JPH05115284A - Variant type human lysozyme - Google Patents

Abstract

PURPOSE:To obtain a new gene useful for producing a protein having human lysozyme activity and cell insertion activity based on an inserted amino acid sequence. CONSTITUTION:A variation type human lysozyme gene encoding a polypeptide wherein an amino acid sequence (e.g. RGDS) having cell adhesion activity is inserted between the 74-position and the 75-position of amino acid sequence of natural type human lysozyme. The gene is prepared by a method described in Japan Pat. Publication No. 1-86,876.

Description

Detailed Description of the Invention

The present invention relates to mutant human lysozyme by recombinant DNA technology and its production. More specifically, according to the present invention, an amino acid sequence having cell adhesion activity was inserted between V at position 74 and N at position 75 in the amino acid sequence of natural human lysozyme (hereinafter, also simply referred to as human lysozyme). Mutant human lysozyme gene encoding mutant human lysozyme, expression vector containing the gene, transformant transformed with the expression vector, human lysozyme activity and insertion using the transformant The present invention also relates to a method for producing a protein having cell adhesion activity based on the amino acid sequence, and a mutant human lysozyme produced in this manner.

[0002]

2. Description of the Related Art Lysozyme is an enzyme protein having antibacterial activity discovered by Fleming in 1922, and acts on peptidoglycan of bacterial cell wall to produce N-acetylglucosamine (NAG) and N-acetylmuramic acid.
Specific cleavage of the β-1,4 bond with (NAM) causes lysis. Its distribution is animals, plants,
It is widely recognized in bacteriophages and found mainly in humans in milk, tears, urine, etc., but its physiological significance has not been completely clarified. Human lysozyme is a simple protein consisting of 130 amino acids and has 4 pairs of disulfide bonds in the molecule. Its three-dimensional structure was analyzed by Artymiuk and Blake (PJ Artymiuk, CCFBlake, J. Mol. Biol., 152
737 1981), and it is known that it has a molecular structure almost equal to that of chicken egg white lysozyme, which has already been subjected to complete X-ray crystallography. On the other hand, it has been already known that by DNA recombination technology, a DNA encoding a protein in which human lysozyme is connected to a signal peptide of egg white lysozyme is chemically synthesized, and this produces a protein identical to human lysozyme in a yeast expression system. (K. Yoshimura et al. BBRC 145 712 1987, Y.
Jigami et al. Gene 43 273 1986). To date, various mutant human lysozymes have been created by this method, and many results have been obtained regarding the structure-function relationship, stability and folding mechanism of proteins using lysozyme (Y. Tan.
iyama et al. BBRC. 152 962 1988, Y. Taniyama et
al. JBC. 265 7570 1990).

On the other hand, fibronectin is collagen,
Similar to elastin, proteoglycan, laminin, vitronectin, and fibrin, it is one of the constituent components of the extracellular matrix that surrounds cells, and it has many parts that interact with cells such as heparin, fibrin, and collagen. It is a functional glycoprotein.
It consists of two subunits of about 220 KDa, and is said to have a molecular weight of about 440 KDa as a whole. The interaction of fibronectin with cells, that is, the function of cell adhesion, is not only essential for the development and morphogenesis of multicellular organisms, but also clinically important in the field of pathologies such as wound healing, thrombus formation, and cancer metastasis. It is known to play various roles. In addition, from the study of Pierschbacher and Ruoslahti using synthetic peptides of fibronectin (MD Pierschbacher & E. Ruoslahti, Nature
309 30 1984), it was shown that the binding site for fibronectin to cells consists of a simple sequence RGD (Arg-Gly-Asp), and this cell adhesion signal is commonly present in fibrinogen, laminin, collagen, vitronectin and the like. And is working. Furthermore, a gene was identified for the cell surface receptor for fibronectin,
It shares homology with other extracellular matrix receptors and is called the integrin family (E. Ruosl.
ahti, Ann. Rev. Biochem. 57 375 1988). In this way, it was extremely interesting that the important biological function of cell adhesion could be converged to the simple sequence of RGD, and information on the three-dimensional structure of this adhesion signal is awaited. Peptides known to inhibit the cell adhesion activity of fibronectin and the binding to the fibronectin receptor (eg GRGDDSP) have cell adhesion activity by themselves. GRGDSP peptide is ¹ H
-It has been reported from NMR spectrum analysis that it has a unique secondary structure containing two β-turns (J. Reed et a
l. EJB. 178 141 1988), the peptide is originally highly fluid, and the peptide is bound to IgG via a linker when measuring the cell adhesion activity, and whether this secondary structure reflects the structure at the time of activity expression. Is doubtful. Moreover, since fibronectin has an extremely large molecular weight, it can be easily inferred that structural analysis is extremely difficult.

