acid numbers 566 to 607 or its corresponding amino acid A protein consisting of a sequence is preferred. More preferably, it begins with any one amino acid of amino acid numbers 235, 242, 249, 252 or the amino acid corresponding thereto in SEQ ID NO: 1, and any one amino acid of amino acid numbers 576, 581, 586, 593, 597, or A protein consisting of a sequence ending with the corresponding amino acid is preferred. Specific examples include a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 24, for example.

The E. coli PBP3 transpeptidase domain starts with any one amino acid of amino acid numbers 205 to 242 or an amino acid corresponding thereto in SEQ ID NO: 25 and ends with any one amino acid of amino acid numbers 559 to 579 or an amino acid corresponding thereto. A protein consisting of More preferably, a protein comprising a sequence starting from any one amino acid of amino acid numbers 215 and 232 in SEQ ID NO: 25 or an amino acid corresponding thereto and ending with the amino acid of amino acid number 569 or an amino acid corresponding thereto is preferable. Specific examples include proteins comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 26 to 27, for example.

<< Shortened loop structure in the H. influenzae PBP3 transpeptidase domain >>
In general, the structure of a loop portion existing in a region composed of a single domain is also unfavorable for crystallization because the structure fluctuates. The present inventors have found that there is a region within the transpeptidase domain of Haemophilus influenzae PBP3 that is likely to be cleaved by proteolytic enzymes, that is, a loop region in which the conformation is presumed to fluctuate. Specifically, when limited digestion was attempted using trypsin, which is a proteolytic enzyme, it was found that cleaved immediately after each amino acid of amino acid numbers 306, 310, 315, and 317 in SEQ ID NO: 1, and this part A modified protein with a reduced length was produced. This modified protein is a polypeptide that begins with any one amino acid corresponding to amino acid numbers 301 to 308 in SEQ ID NO: 1 and ends with any one amino acid corresponding to amino acid numbers 315 to 322. It is a modified protein of the Haemophilus influenzae PBP3 transpeptidase domain, which is substituted with a polypeptide consisting of any amino acid having 3 to 5 residues. More preferably, the Haemophilus influenzae PBP3 transpeptidase, wherein the polypeptide consisting of the sequence corresponding to amino acid numbers 306 to 317 in SEQ ID NO: 1 is substituted with a polypeptide consisting of any amino acid having 3 to 5 residues. A domain-modified protein, more preferably a polypeptide consisting of a sequence corresponding to amino acids 306 to 317 in SEQ ID NO: 1 is replaced with a polypeptide consisting of 4 residues of amino acids such as glycine, threonine, glycine and glycine. It is a modified protein of the Haemophilus influenzae PBP3 transpeptidase domain. Specific examples include proteins comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 22 to 24, for example.

In the present invention, a primer for constructing the above-described modified protein expression system is disclosed. By using the primer corresponding to the N-terminal part of the transpeptidase domain represented by the primer selected from the group consisting of SEQ ID NOs: 34 to 37 and 43 and the primer described in SEQ ID NO: 28 in the gene amplification reaction method, A restriction enzyme Kpn encoding glycine or threonine at the end of DNA encoding a sequence starting from any one amino acid of amino acid numbers 225 to 262 or the amino acid corresponding thereto and ending with the amino acid of amino acid number 305 or the amino acid corresponding thereto in 1 I Can amplify fragments with added recognition sequences. Further, a gene corresponding to a primer corresponding to the C-terminal portion of a transpeptidase domain represented by a primer selected from the group consisting of SEQ ID NOs: 38 to 42 and 44 and a primer selected from the group consisting of SEQ ID NOs: 29 to 31 By using the method, at the end of DNA encoding a sequence starting from the amino acid of amino acid number 318 or its corresponding amino acid in SEQ ID NO: 1 and ending with any one amino acid of amino acid numbers 566 to 607 or its corresponding amino acid A fragment to which a restriction enzyme Kpn I recognition sequence encoding glycine and threonine and a DNA sequence encoding a linker consisting of glycine having 1 to 3 amino acid residues can be amplified. By combining the above two DNA fragments and introducing them into an expression vector, which will be described later, a recombinant vector necessary for the production of the Haemophilus influenzae PBP3 transpeptidase domain with a shortened loop structure can be produced.

<< Polynucleotide Encoding PBP3 Transpeptidase Domain >>
According to the present invention, polynucleotides encoding H. influenzae and E. coli PBP3 transpeptidase domains, homologous proteins and modified proteins thereof are provided. Given the amino acid sequence of a protein, the base sequence encoding it can be easily determined, and therefore various base sequences encoding the protein of the present invention can be selected. In the present specification, the term “polynucleotide” includes both DNA and RNA, and DNA is preferred.

<< Expression of PBP3 soluble part and transpeptidase domain >>
The PBP3 soluble portion and the transpeptidase domain are in the form of a DNA molecule, particularly a DNA expression vector, containing the DNA fragment that encodes it in a state in which it can be replicated and expressed in the host cell. It is obtained by performing transformation and culturing the transformant. This DNA molecule can be obtained by incorporating a DNA fragment encoding a PBP3 soluble portion or transpeptidase domain into a vector molecule. According to a preferred embodiment of the invention, this vector is a plasmid. The vector used in the present invention can be appropriately selected from viruses, plasmids, cosmid vectors and the like in consideration of the type of host cell to be used. For example, when the host cell is Escherichia coli, pUC and pBR type plasmids, when Bacillus subtilis is used, pUB type plasmids are used, and when yeast is used, YEp, YRp and YCp type plasmid vectors are used. Any host cell can be used as long as a host-vector system has been established, and Escherichia coli is preferable. A transformant obtained by transformation of a host cell can be cultured under appropriate conditions, and a cell extract of the obtained transformant can be recovered according to a conventional method. Here, it is also possible to express a polypeptide additional to the PBP3 soluble part or transpeptidase domain, such as glutathione-S-transferase (GST) or a polypeptide rich in histidine residues (His-tag) as a fusion protein. Is possible.

<< PBP3 soluble part and purification of transpeptidase domain >>
The PBP3 soluble portion and transpeptidase domain expressed in E. coli can be purified by conventional chromatographic techniques. When expressed by adding His-tag, purification is possible with an affinity column to which nickel ions are immobilized, and even when no tag is added, an ion exchange column, a blue column using a dye such as Cibacron Blue, It can be purified by combining a hydroxyapatite column, a hydrophobic interaction column, a gel filtration column, or the like.