Maeda et al.
DS tetrapeptide is a truncated form of protein A
(Truncated type) and succeeded in creating a novel IgG-binding cell adhesion protein, paving the way for the creation of a multifunctional protein (T. Maeda et al. JBC. 264 15165 1989). Nevertheless, this protein A-RGDS chimeric protein has a molecular weight of 30 KDa or more, and its structure has not been analyzed. Shimojo et al. Transplanted the RGDS peptide into domain 1 of calpastatin, which is a calpain-specific inhibitor of the Ca ²⁺ -dependent thiol protease.
(Tomoko Shimojo et al., 2nd Annual Meeting of the Protein Society of Japan p66199
0). This calpastatin-RGDS chimeric protein has cell adhesion activity and has a relatively small molecular weight of 15 KDa,
It seems to be suitable for structural analysis. However, the structure of calpastatin domain 1 was determined by NMR spectrum, CD
When analyzed by spectrum, it was confirmed that a solid three-dimensional structure was not formed.

[0005]

From the above viewpoints, the present inventors have aimed to create a chimeric protein capable of analyzing the higher-order structure of a cell adhesion functional site containing an RGD sequence, and A multifunctional chimeric protein having a peptide containing an RGD sequence inserted was created. The present inventors have already completed a precise X-ray crystallographic analysis of human lysozyme, and the whole three-dimensional structure thereof has been clarified.
If the human lysozyme-RGD chimeric protein has a similar three-dimensional structure to that of human lysozyme, it is considered that structural analysis of the inserted peptide portion containing the RGD sequence will be extremely easy. Furthermore, if information on this three-dimensional structure is obtained, agonists and antagonists for cell adhesion signals having RGD sequences could be designed with peptides or low molecular weight organic compounds. Moreover, since the phenomenon of cell adhesion is closely related to wound healing, thrombus formation, and cancer metastasis, it is considered that there is a good possibility that the designed agonists and antagonists will be used in clinical applications. Further, regarding the human lysozyme-RGD chimeric protein itself, the antibacterial action derived from lysozyme and RGD
Since it also has a wound healing action derived from the sequence, it can be expected to be clinically applied as a therapeutic drug having a bactericidal action at the time of corneal injury, for example.

Regarding the insertion position of the peptide containing RGD, the present inventors have determined that the amino acid sequence of human lysozyme indicates R
A site that is easily converted into a GD sequence and a site that is exposed on the surface of the molecule due to its three-dimensional structure and does not affect the lytic activity were selected. As a result, the 21 to 24 position RG I S RG D
Mutant converted to S (21RGD4), V at position 74 and 75
Mutant in which RGDS was inserted between N at position (74RGD
4), 101-104 of variants of R DPQ was converted into R GDS of (101RGD4), and 119 to 121 of the RDV was converted to R G DV variant (119RGD4)
Were produced using a yeast secretion expression system. Of these, 21
Three mutants except RGD4 were secreted into the medium.
Cell adhesion activity was not observed with 101RGD4, 119
It was also extremely low in RGD4, but 74RG
A fairly strong activity was observed in D4. So next, 7
A mutant in which a peptide having a sequence around the active site of fibronectin added between V in position 4 and N in position 75, that is, a mutant in which GRGDSP is inserted (74RGD
6), a mutant in which TGRGDSPA was inserted (74RGD
8), a mutant in which VTGRGDSPAS was inserted (74RG
D10) and a mutant (74RGD12) in which AVTGRGDSPASS was inserted were produced, and the cell adhesion activity was 74RGD4 <74RG while the lytic activity was retained.
D6 <74RGD8 <74RGD10 <74RGD12
It increased in order. Thus, the present inventors have completed the present invention by creating a novel multifunctional protein in which human lysozyme is newly imparted with RGD-derived cell adhesion activity, and at the same time, the three-dimensional structure analysis of the RGD sequence was performed. Opened the way.