<< Crystallization of PBP3 transpeptidase domain >>
The protein purified by the above method can be used for crystallization. The purified protein can be used for crystallization screening by a vapor diffusion method or a microbatch method using a crystallization reagent commercially available from Hampton Research or Qiagen. In some cases, by further optimizing the conditions such as the concentration of the crystallizing agent, the pH, the kind and concentration of the salt, and the additive, a high-quality crystal that can be used for X-ray crystal structure analysis can be obtained. In addition, formation of crystal nuclei and crystal growth can be controlled by overlaying oil on a seeding method or a reservoir solution. The crystallization temperature is generally 4 ° C to 20 ° C. As the protein used for crystallization, any protein selected from the group consisting of SEQ ID NOs: 2 to 24 and 26 to 27 may be used. In addition, selenomethionine substitution products in which the methionine residue in the protein is substituted with selenomethionine and modified proteins in which lysine residues are reduced and dimethylated are also used for crystallization. The concentration of the purified protein used for crystallization is preferably about 4 to 20 mg / mL. Then, crystallization is carried out by adding an appropriate amount of a crystallization agent, salts, buffer solution, additive or the like to the protein solution. Examples of the crystallization agent used for crystallization of PBP3 of the present invention include inorganic salts such as ammonium sulfate, water-soluble polymers such as polyethylene glycol, and organic solvents such as isopropanol and ethanol. As the buffer solution, any known buffer solution can be used. Furthermore, various metal ions, organic solvents, reducing agents, and the like can be used as additives. In the case of a selenomethionine substituted product, a crystal can often be produced under the peripheral conditions in the case of an unsubstituted product, but it is preferable to add a reducing agent such as dithiothreitol in order to suppress oxidation of the selenium atom.

<< Enzyme assay using PBP3 transpeptidase domain >>
The above-mentioned purified protein should be used for an enzyme assay by an existing method using a labeled compound having affinity for PBP, such as BOCILLIN FL (fluorescently labeled benzylpenicillin, manufactured by Molecular Probes) or [ ³ H] benzylpenicillin. Can do. Existing methods are described in Antimicrob. Agents Chemother. 43 (5) 1122-1128 (1999), published patent publication 2004-290070, and the like.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited by an Example. In addition, production of various vectors, protein expression, etc. are described in Molecular Cloning, A Laboratory Manual, 3rd edition (written by Sambrook and Russell, published by Cold Spring Laboratories Press (2001)) unless otherwise specified. It can be carried out according to the method.

<< Construction of gene encoding soluble part of Haemophilus influenzae PBP3 >>
The following primers were designed to isolate DNA encoding the soluble portion of PBP3 excluding the membrane binding region.

HI-PBP3-67-5: 5′-CGCTTAATTAAACATATGACCGGATCCCTCTATTAATGCCGATACGTT-3 ′ (SEQ ID NO: 32)
HI-PBP3-610-3: 5′-TTAGTAGTAGTACGGATCCCCCTGAGTTAATTTCACTTTTTGATTCTTTGT-3 ′ (SEQ ID NO: 33)

A polymerase chain reaction (PCR) was performed with Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) according to the attached instructions using Haemophilus influenzae Rd strain genomic DNA as a template and the primers described above. The amplified DNA fragment was digested with BamHI and XhoI. This fragment was subcloned into a vector pET-30a (+) (manufactured by Novagen) previously cleaved with BamHI and XhoI using T4 DNA Ligase (manufactured by Invitrogen) according to the method of the attached manual. The obtained recombinant plasmid was introduced into Escherichia coli COMPETENT high DH5α (manufactured by TOYOBO) according to the method described in the attached instructions to obtain a transformant. The transformant was cultured overnight at 37 ° C. on an LB agar plate containing 30 μg / mL kanamycin to obtain kanamycin resistant colonies. A recombinant plasmid (pET-30a (+) _ HI_67-610) was prepared from the obtained colonies.

<< Expression of soluble part of Haemophilus influenzae PBP3 >>
pET-30a (+) _ HI_67-610 was transformed into Escherichia coli BL21 (DE3) (manufactured by Novagen), and the resulting transformant was overnight at 37 ° C. in LB medium (100 mL) containing 30 μg / mL kanamycin. Cultured with shaking. 20 mL of the culture solution was added to 1 L of LB medium containing 30 μg / mL kanamycin, and cultured with shaking at 37 ° C. for 3 hours, and isopropyl-β-D-thiogalactopyranoside (IPTG) having a final concentration of 1 mM was added. After induction at 3 ° C. for 3 hours, the cells were collected by a centrifuge, the cells were suspended in phosphate buffered saline (PBS), collected again by a centrifuge, and stored frozen at −20 ° C. The cells are suspended in a cell disruption buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 5 mM imidazole, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg / mL lysozyme), and cells are sonicated. Was crushed. The sonication residue was removed by centrifugation followed by a 0.2 μm filter to obtain a cell extract.

<< Purification of H. influenzae PBP3 soluble part >>
(1) Purification by affinity chromatography All the following purification operations were performed at 4 ° C. The cell extract obtained by the method of Example 2 was purified by affinity chromatography. As the affinity chromatography column, Ni-NTA (manufactured by QIAGEN) was used as a carrier, and a column with a column capacity of 30 ml was prepared by the method described in the manual. After equilibration with an equilibration buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 5 mM imidazole, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride), the cell extract was applied and equilibrated with 3 column volumes. After washing with 50 mM HEPES pH 7.5, 500 mM sodium chloride, 1 mM 2-mercaptoethanol, eluting with a linear gradient of 5-250 mM imidazole (20 column volumes) in 1 mM phenylmethylsulfonyl fluoride, Collected together.

(2) Purification by hydrophobic interaction chromatography The PBP3 soluble part obtained in (1) above was mixed with 50 mM HEPES pH 7.5, 3.5 M ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol solution. The final concentration of ammonium sulfate was 2M. RESORCEPHE 6 ml (GE Healthcare Bio), a hydrophobic interaction column in which the prepared PBP3 soluble part was equilibrated with an equilibration buffer (50 mM HEPES pH 7.5, 2 M ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). Applied to Science) at a flow rate of 5 mL / min. After washing with 3 column volumes of equilibration buffer, elute with a linear gradient (150 mL) of 2-0 M ammonium sulfate in 50 mM HEPES pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol solution. Summed up the minutes.