That is, the present invention provides a mutation encoding a polypeptide in which an amino acid sequence having cell adhesion activity is inserted between V at position 74 and N at position 75 of the amino acid sequence of natural human lysozyme. Provides a human lysozyme gene. The inserted amino acid sequence is R
GDS, GRGDSP, TGRGDSPA, VTGRG
Examples include DSPAS and AVTGRG DSPASS. The present invention also provides an expression vector containing the above-mentioned mutant human lysozyme gene. The present invention further provides a host cell transformed with the above expression vector and capable of producing and secreting mutant human lysozyme. Furthermore, the present invention comprises culturing the above-mentioned transformed host cell, isolating a protein having a human lysozyme activity secreted into the culture medium and a cell adhesion activity based on the inserted amino acid sequence, and purifying it if desired. The present invention provides a method for producing mutant human lysozyme and a mutant human lysozyme produced by this method.

In the present specification, amino acids are described by the one-letter code system shown below. Q Glutamine F Phenylalanine M Methionine P Proline N Asparagine H Histidine D Aspartic Acid G Glycine I Isoleucine K Lysine T Threonine A Alanine L Leucine R Arginine S Serine C Cystine Y Tyrosine W Tryptophan V E

Preparation of the mutant human lysozyme gene of the present invention, construction of an expression vector containing the gene, transformation of host cells with the expression vector, and production of mutant human lysozyme using the transformant obtained All were performed according to the method disclosed in Japanese Patent Application Laid-Open No. 1-86876 filed by the present applicant.

That is, as shown in FIG. 1, a known plasmid pERI8602 containing a synthetic gene encoding the amino acid sequence of natural human lysozyme (JP-A-1-8).
No. 6876), the XhoI-SmaI restriction fragment was excised. On the other hand, a double-stranded synthetic oligomer encoding the amino acid sequence part to be inserted was synthesized, and M13mp19
hinc of hLZM RF DNA (JP-A-1-86876)
M13mp19 74RGD4 RF inserted into the II site
DNA, M13mp19 74RGD6 RF DNA, M
13mp19 74RGD8 RF DNA, M13mp19
74RGD10 RF DNA and M13mp19 74
Obtain RGD12RF DNA. This is XhoI and Sm
XhoI-S in which a nucleotide sequence encoding a desired amino acid sequence was inserted by digestion with aI
Obtain the maI restriction fragment. This XhoI-SmaI fragment was pER
Ligated with the XhoI-SmaI fragment of I8602,
Expression plasmid, pGEL74RGD4, pGEL74R
GD6, pGEL74RGD8, pGEL74RGD10
And pGEL74RGD12 are constructed. All of these plasmids were constructed in the same way, but typically pG
A schematic diagram of the construction of EL74RGD4 is shown in FIG.

The individual operations in the series of operations outlined above are well known to those skilled in the art. For example, transformation of E. coli, such as in DNA cloning, is described by Cohen.
(Cohen) et al. [Cohen, SN et al., Proceeding of the National Academy of Science (Prohen
c.Natl.Acad.Sci.USA) 69 2110 (1972)]. Examples of E. coli hosts include E. coli T
G-1, E. coli DH-1, E. coli 294 and the like can be used.

Further, in order to isolate the plasmid DNA having the desired gene inserted therein from the transformant, an alkaline extraction method [Birnboim, HC and Doly, J., Nucleic Acids Res. 7 1513 (1979)]
Etc. can be used. Then plasmid DNA
Is treated with an appropriate restriction enzyme to excise the inserted gene, and the gene is isolated by, for example, agarose gel electrophoresis or polyacrylamide electrophoresis. These series of operations are known, and are referred to in literatures such as "Molecular Cloning".
(1982), Cold Spring Harbor Laboratory ”.

Chemical synthesis of DNA can be carried out, for example, by the method of Crea et al.
[Crea, R. et al., Proceeding of the National
Academy of Science (Proc.Natl.Acad.Sci.US
A) 75 765 (1978)] and the like.

Further, a commercially available kit (for example, a kit manufactured by Amersham) is used to specifically mutate a desired site of DNA, and a dideoxynucleotide synthetic chain termination is used to confirm the sequence. Law [Sanger, F.
Et al., Proc. Natl. Acad. Sci. USA 74 54
63 (1977)] is used.

In the present specification, an example of construction of the expression plasmid of the present invention using the expression plasmid pERI8602 encoding natural human lysozyme was shown, but the present invention is not limited thereto.

In the case of yeast host, examples of promoters include PHO5 promoter, GLD promoter, PGK promoter, ADH promoter, PHO.
81 promoter, GAL1 promoter, GAL10
When the promoter is an animal cell host,
As the promoter, for example, SV40 early gene promoter, metallothionein promoter, heat shock promoter, etc. can be used. The use of enhancers for expression is also effective.

The plasmid pGEL74RGD of the present invention
4, pGEL74RGD6, pGEL74RGD8, pG
EL74RGD10 and pGEL74RGD12 are
It is suitable for expressing and secreting mutant human lysozyme in a yeast host. A particularly preferred host is Saccharomyces cerevisiae AH.
22.

By inserting the mutant human lysozyme gene of the present invention into a vector for animal cells, mouse L cells,
Chinese hamster ovary cells (CHO), as well as other eukaryotic cells, can be used as hosts.

Methods for transforming eukaryotic cells are known to those skilled in the art. For example, yeast can be transformed by the method of Hinnen et al.
[Procedures of the National Academy of Science (Proc.Natl.Acad.Sci.USA) 75 19
27 (1978)], and transformation of eukaryotic cells was carried out by the method described in "Protein / Nucleic Acid / Enzyme, Vol. 28, 1983," Introduction and Expression of Recombinant Gene into Cell "(Kyoritsu Shuppan). It can be carried out.

Cultivation of the transformant can be carried out by any method known to those skilled in the art. When yeast is used, the medium may be, for example, Burkholde.
r) Minimum medium [Bostian, KL, et al., Procedures of the National Academy of Science USA, 77 4505 (1980)]. Culturing is usually at 15 ° C to 40 ° C, preferably 24 ° C to 37 ° C for 10
It is performed for 144 hours, preferably 24 to 96 hours, and aeration and stirring may be added if necessary.

When a transformant in which a eukaryotic cell such as an animal cell is used as a host, the medium is, for example, MEM [H. Eagle, Science 1 ] of Eagle.
30 432 (1959)], Modified Eagle's Medium of Dulbecco [Orgad Laub and Will
iam J. Rutter, Journal of Biological Chemistry (J. Biol. Chem.) 258 6043 (1983)]. Culturing is usually 30 to 42 ° C, preferably 35 ° C to
Perform at 37 ° C. for about 1-10 days.