(3) Purification by gel filtration chromatography The PBP3 soluble part obtained in (2) above is concentrated using Amicon Ultra-4 30,000 MWCO (Millipore), which is an ultrafiltration membrane having a retained molecular weight of 30,000 or more. , Gel filtration column Hiload 16/60 Superdex75 prep grade (GE Healthcare Biosciences) manufactured by equilibration with gel filtration buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) ) At a flow rate of 1 mL / min. The main fractions were combined and concentrated to a protein concentration of 10 mg / mL using the ultrafiltration membrane described above.

<< Estimation of transpeptidase domain by trypsin-limited degradation >>
The H. influenzae PBP3 soluble portion purified in Example 3 was diluted with 50 mM HEPES pH 7.5, 500 mM sodium chloride, 2 mM dithiothreitol to a final protein concentration of 2 mg / mL. Next, 10 μL of the diluted solution was mixed with 10 μL of 0.001-0.003 mg / mL trypsin (Trypsin TPCK treated From Bovine Pancreas, manufactured by Sigma) solution containing 2 mM EDTA, and reacted at 26 ° C. for 1 hour. Divided equally. One was mixed with an equal volume of SDS-PAGE sample buffer containing 1 mM PMSF and a reducing agent, boiled for 3 minutes, applied to SDS-PAGE, migrated, transferred to a PVDF membrane, and a major band was excised. Were used to analyze the N-terminal amino acid sequence. The remaining one was added with one-tenth amount of 10% acetic acid, purified using ZipTipC4 (manufactured by Millipore), and the molecular weight was measured with a MALDI-TOF mass spectrometer. As a result, major bands having molecular weights of 39 k and 31 k were confirmed by SDS-PAGE. As a result of the N-terminal amino acid sequence, the N-terminal amino acid sequence of the band near 39k was NIVA, and the band near 31k was a mixture of VGV, SELMR, NRAIT and AITDTF. Many peaks were observed in mass spectrometry, but peaks exist in the vicinity of molecular weights of 39000 and 31000. From the above results, the 39-k band in SDS-PAGE is the amino acid number shown in SEQ ID NO: 1. The bands 235-593, 31k were found to correspond to the mixtures 307-593, 311-593, 316-593 and 318-593. Therefore, amino acid numbers 235 and 307 to 318 were estimated as N-terminal candidates for the transpeptidase domain region, and amino acid number 593 was estimated as a C-terminal candidate. In E. coli, from the similarity of amino acid sequences, near amino acid number 215 was selected as an N-terminal candidate of the transpeptidase domain region, and near amino acid number 569 was selected as a C-terminal candidate.

<< Construction of genes encoding H. influenzae and E. coli PBP3 transpeptidase domains >>
The following primers were designed to isolate DNA encoding H. influenzae and E. coli PBP3 transpeptidase domains.

HI-PBP3-235-5: 5'-cacccatatggggaattattgtcacatatc-3 '(SEQ ID NO: 34)
HI-PBP3-242-5: 5'-cacccatatgtccgagagaaaaatagatgc-3 '(SEQ ID NO: 35)
HI-PBP3-249-5: 5′-cacccatatggcacaagatgtacttattattacg-3 ′ (SEQ ID NO: 36)
HI-PBP3-252-5: 5'-cacccatatggttaccttagattagattagaaaaattgc-3 '(SEQ ID NO: 37)
HI-PBP3-593-3: 5'-cccctcgagtttttcgtttgttgtttttcagc-3 '(SEQ ID NO: 38)
HI-PBP3-597-3: 5'-cccctcgagttaacgtttgtacactttcgttg-3 '(SEQ ID NO: 39)
HI-PBP3-586-3: 5′-cccctcgagttatgcttcagcatctgcggagatag-3 ′ (SEQ ID NO: 40)
HI-PBP3-581-3: 5′-cccctcgagtacgggaatagcatttgcagg-3 ′ (SEQ ID NO: 41)
HI-PBP3-576-3: 5′-cccctcgagttaacgtataacggcatcccataatgttag-3 ′ (SEQ ID NO: 42)
HI-PBP3-235-5-2: 5′-gggaattccatggggaattttgtcacatatc-3 ′ (SEQ ID NO: 43)
HI-PBP3-593-3-2: 5′-ccgctcgagtttcgttgttgtgtttcacc-3 ′ (SEQ ID NO: 44)
HI-PBP3-307-5: 5′-gggaattccatggtcggcgtgaaaatcagatta-3 ′ (SEQ ID NO: 45)
EC-PBP3-232-5: 5'-catgcttagcctggcgctgagattattagaacg-3 '(SEQ ID NO: 46)
EC-PBP3-215-5: 5'-catgcttagcggtcgcgtataattgaagaattatt-3 '(SEQ ID NO: 47)
EC-PBP3-569-3: 5'-gttctcgagtttagtcaggcgccccggctc-3 '(SEQ ID NO: 48)

The polymerase chain reaction (PCR) using Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) with the appropriate combination of the primers, using Haemophilus influenzae Rd strain genomic DNA, mutagenized strain genomic DNA, Escherichia coli cell extract or already prepared plasmid DNA as a template. ) Was carried out according to the attached instructions. H. influenzae mutation-introduced strains were prepared by methods described in the literature (Antimicrob. Agents Chemother. 49 (7) 2834-2839 (2005). The amplified DNA fragments were digested with restriction enzymes, and these fragments were previously digested with restriction enzymes. Instructions attached to the cleaved vectors pET-21b (+), pET-28b (+) or pET-30a (+) (manufactured by Novagen) with Ligation high (manufactured by TOYOBO) or T4 DNA Ligase (manufactured by Invitrogen) The obtained recombinant plasmid was introduced into Escherichia coli COMPETENT high DH5α (manufactured by TOYOBO) according to the method described in the attached instruction to obtain a transformant. Anne Drug-resistant colonies were obtained by culturing overnight at 37 ° C. on LB agar plates containing syrin or 30 μg / mL kanamycin, and a recombinant plasmid was prepared from the obtained colonies. The combinations of primers, vectors and restriction enzymes are shown in Table 1.