After completion of the culture, the cells and the supernatant are separated by a method known to those skilled in the art. According to the expression vector of the present invention, the produced mutant human lysozyme is efficiently secreted and thus can be obtained from the supernatant, but when it remains in the cell, a usual method in the art, for example, an ultrasonic disruption method is used. , Crushing method using French press, mechanical crushing method such as grinding,
The cells are disrupted by a disruption method using a cytolytic enzyme and then extracted. If necessary, a surfactant such as Triton-X100 or deoxycholate is added to extract the produced mutant human lysozyme. The obtained mutant human lysozyme is subjected to a conventional protein purification method such as salting out,
Isoelectric point precipitation, gel filtration, ion exchange chromatography, high performance liquid chromatography (HPLC, FPLC
Etc.) and the like.

The culture supernatant separated from the thus obtained transformant culture and human lysozyme in the cells were purified and isolated, and their activity was measured by the assay method described below. It was found that human lysozyme expresses strong cell adhesion activity while retaining lytic activity.

That is, the expression vector containing the mutant human lysozyme gene of the present invention is introduced into an appropriate host, the resulting transformant is cultured under appropriate conditions, and the culture supernatant of the protein having human lysozyme activity. Can be isolated and, if desired, purified by a conventional method to obtain a mutant human lysozyme having lytic activity and cell adhesion activity.

Hereinafter, the present invention will be described in more detail with reference to examples. The following examples are merely examples,
It does not limit the invention in any way.

Example 1 Preparation of Synthetic Oligonucleotides Corresponding to Peptides Containing RGD Sequence The 10 kinds of oligonucleotides shown in Table 1 were synthesized respectively using an Applied Biosystems DNA Synthesizer 380B to prepare a trityl group. Was obtained in a deprotected state.

[0027]

[Table 1]

This oligonucleotide was treated with ammonia at 55 ° C. for 6 hours and dried to give 5 OD ₂₆₀ in 50
Loaded on 10% polyacrylamide gel electrophoresis with% urea. After migration, remove the glass plate and TLC plate
U on (Merck Art5554DC-Alutolien Kieselgel-60F ₂₅₄ )
Irradiate the V lamp to cut out the desired band, and
5M triethylammonium hydrogen carbonate solution (0.5M T
The sample was immersed in EAB (for Wako nucleic acid synthesis) (5 ml) at room temperature overnight to elute from the gel. Next, the eluted oligonucleotide was loaded on a SEP-PAK ^R C18 cartridge (manufactured by Waters) and washed with 25 mM TEAB (15 ml), and then 17% acetonitrile-0.1 M TEAB (4 m
It was eluted with l) and dried. The 5'-phosphorylation of the oligonucleotide is 50 mM Tris-HCl (pH 8.0) -10 mM.
The amount of purified synthetic oligonucleotide 0.055 OD _{260 in} MgCl ₂ -5 mM DTT solution (20 μl) was added with T4 polynucleotide kinase (Takara Shuzo) (10 unit) at 37 ° C. for 45 minutes.
The kinase was inactivated by heat treatment at 95 ° C for 5 minutes.

Example 2 M13mp19hLZM RF D
Preparation of HincII digested fragment from NA 50 mM Tris-HCl (pH 7.5) -10 mM MgCl ₂ −
M13mp19h into which wild type human lysozyme gene was inserted in 1 mM DTT-100 mM NaCl solution (70 μl)
HincII (manufactured by Takara Shuzo) on LZM RF DNA (15 μg)
(60 units) was allowed to act at 37 ° C for 1 hour. Next, 1 M Tris-HCl (pH 8.0) (8 μl) and alkaline phosphatase (derived from E. coli C75) (0.5 unit) were added to the reaction solution, and the mixture was reacted at 55 ° C. for 30 minutes. After phenol treatment with phenol / chloroform / isoamyl alcohol (24/24/1), gel electrophoresis was performed using 0.8% low melting point agarose (SeaPlaque ^R agarose: manufactured by FMC) to remove HincII digested fragments. Purified.