<< Expression of H. influenzae and E. coli PBP3 transpeptidase domains >>
The recombinant vector prepared in Example 5 was transformed into E. coli BL21 (DE3) or B834 (DE3) (manufactured by Novagen), and the resulting transformant was 50-100 μg / mL ampicillin or 30 μg / mL kanamycin. SB medium (1.2% (w / v) Bacto Tryptone, 2.4% (w / v) Yeast Extract, 0.5% (v / v) glycerol, 0.072M dipotassium hydrogen phosphate, 0% 0. 28M potassium dihydrogen phosphate) at 600 nm. D. Until approximately 1 was reached. A final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and after induction overnight at 20 ° C., the cells were collected by a centrifuge. The cells were suspended in the cell disruption buffer, subjected to ultrasonic treatment, and the cells were disrupted. The sonication residue was removed by centrifugation to obtain a cell extract. As a result of SDS-PAGE analysis, H. influenzae PBP3 consisting of the sequence shown in SEQ ID NO: 49, which starts with amino acid 307 in SEQ ID NO: 1 and ends with amino acid 593, has protein expression only in the insoluble fraction. confirmed. Regarding other proteins, some of the protein was expressed in the insoluble fraction, but it was confirmed that the protein was expressed in the soluble fraction in all combinations. Therefore, amino acid numbers 307 to 318 in SEQ ID NO: 1 were inappropriate as the N-terminal site of the transpeptidase domain, and this region was presumed to have a loop structure in the domain. The combinations of the recombinant vector, host, tag, and disruption buffer are shown in Table 2.

<< Construction of a gene encoding a modified protein with a shortened loop structure present in the H. influenzae PBP3 transpeptidase domain >>
In order to isolate DNA encoding a modified protein with a shortened loop structure, the following primers were designed.

HI-PBP3-305-Gly-Thr-3: 5′-cggggtaccgttgtttggattagaagaggggcgcagtcgc-3 ′ (SEQ ID NO: 28)
HI-PBP3-Gly-Thr-Gly-318-5: 5'-cggggtaccgggtcacatattacgtattttgaggccagtt-3 '(SEQ ID NO: 29)
HI-PBP3-Gly-Thr-Gly2-318-5: 5'-cggggtaccgggtggtgcaattattaccattattttgaggca-3 '(SEQ ID NO: 30)
HI-PBP3-Gly-Thr-Gly3-318-5: 5′-cggggtaccgggtggtgggtgatattaccatatttttgag-3 ′ (SEQ ID NO: 31)
HI-PBP3-235-5-2: 5′-gggaattccatggggaattttgtcacatatc-3 ′ (SEQ ID NO: 43)
HI-PBP3-593-3-2: 5′-ccgctcgagtttcgttgttgtgtttcacc-3 ′ (SEQ ID NO: 44)
HI-PBP3-252-5: 5'-cacccatatggttaccttagattagattagaaaaattgc-3 '(SEQ ID NO: 37)
HI-PBP3-586-3: 5′-cccctcgagttatgcttcagcatctgcggagatag-3 ′ (SEQ ID NO: 40)
HI-PBP3-576-3: 5′-cccctcgagttaacgtataacggcatcccataatgttag-3 ′ (SEQ ID NO: 42)

Using the recombinant vector pET-30a (+) _ HI — 235-593 described in Example 5 as a template, the above primers are appropriately combined, and the polymerase chain reaction (PCR) is performed with Pyrobest DNA Polymerase (Takara Bio Inc.). It was carried out according to the manual. Table 3 shows combinations of amplified DNA fragments and primers.

The amplified DNA fragment 1 was digested with restriction enzymes NdeI and KpnI, and DNA fragment 3 was digested with KpnI and XhoI. These fragments were subcloned into a vector pET-30a (+) (manufactured by Novagen) previously cleaved with NdeI and XhoI using T4 DNA Ligase (manufactured by Invitrogen) according to the method of the attached instruction. The obtained recombinant plasmid was introduced into Escherichia coli COMPETENT high DH5α (manufactured by TOYOBO) according to the method described in the attached instructions to obtain a transformant. The transformant was cultured overnight at 37 ° C. on an LB agar plate containing 30 μg / mL kanamycin to obtain kanamycin resistant colonies. A recombinant plasmid (pET-30a (+) _ HI_235-305-GTGG-318-593) was prepared from the obtained colonies.

Next, using the recombinant vector pET-30a (+) _ HI_235-305-GTGG-318-593 as a template, primers HI-PBP3-252-5 and HI-PBP3-576-3, or HI-PBP3-252-5 And HI-PBP3-586-3 were used in combination, and the polymerase chain reaction (PCR) was carried out with Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) according to the attached instructions to obtain DNA fragments 5 and 6, respectively. Amplified DNA fragments 5 and 6 were digested with restriction enzymes NdeI and XhoI. These fragments were subcloned into a vector pET-21b (+) (manufactured by Novagen) previously cleaved with NdeI and XhoI using Ligation High (manufactured by TOYOBO) according to the method of the attached instruction. The obtained recombinant plasmid was introduced into Escherichia coli COMPETENT high DH5α (manufactured by TOYOBO) according to the method described in the attached instructions to obtain a transformant. Transformants were cultured overnight at 37 ° C. on LB agar plates containing 100 μg / mL ampicillin to obtain ampicillin resistant colonies. Recombinant plasmids (pET-21b (+) _ HI_252-305-GTGG-318-576 and pET-21b (+) _ HI_252-305-GTGG-318-586) were prepared from the obtained colonies.

<< Expression of modified protein with a shortened loop structure present in the H. influenzae PBP3 transpeptidase domain >>
The recombinant vector prepared in Example 7 was transformed into Escherichia coli B834 (DE3) (manufactured by Novagen), and the obtained transformant was SB medium containing 100 μg / mL ampicillin or 30 μg / mL kanamycin (1. 2% (w / v) Bacto Tryptone, 2.4% (w / v) Yeast Extract, 0.5% (v / v) glycerol, 0.072M dipotassium hydrogen phosphate, 0.028M potassium dihydrogen phosphate ) At 600 nm. D. Until approximately 1 was reached. A final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and after induction overnight at 20 ° C., the cells were collected by a centrifuge. The cells were suspended in the cell disruption buffer, subjected to ultrasonic treatment, and the cells were disrupted. The sonication residue was removed by centrifugation to obtain a cell extract. When analyzed by SDS-PAGE, some of the protein was expressed in the insoluble fraction, but it was confirmed that all the combinations of the target proteins were expressed in the soluble fraction. Table 4 shows combinations of recombinant vectors, hosts, tags, and disruption buffers.

<< Expression of selenomethionine substitutes of H. influenzae and E. coli PBP3 transpeptidase domains >>
(1) Expression of selenomethionine substitution product of Haemophilus influenzae PBP3 transpeptidase domain modified protein (amino acid SEQ ID NO: 22) In order to prepare a heavy atom derivative crystal used for X-ray crystal structure analysis by MAD method, We tried to express a selenomethionine-substituted product substituted with selenium, a heavy atom. The transformant (E. coli B834 (DE3) / pET-30a (+) _ HI_235-305-GTGG-318-593) obtained in Example 8 was used at 37 ° C. in 100 mL of LB medium containing 30 μg / mL kanamycin. The cells were cultured overnight, collected in a centrifuge, suspended in 100 ml of LeMaster medium (composition shown in Table 5), collected again in a centrifuge, and resuspended in 100 ml of LeMaster medium. This suspension was inoculated into 5 L of LeMaster medium containing 30 μg / mL kanamycin and O.D. D. Until approximately 1 was reached. A final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and after induction at 20 ° C. for 10 hours, the cells were collected by a centrifuge and the cells were collected in phosphate buffered saline (PBS). After the suspension, the cells were collected again by a centrifuge and stored frozen at -20 ° C. Cell disruption buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 5 mM imidazole, 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin, 1 μM pepstatin A, 0.1 mg / mL The cells were suspended in lysozyme) and disrupted by sonication. The sonication residue was removed by centrifugation followed by a 0.2 μm filter to obtain a cell extract.

(2) Expression of selenomethionine substitution product of E. coli PBP3 transpeptidase domain (amino acid SEQ ID NO: 26) In order to prepare a heavy atom derivative crystal used for X-ray crystal structure analysis by MAD method, the sulfur atom of methionine is a heavy atom. An attempt was made to express a selenomethionine-substituted product substituted with a certain selenium. The transformant (E. coli B834 (DE3) / pET-21b (+) _ EC — 215-569) obtained in Example 6 was cultured overnight at 37 ° C. in 100 mL of LB medium containing 50 μg / mL ampicillin. Was collected in a centrifuge, suspended in 50 ml of LeMaster medium containing 50 μg / mL ampicillin, collected again by a centrifuge, and resuspended in 50 ml of LeMaster medium containing 50 μg / mL ampicillin. 24 mL of this suspension was inoculated into 4.8 L of LeMaster medium containing 50 μg / mL ampicillin, and O.D. D. Until approximately 1 was reached. A final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and after induction overnight at 20 ° C., the cells were collected by a centrifuge and the cells were collected in 200 ml of phosphate buffered saline (PBS). Then, the cells were collected again by a centrifuge and stored frozen at -20 ° C. The cells are suspended in a cell disruption buffer (50 mM sodium phosphate buffer pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin, 1 μM pepstatin A, 0.1 mg / mL lysozyme). The cells were disrupted by sonication. The sonication residue was removed by centrifugation followed by a 0.2 μm filter to obtain a cell extract.

<< Purification of H. influenzae and E. coli PBP3 transpeptidase domains >>
(1) Purification of Haemophilus influenzae PBP3 transpeptidase domain (amino acid SEQ ID NO: 2) (1-1) Purification with nickel column All the following purification operations were performed at 4 ° C. The cell extract obtained by the method of Example 6 from the recombinant E. coli culture solution (2 L) of BL21 (DE3) / pET-30a (+) _ HI_235-593 was purified by affinity chromatography. As the affinity chromatography column, Ni-NTA (manufactured by QIAGEN) was used as a carrier, and a column with a column capacity of 30 ml was prepared by the method described in the manual. After equilibration with an equilibration buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol), the cell extract was applied at a flow rate of 3 mL / min and equilibrated. After washing with the crystallization buffer, elution was performed with a linear gradient of 5-250 mM imidazole (3 column volumes) in 50 mM HEPES pH 7.5, 500 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol solution. The main fractions were collected together.

(1-2) Purification by hydrophobic interaction column The sample obtained in (1-1) above was mixed with 50 mM HEPES pH 7.5, 3.5 M ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol solution. The final concentration of ammonium sulfate was 2M. In 6 ml of RESOURCECHE, a hydrophobic interaction column equilibrated with an equilibration buffer (50 mM HEPES pH 7.5, 2 M ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) (manufactured by GE Healthcare Biosciences) Samples were applied at a flow rate of 5 mL / min. After washing with 3 column volumes of equilibration buffer, elute with a linear gradient (150 mL) of 2-0 M ammonium sulfate in 50 mM HEPES pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol solution. Summed up the minutes.

(1-3) Purification by gel filtration column The sample obtained in (1-2) above was concentrated using an ultrafiltration membrane, and gel filtration buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 1 mM fluoride). The gel filtration column Hiload 16/60 Superdex75 prep grade (manufactured by GE Healthcare Biosciences) equilibrated with phenylmethylsulfonyl and 1 mM dithiothreitol was applied at a flow rate of 1 mL / min. Each major fraction was combined and concentrated using an ultrafiltration membrane to a protein concentration of 10 mg / mL under conditions of both final concentrations of 500 mM or 100 mM sodium chloride. As a result, no precipitation occurred under any salt concentration. Further, after mixing with BOCILLIN FL (fluorescently labeled benzylpenicillin, manufactured by Molecular Probes) solution (final concentration 10 μM), the mixture was reacted at 37 ° C. for 20 minutes, and SDS-PAGE was performed. As a result of irradiating a laser having an excitation wavelength of 473 nm and detecting fluorescence having a wavelength of 520 nm or more, it was confirmed that the purified PBP3 transpeptidase domain retains the binding ability to BOCILLIN FL.