Example 3 M13mp19 74RGD4 R
Preparation of F DNA M13mp19hLZM RF HincI obtained in Example 2
I digested fragment (5 μg) and 74 obtained in Example 1
For RGD4, ethanol precipitation was carried out after adding 0.055 OD ₂₆₀ amounts of each two kinds of synthetic oligonucleotides. Next, T4 DNA ligase (Takara Shuzo) (350unit)
66 mM Tris-HCl (pH 7.6) -6.6 mM
MgCl ₂ -10 mM DTT-0.1 mM ATP solution (20
μl) at 16 ° C. overnight, and then E. coli TG-1
The strain was transfected. M13mp RF DNA was prepared from the obtained clones by the alkali extraction method.
Screening by HphI digestion was performed from among these clones, and finally, a desired clone was selected by DNA sequencing using a 7-Deaza Seakenes Kit manufactured by Toyobo.

Example 4 Expression vector pGEL74R
Preparation of GD4 10 mM Tris-HCl (pH 8.0) -7 mM MgCl ₂ -2
0 mM KCl-7 mM 2-mercaptoethanol solution (1
5 μl), M13mp1974RG obtained in Example 3
D4 RF DNA (2 μg each) with SmaI (Takara Shuzo) (10un
it) was allowed to act for 45 minutes at 30 ° C. Further 1.5 M NaCl (1.3 μl) and XhoI (Takara Shuzo) (10 u were added to this reaction solution).
nit) and add 20 μl with distilled water, then at 37 ℃ for 45
Let react for minutes. The obtained XhoI-SmaI fragment was purified by 1% agarose electrophoresis, and then 4000 xg using Millipore Ultra Free C3HV,
Centrifugation was carried out twice for 10 minutes and collected. Similarly pE
RI8602 (3 μg) was digested with XhoI-SmaI, and the XhoI-SmaI fragment of the resulting vector was purified by 0.8% low melting point agarose gel electrophoresis. Xho of M13mp1974RGD4 RF DNA thus obtained
The I-SmaI fragment (100 ng) and the XhoI-SmaI fragment (300 ng) of the pERI8602 vector were ligated using the DNA ligation kit manufactured by Takara Shuzo, and then E. coli DH
-1 strain competent cells were transfected. The obtained clones were screened by restriction enzyme digestion to obtain the desired plasmid DNA (pGEL74RGD
4) was obtained.

Example 5 Preparation of yeast transformant Expression plasmid pGEL74RGD4 obtained in Example 4
Saccharomyces cerevisiae AH22 strain (leucine auxotrophic) was transformed with S. cerevisiae AH2.
2 / pGEL74RGD4 was obtained. Secretion expression of mutant human lysozyme (74RGD4) in transformants was
It was confirmed by measuring halo activity on agarose plates containing icrococcus cells and lytic activity in the culture supernatant in liquid culture. Transformant S. cerev
isiae AH22 / pGEL74RGD4 as well as S. cerevisiae AH22 / pGEL74RGD obtained in a similar manner
8 and S. cerevisiae AH22 / pGEL74RGD
No. 12 has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology (accession number: Micromachine Research Institute No. 12450,
No. 12451, No. 12452, Contract date: August 1991
27th of a month).

EXAMPLE 6 Cultivation of yeast transformant S. cerevisiae obtained in Example 5 cerevisiae AH2
2 / pGEL74RGD4 in Burkholder's Modified Medium I
II (per 1 L, KH ₂ PO ₄ (0.4 g), glucose (1
0 g), asparagine (5 g) and sucrose (80 g)) (5 ml) were inoculated into the test tube, and cultured at 30 ° C. for 3 days with shaking. The obtained culture solution (4 ml) was added to the same medium III (1
It was transferred to a 200 ml volume Kolben containing 6 ml) and cultured at 30 ° C. for 1 day with shaking. This culture solution (20 ml) was added to the same medium III
(180 ml) and transferred to a 500 ml Kolben and cultured at 30 ° C. for 4 days with shaking.

Example 7 Mutant Human Lysozyme (74R
Purification of GD4) From the culture solution obtained in Example 6, a culture supernatant was obtained by centrifugation. Next, 50 mM sodium phosphate buffer (pH)
CM-Toyopearl 650M column equilibrated in 6.5)
(1.6 cmφ × 5 cm, 10 ml) was loaded with the culture supernatant at a flow rate of 20 ml / h. After washing with the same buffer (50 ml),
6M NaCl-50 mM sodium phosphate buffer (pH 6.
Elution was performed using 5) at a flow rate of 10 ml / h. This eluate was obtained by a diaflow device using a YM5 membrane.
(10 ml) was concentrated and desalted to finally give a sample solution of 50 mM sodium phosphate buffer (pH 6.5) (1 ml). Furthermore, Asahipak ES-502C column (7.6 mmφ × 10
High performance liquid chromatography using (cm) was performed under the following conditions. Solution A: 50 mM sodium phosphate (pH 6.5),
Solution B: 0.6M Na ₂ SO ₄ -50 mM sodium phosphate (p
H6.5), elution conditions: 0 minutes (% A85,% B15) -45 minutes
(% A70,% B30) -50 minutes (% A0,% B100), flow rate: 1 ml / min. Under these conditions, mutant human lysozyme 74
RGD4 was eluted in 30-40 minutes. This fraction was collected, dialyzed thoroughly against distilled water, and then freeze-dried to obtain a final preparation. 74RGD4 and 74RGD8 S
DS-polyacrylamide gel electrophoresis (gel concentration 15
%) Shows the migration pattern in FIG. In FIG. 2, (A) was performed under reducing conditions, (B) was performed under non-reducing conditions, lane 1 was a molecular weight marker (2 μg), lane 2 was natural human lysozyme (3 μg), lane 3 was 74RGD4 (3 μg),
Lane 4 is the migration pattern of 74RGD8 (3 μg).

Example 8 Mutant Human Lysozyme 74RG
Measurement of lytic activity of D4, 74RGD6, 74RGD874RGD10 and 74RGD12 In the same manner as in Examples 2 to 7, mutant lysozyme 74RG.
D6, 74RGD8, 74RGD10 and 74RGD
12 was prepared, and 0.1M potassium phosphate buffer (pH 6.
2) (2 ml) Micrococcus luteus (manufactured by Sigma) (0.4
(mg) was used as a substrate for reaction at 25 ° C. for 3 minutes, and the decrease in absorbance at 450 nm was measured to measure the lytic activity. As the standard human lysozyme, a human urine-derived natural human lysozyme manufactured by Green Cross was used. The amount of human lysozyme protein in the sample solution was determined based on the absorbance at 280 nm. The results obtained by measuring the lytic activity three times were as shown in Table 2 below.

[0036]

[Table 2]

Example 9 Mutant Human Lysozyme 74RG
Measurement of cell adhesion activity of D4, 74RGD6, 74RGD874RGD10 and 74RGD12 1) Preparation of sample The sample was diluted with 0.1M NaHCO ₃ and 400 μl per well was added to a Terumo 24-well flat plate and adsorbed at 4 ° C. overnight. Let After washing with PBS, PB containing 1% BSA
After adding S (500 μl) and incubating at room temperature for 3 to 4 hours, it was washed with PBS and used for measurement of adhesive activity.

2) Preparation of cell suspension BHK cells in the subculture were treated with 0.25% trypsin, 0.2%.
Incubate for 2 minutes at 37 ° C in PBS containing 02% EDTA, peel and then ice-cool DME / HEPE.
Suspended in S saline (1: 1) (10 ml). here,
DME represents Dulbecco's minimal medium and HEPES saline represents 20 mM HEPES buffer (pH 7.1) containing 137 mM NaCl and 3 mM KCl.
The supernatant was removed by centrifugation and ice-cooled DME / HEPES containing STI (soybean trypsin inhibitor) (1 mg) was added.
The cells were suspended in physiological saline (10 ml). After centrifugation and washing with STI-free ice-cold DME / HEPES physiological saline, cells were suspended in the same solution at 1 × 10 ⁵ cells / ml.