(2) Purification of Haemophilus influenzae PBP3 transpeptidase domain (amino acid sequence numbers 3 to 5, 8, and 9) (2-1) Purification with nickel column The following purification was performed at 4 ° C. B834 (DE3) / pET-28b (+) _ HI_235-597, B834 (DE3) / pET-28b (+) _ HI_242-597, B834 (DE3) / pET-28b (+) _ HI_242-593, B834 (DE3) / The cell extract obtained by the method of Example 6 from the recombinant Escherichia coli culture solution (400 mL) of pET-28b (+) _ HI_249-597 and B834 (DE3) / pET-28b (+) _ HI_249-593 was subjected to affinity chromatography. Purified with Nickel was bound to HiTrap Chelating column 5 mL (manufactured by GE Healthcare Bioscience) by the method described in the manual, and then equilibrated with equilibration buffer (20 mM sodium phosphate buffer pH 7.4, 500 mM sodium chloride, 20 mM imidazole). did. The cell extract was applied thereto at a flow rate of 2 mL / min, washed with 5 column volumes of equilibration buffer at a flow rate of 4 mL / min, and then 20 mM sodium phosphate buffer pH 7.4, 20 mM-500 mM in a 500 mM sodium chloride solution. Elution was performed with a linear gradient of imidazole (10 column volumes) and the main fractions were collected together.

(2-2) Removal of His-tag The sample purified in (2-1) above was dialyzed against a dialysis buffer (20 mM Tris-HCl pH 7.9, 200 mM sodium chloride, 1 mM EDTA), and a final concentration of 8 unit / mL thrombin (manufactured by Sigma) was added and reacted at 8 ° C. overnight, and then phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM to stop the reaction. Next, imidazole was added to a final concentration of 20 mM, and Ni Sepharose High Performance (GE Healthcare Biosciences) was equilibrated with an equilibration buffer (20 mM Tris-HCl pH 7.9, 200 mM sodium chloride, 1 mM EDTA). ) A carrier was added. After mixing for 1 hour at 4 ° C., the supernatant was collected by centrifugation.

(2-3) Purification by blue column The sample obtained in (2-2) above was purified using 5 mL of HiTrap Blue (manufactured by GE Healthcare Bioscience). The flow rate was 5 mL / min, the sample was applied to a column equilibrated with equilibration buffer (20 mM Tris-HCl pH 7.9, 200 mM sodium chloride, 1 mM EDTA), washed with 2 column volumes of equilibration buffer, Elution was performed with a linear gradient (20 column volumes) of 200-2000 mM sodium chloride in 20 mM Tris-HCl pH 7.9, 1 mM EDTA solution. SDS-PAGE was performed and the major elution fractions were summarized.

(2-4) Purification by cation exchange column The sample obtained in (2-3) above was purified using MonoS HR10 / 10 (manufactured by GE Healthcare Biosciences). The sample was dialyzed against equilibration buffer (20 mM HEPES pH 7.5, 50 mM sodium chloride, 1 mM EDTA). Samples were applied to a column equilibrated with equilibration buffer at a flow rate of 4 mL / min, washed with 2 column volumes of equilibration buffer, and then a linear gradient of 50-500 mM sodium chloride in 20 mM HEPES pH 7.5, 1 mM EDTA solution. Elute with (20 column volumes). Conduct SDS-PAGE, collect the main fractions, and use Amicon Ultra-4 30,000 MWCO (Millipore), which is an ultrafiltration membrane with a retention molecular weight of 30,000 or more, under a final concentration of 200 mM sodium chloride. To a protein concentration of 10 mg / mL. The concentrated sample was mixed with BOCILLIN FL (fluorescently labeled benzylpenicillin, manufactured by Molecular Probes) solution (final concentration 10 μM), reacted at room temperature for 20 minutes, and subjected to SDS-PAGE. As a result of irradiating a laser having an excitation wavelength of 473 nm and detecting fluorescence having a wavelength of 520 nm or more, it was confirmed that the purified sample retained the binding ability to BOCILLIN FL.

(3) Purification of Haemophilus influenzae PBP 3-point mutant transpeptidase domain (amino acid sequence numbers 15, 16, and 19) (3-1) Purification with nickel column The following purification was performed at 4 ° C. BL21 (DE3) / pET-30a (+) _ HI_235-593 (M377I, S385T, R517H), BL21 (DE3) / pET-30a (+) _ HI_235-593 (S385T, N526K) and B834 (DE3) / pET-30a The cell extract obtained by the method of Example 6 from the recombinant E. coli culture solution (400 mL) of (+) _ HI_235-593 (M377I, S385T, L389F, N526K) was purified by affinity chromatography. Nickel was bound to HiTrap Chelating column 5 mL (manufactured by GE Healthcare Bioscience) by the method described in the manual, and then equilibrated with equilibration buffer (20 mM sodium phosphate buffer pH 7.4, 500 mM sodium chloride, 20 mM imidazole). did. The cell extract was applied thereto at a flow rate of 2 mL / min, washed with 5 column volumes of equilibration buffer at a flow rate of 4 mL / min, and then 20 mM sodium phosphate buffer pH 7.4, 20 mM-500 mM in a 500 mM sodium chloride solution. Elution was performed with a linear gradient of imidazole (10 column volumes) and the main fractions were collected together.

(3-2) Purification by blue column The sample purified in (3-1) above was dialyzed against dialysis buffer (20 mM Tris-HCl pH 7.9, 50 mM sodium chloride, 1 mM EDTA), and then HiTrap Blue 5 mL (GE). It refine | purified using Healthcare Bioscience. The flow rate was 5 mL / min, the sample was applied to a column equilibrated with equilibration buffer (20 mM Tris-HCl pH 7.9, 200 mM sodium chloride, 1 mM EDTA), washed with 2 column volumes of equilibration buffer, Elution was performed with a linear gradient (20 column volumes) of 200-2000 mM sodium chloride in 20 mM Tris-HCl pH 7.9, 1 mM EDTA solution. Conduct SDS-PAGE, collect the major elution fractions, and use Amicon Ultra-4 30,000 MWCO (Millipore), which is an ultrafiltration membrane with a retained molecular weight of 30,000 or more, and a final concentration of 200 mM sodium chloride. The solution was concentrated to a protein concentration of 10 mg / mL.