3) Measurement of adhesive activity Cell suspension (300 μm) was applied to the plate on which the above sample was adsorbed.
l) was added and incubated at 37 ° C. for 1 hour. 37 ° C
Non-adsorbed cells were removed by washing 3 times with DME / HEPES physiological saline solution, and the remaining cells were fixed with 4% formalin / PBS, and cell extension was observed under a microscope.

The measurement results are shown in FIG. The horizontal axis represents the concentration of the sample protein added to the measurement system, and the vertical axis represents the number of cells in which spreading was observed. From FIG. 3, the cell adhesion activity was 74RGD4 <74RGD6 <74RGD8 <74RG.
It was observed that the order of increase was D10 <74RGD12.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the construction of a vector pGEL74RGD4 for expressing mutant human lysozyme in yeast.

FIG. 2 Mutant human lysozyme 74RGD4 and 8
FIG. 3 is a copy diagram showing the results of SDS-polyacrylamide gel electrophoresis of the above.

FIG. 3 Mutant human lysozyme 74RGD4, 74R
GD6, 74RGD8, 74RGD10 and 74RG
It is a graph which showed the measurement result of the cell adhesion activity of D12.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 865)

Claims (20)

[Claims] 1. A mutant human lysozyme gene encoding a polypeptide in which an amino acid sequence having cell adhesion activity is inserted between V at position 74 and N at position 75 of the amino acid sequence of natural human lysozyme. .. 2. The mutant human lysozyme gene according to claim 1, wherein the inserted amino acid sequence having cell adhesion activity is RGDS. 3. The mutant human lysozyme gene according to claim 1, wherein the inserted amino acid sequence having cell adhesion activity is GRGDSP. 4. The mutant human lysozyme gene according to claim 1, wherein the inserted amino acid sequence having cell adhesion activity is TGRGDSPA. 5. The mutant human lysozyme gene according to claim 1, wherein the inserted amino acid sequence having cell adhesion activity is VTGRGDSPAS. 6. The mutant human lysozyme gene according to claim 1, wherein the inserted amino acid sequence having cell adhesion activity is AVTGRGDSPASS. 7. An expression vector containing the mutant human lysozyme gene according to claim 1 and capable of autonomously replicating in a eukaryote. 8. The expression vector according to claim 7, which is the plasmid pGEL74RGD4. 9. The expression vector according to claim 7, which is the plasmid pGEL74RGD6. 10. The expression vector according to claim 7, which is the plasmid pGEL74RGD8. 11. The expression vector according to claim 7, which is the plasmid pGEL74RGD10. 12. The expression vector according to claim 7, which is the plasmid pGEL74RGD12. 13. A host cell capable of producing and secreting a mutant human lysozyme transformed with the expression vector according to claim 7. 14. A transformant Saccharomyces cerevisia.
14. The host cell of claim 13, which is eAH22 / pGEL74RGD4. 15. Transformant Saccharomyces cerevisia
14. The host cell of claim 13, which is eAH22 / pGEL74RGD6. 16. A transformant Saccharomyces cerevisia
14. The host cell of claim 13, which is eAH22 / pGEL74RGD8. 17. A transformant Saccharomyces cerevisia.
e AH22 / pGEL74RGD10.
The host cell according to. 18. A transformant Saccharomyces cerevisia
e AH22 / pGEL74RGD12.
The host cell according to. 19. The transformant according to any one of claims 13 to 18 is cultured, and a protein having a human lysozyme activity secreted into the culture medium and a cell adhesion activity based on the inserted amino acid sequence is isolated. A method for producing a mutant human lysozyme, which comprises purifying if desired. 20. A mutant human lysozyme produced by the method according to claim 19.