(4) Purification of a selenomethionine substitution product of a modified protein (amino acid SEQ ID NO: 22) with a shortened loop structure existing in the H. influenzae PBP3 transpeptidase domain (4-1) Purification with a nickel column All the following purification operations were performed at 4 ° C. I went there. The cell extract obtained by the method of Example 9 (1) was purified by affinity chromatography. As the affinity chromatography column, Ni-NTA (manufactured by QIAGEN) was used as a carrier, and a column with a column capacity of 30 ml was prepared by the method described in the manual. After equilibration with an equilibration buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol, 1 μM leupeptin, 1 μM pepstatin A), the cell extract was flowed at a flow rate of 3 mL. / Min and washed with equilibration buffer, followed by elution buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 500 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol, 1 μM leupeptin, 1 μM pepstatin A ).

(4-2) Purification by hydrophobic interaction column The sample obtained in (4-1) above is mixed with 50 mM HEPES pH 7.5, 3.5 M ammonium sulfate 5 mM dithiothreitol solution, and the final concentration of ammonium sulfate is reduced. It was prepared to be about 1.5M. The prepared sample was equilibrated with an equilibration buffer (50 mM HEPES pH 7.5, 2 M ammonium sulfate, 5 mM dithiothreitol) in 6 ml of RESOURCECHE HE (manufactured by GE Healthcare Bioscience) at a flow rate of 5 mL / Applied in min. After washing with 3 column volumes of equilibration buffer, elution was performed with a linear gradient of 2-0 M ammonium sulfate (150 mL) in 50 mM HEPES pH 7.5, 5 mM dithiothreitol solution, and the main fractions were combined.

(4-3) Purification by gel filtration column The sample obtained in (4-2) above was concentrated with an ultrafiltration membrane, and gel filtration buffer (50 mM HEPES pH 7.5, 500 mM sodium chloride, 5 mM dithiothreitol). And applied to a gel filtration column Hiload 16/60 Superdex75 prep grade (manufactured by GE Healthcare Bioscience) at a flow rate of 1 mL / min, and the main fractions were collected.

(4-4) Purification by cation exchange column The sample obtained in (4-3) above was purified using MonoS HR10 / 10 (manufactured by GE Healthcare Biosciences). Samples were dialyzed against equilibration buffer (50 mM HEPES pH 7.5, 50 mM sodium chloride, 5 mM dithiothreitol). The sample was applied to the column equilibrated with the equilibration buffer at a flow rate of 2 mL / min, washed with 3 column volumes of the equilibration buffer at a flow rate of 3 mL / min, and then washed with 50 mM in 20 mM HEPES pH 7.5, 5 mM dithiothreitol solution. Elute with a linear gradient of -500 mM sodium chloride (75 mL). The elution fraction was mixed with BOCILLIN FL (fluorescently labeled benzylpenicillin, manufactured by Molecular Probes) solution (final concentration 10 μM), and reacted at 37 ° C. for 20 minutes to carry out SDS-PAGE. As a result of irradiating a laser having an excitation wavelength of 473 nm and detecting fluorescence having a wavelength of 520 nm or more, it was confirmed that the purified sample retained the binding ability to BOCILLIN FL. Using an ultrafiltration membrane, the protein was concentrated to a protein concentration of 10 mg / mL under the condition of a final concentration of 50 mM sodium chloride.

(5) Purification of selenomethionine-substituted product of E. coli PBP3 transpeptidase domain (amino acid SEQ ID NO: 26) (5-1) Purification by anion exchange column The following purification operations were all performed at 4 ° C. The cell extract obtained by the method of Example 9 (2) was purified by anion exchange chromatography. Using Q Sepharose HP (GE Healthcare Bioscience) as a carrier, a column with a column volume of 114 mL was prepared by the method described in the manual. After equilibration with an equilibration buffer (50 mM sodium phosphate buffer pH 6.0, 1 mM EDTA, 5 mM 2-mercaptoethanol), the sample was applied at a flow rate of 2.5 mL / min, and about 2 column volumes at a flow rate of 5 mL / min. After washing with equilibration buffer, elute with a linear gradient (720 mL) of 0-1 M sodium chloride in 50 mM sodium phosphate buffer pH 6.0, 1 mM EDTA, 5 mM 2-mercaptoethanol solution, and the major fractions by SDS-PAGE Were collected together.

(5-2) Purification by blue column The sample purified in (5-1) above was dialyzed against a dialysis buffer (20 mM Tris-HCl pH 8.5, 1 mM EDTA, 5 mM 2-mercaptoethanol), and then HiTrap Blue 1 mL. Purified using (GE Healthcare Bioscience). Purification was performed in 9 divided samples. The flow rate was 1 mL / min, the sample was applied to a column equilibrated with equilibration buffer (20 mM Tris-HCl pH 8.5, 1 mM EDTA, 5 mM 2-mercaptoethanol), and washed with 2 column volumes of equilibration buffer. Then, the main elution fractions were collected by eluting with a linear gradient (17.5 column volume) of 0-2M sodium chloride in 20 mM Tris-HCl pH 8.5, 1 mM EDTA, 5 mM 2-mercaptoethanol solution.

(5-3) Purification by gel filtration column The sample obtained in the above (5-2) is concentrated using Amicon Ultra-15 30,000 MWCO (Millipore), which is an ultrafiltration membrane having a retention molecular weight of 30,000 or more. And a gel filtration column Hiload 16/60 Superdex75 prep grade (manufactured by GE Healthcare Bioscience) equilibrated with a gel filtration buffer (20 mM Tris hydrochloric acid pH 7.9, 150 mM sodium chloride, 1 mM EDTA, 1 mM dithiothreitol). Application was performed at a flow rate of 1 mL / min. Purification was performed in two portions. The main fractions were collected, and buffer substitution and concentration were performed using Amicon Ultra-15 10,000 MWCO (Millipore), which is an ultrafiltration membrane having a retention molecular weight of 10,000 or more, and 10 mM Tris hydrochloric acid pH 7.9, 150 mM. The protein concentration was adjusted to 10 mg / mL under sodium chloride and 1 mM dithiothreitol conditions.

<< Reductive dimethylation of Haemophilus influenzae PBP3 transpeptidase domain >>
The lysine residue of the selenomethionine substitution product of Haemophilus influenzae PBP3 transpeptidase domain (amino acid SEQ ID NO: 22) purified in Example 10 (4) was reported in the literature (Biochem. Biophys. Res. Commun. 2000, 275 (2), 549- 552) and reduced dimethylated according to the method described. The obtained sample was purified using MonoS HR10 / 10 (manufactured by GE Healthcare Bioscience) which is a cation exchange column. The flow rate was 4 mL / min, the sample was applied to a column equilibrated with equilibration buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 50 mM sodium chloride), washed with 2 column volumes of equilibration buffer, and then 20 mM. Elution with a linear gradient (20 column volumes) of 50-525 mM sodium chloride in HEPES pH 7.5, 1 mM EDTA. SDS-PAGE was performed and the main elution fractions were combined, adjusted to a final sodium chloride concentration of 0.2 M, and concentrated to a protein concentration of 10 mg / mL. As a result of analysis by a mass spectrometer using an electrospray ionization method, a main peak was confirmed at a molecular weight of 39798. Therefore, this reduced dimethylated protein has a calculated molecular weight of 39807, 23 lysine residues and N It was considered that 22 sites were dimethylated in a total of 24 sites of dimethylation modification candidates at the end.

<< Crystallizing screening of H. influenzae PBP3 soluble portion, H. influenzae and E. coli PBP3 transpeptidase domains >>
The samples prepared in Examples 3, 10 and 11 were subjected to crystallization screening by the sitting drop vapor diffusion method. After mixing 0.5 μL of the protein solution and 0.5 μL of the crystallization agent to form a crystallization drop and dispensing 50-100 μL of the crystallization agent as a reservoir solution, the sealed plate is placed in a constant temperature bath at 4 ° C. to 20 ° C. I put it. As a crystallization agent, a reagent commercially available from Qiagen, Inc. was used.

The PBP3 transpeptidase domain of the present invention is a novel protein and is extremely useful industrially because it can be used for the production of high-quality crystals that can be used for obtaining crystal structure information effective in the efficient design of inhibitors and the like.

Claims (19)

A protein having transpeptidase activity selected from the group consisting of:
(A) Haemophilus influenzae penicillin binding protein (PBP) comprising a sequence beginning with any one of amino acids 225 to 262 of SEQ ID NO: 1 and ending with any one of amino acids 566 to 607 A protein consisting of three transpeptidase domains.
(B) any one of the amino acid numbers corresponding to amino acid numbers 566 to 607 starting from any one amino acid corresponding to amino acid numbers 225 to 262 of the amino acid sequence having at least 85% homology in SEQ ID NO: 1 A homologous protein consisting of a sequence ending with one amino acid.
(C) starting from any one of the amino acid numbers corresponding to amino acid numbers 225 to 262 of the amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted in SEQ ID NO: 1, and amino acid number 566 A modified protein consisting of a sequence ending with any one of the amino acid numbers corresponding to 607. A polypeptide having 3 to 5 residues starting from any one amino acid corresponding to amino acid numbers 301 to 308 in SEQ ID NO: 1 and ending with any one amino acid corresponding to amino acid numbers 315 to 322 2. The protein according to claim 1, wherein the protein is substituted with a polypeptide comprising any amino acid. The protein according to claim 1, wherein a polypeptide consisting of the sequence corresponding to amino acid numbers 306 to 317 in SEQ ID NO: 1 is substituted with a polypeptide consisting of any amino acid having 3 to 5 residues. The polypeptide consisting of the sequence corresponding to amino acid numbers 306 to 317 in SEQ ID NO: 1 is substituted with a polypeptide consisting of 4-residue amino acids which are glycine, threonine, glycine and glycine. The protein described. It begins with any one amino acid of amino acid numbers 235, 242, 249, 252 or the amino acid corresponding thereto in SEQ ID NO: 1, and any one amino acid of amino acid numbers 576, 581, 586, 593, 597 or an amino acid corresponding thereto The protein according to any one of claims 1 to 4, which ends. The protein according to any one of claims 1 to 5, comprising a sequence selected from the group consisting of SEQ ID NOs: 2 to 24. A protein having transpeptidase activity selected from the group consisting of:
(A) Escherichia coli penicillin binding protein (PBP) 3 consisting of a sequence starting from any one amino acid of amino acid numbers 205 to 242 of SEQ ID NO: 25 and ending with any one amino acid of amino acid numbers 559 to 579 A protein consisting of the transpeptidase domain.
(B) any one of the amino acid numbers corresponding to amino acid numbers 559 to 579, starting from any one amino acid corresponding to amino acid numbers 205 to 242 of the amino acid sequence having at least 85% homology in SEQ ID NO: 25 A homologous protein consisting of a sequence ending with one amino acid.
(C) starting from any one amino acid corresponding to amino acid numbers 205 to 242 of the amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted in SEQ ID NO: 25; A modified protein consisting of a sequence ending with any one of the amino acid numbers corresponding to 579. The protein according to claim 7, which consists of a sequence beginning with any one amino acid of amino acid numbers 215 and 232 or an amino acid corresponding thereto in SEQ ID NO: 25 and ending with an amino acid of amino acid number 569 or an amino acid corresponding thereto. The protein according to any one of claims 7 and 8, which consists of a sequence selected from the group consisting of SEQ ID NOs: 26 and 27. A protein in which at least one methionine residue in the protein according to any one of claims 1 to 9 is substituted with selenomethionine. A protein in which at least one lysine residue in the protein according to any one of claims 1 to 10 is reductively dimethylated. The polynucleotide which codes the protein as described in any one of Claims 1-10. A recombinant vector containing the polynucleotide according to claim 12. A host cell transformed with the recombinant vector according to claim 13. A method for producing a protein comprising the steps of culturing the host cell according to claim 14, and the step of collecting the protein according to any one of claims 1 to 11 from the host cell obtained by the culture or a culture thereof. A method for assaying a compound that binds to Haemophilus influenzae or E. coli PBP3, comprising contacting the protein according to any one of claims 1 to 11 with the compound, and then detecting whether the compound binds to PBP3. Method. A method for producing a crystal of a transpeptidase domain of Haemophilus influenzae or E. coli PBP3, comprising the step of producing the protein according to any one of claims 1 to 11, and the produced protein and a crystallizing agent. Depositing crystals of the transpeptidase domain of H. influenzae or E. coli PBP3. A primer having the sequence of any one of SEQ ID NOs: 28 to 31. A method for producing a modified protein in which a large fluctuation loop structure present in a protein of Haemophilus influenzae PBP3 transpeptidase domain is shortened, comprising the step of using the primer according to claim 18 in a gene amplification reaction method. The method to manufacture the protein as described in any one of 2-4.