CN113528482B - High-activity heparin skeleton synthase PmHS2 mutant and application thereof - Google Patents

High-activity heparin skeleton synthase PmHS2 mutant and application thereof Download PDF

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CN113528482B
CN113528482B CN202110881483.0A CN202110881483A CN113528482B CN 113528482 B CN113528482 B CN 113528482B CN 202110881483 A CN202110881483 A CN 202110881483A CN 113528482 B CN113528482 B CN 113528482B
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CN113528482A (en
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生举正
孙柳
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Shandong University
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract

The invention relates to a high-activity heparin skeleton synthase PmHS2 mutant and application thereof. The mutant is any one of position mutation of 130 th serine, 135 th serine, 197 th phenylalanine, 326 th threonine, 344 th threonine and 476 th isoleucine of the heparin skeleton synthase PmHS 2. The heparin skeleton synthase PmHS2 mutant provided by the invention has good stability, and can still keep more than 40% of enzyme activity after being placed at 37 ℃ for 72 hours, which is more than 2 times of the existing heparin skeleton synthase PmHS 2. In particular to a heparin skeleton synthase mutant PmHS2 (F197Y), a heparin skeleton synthase mutant PmHS2 (S135A) and a protein expressed by the heparin skeleton synthase mutant PmHS2 (T344S), which can still keep more than 80 percent of enzyme activity after being placed for 72 hours at 37 ℃ and is 5 to 7 times of the existing heparin skeleton synthase PmHS 2.

Description

High-activity heparin skeleton synthase PmHS2 mutant and application thereof
Technical Field
The invention relates to a high-activity heparin skeleton synthase PmHS2 mutant and application thereof, belonging to the technical field of biology.
Background
Glycosaminoglycans (GAGs) are a complex biological macromolecule, a linear unbranched acidic polysaccharide formed by a regular arrangement of repeating disaccharide units consisting of hexuronic acid or hexose and hexosamine. Glycosaminoglycans are widely distributed and varied, and are classified into sulfate-free glycosaminoglycans and sulfate-modified glycosaminoglycans according to the degree of sulfation, with only one type of hyaluronic acid, whereas sulfate-modified glycosaminoglycans include dermatan sulfate/chondroitin sulfate, heparan sulfate/heparin, keratan sulfate, and the like.
Heparin (HP) is an important glycosaminoglycan, and is composed of D-beta-glucuronic acid (or L-alpha-iduronic acid) and N-acetylglucosamine to form a repeating disaccharide unit. It is named as it was first discovered from the liver that heparin has the activity of enhancing the natural serine protease inhibitor antithrombin iii, catalyzes the inhibition of all serine proteases of the intrinsic coagulation pathway, and thus has an anticoagulant antithrombotic effect, and can also mediate a variety of important physiological processes including cell adhesion, lipid metabolism and growth factor regulation by binding other proteins. Can also be used for treating angina pectoris, nephrotic syndrome, serious burn, rheumatoid arthritis, etc., and the demand is internationally in the forefront of biotechnology pharmaceutical field throughout the year.
Heparin sources are mainly classified into animal extraction, chemical synthesis and chemoenzymatic synthesis. Animal heparin is mainly extracted from small intestine mucous membranes of pigs and cattle, but the heparin product has different structure, configuration and molecular weight due to the difference of extraction methods and different numbers of basic units of heparin from animal sources, which may cause heparin pollution. In addition, heparin has a complex structure, and when the sugar chain is prolonged by using a chemical synthesis method, the steps of protecting, deprotecting, activating, coupling and the like on an unstable group are considered, and the conditions of regioselectivity, stereoselectivity and the like are also considered, so that the difficulty is high and the yield is low. The chemical enzymatic synthesis of HP is a method combining a chemical method and an enzymatic method, and the enzymatic reaction condition required in the synthesis process is mild and the specificity is strong. Compared with chemical synthesis, the method has simple steps and high efficiency. The enzymes commonly used for synthesizing the heparin skeleton sugar chain by the chemical enzyme method at present comprise heparin skeleton synthase KfiA and heparin skeleton synthase PmHS2, wherein the heparin skeleton synthase PmHS2 is a bifunctional enzyme with N-acetylglucosamine transferase activity and glucuronic acid transferase activity, and the enzyme has wide application in the chemical enzyme method synthesis, and the improvement of the activity of the enzyme is helpful for the efficient synthesis of the heparin skeleton sugar chain.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a high-activity heparin skeleton synthase PmHS2 mutant and application thereof.
Description of the terminology:
GlcA-pNP: chinese is known as 4-nitrophenyl- β -D-glucuronic acid, which acts as an initial acceptor substrate for heparin oligosaccharide synthesis;
UDP-GlcNAc: chinese is called uridine diphosphate-N-acetamido glucose, which serves as a donor to provide acetylglucosamine for heparin oligosaccharide synthesis;
the technical scheme of the invention is as follows:
the mutant of the high-activity heparin skeleton synthase PmHS2 is any one of 130 th serine, 135 th serine, 197 th phenylalanine, 326 th threonine, 344 th threonine and 476 th isoleucine of the heparin skeleton synthase PmHS 2.
According to the invention, the mutant is a heparin skeleton synthase PmHS2 mutant (S130T), the amino acid sequence of the mutant is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1; the 130 th serine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into threonine.
According to the invention, the mutant is a heparin skeleton synthase PmHS2 mutant (S135A), the amino acid sequence of the mutant is shown as SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 3; the 135 th serine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into alanine.
According to the invention, the mutant is a heparin skeleton synthase PmHS2 mutant (F197Y), the amino acid sequence of the mutant is shown as SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 5; the 197 th phenylalanine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into lysine.
According to the invention, the mutant is a heparin skeleton synthase PmHS2 mutant (T326S), the amino acid sequence of the mutant is shown as SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 7; the 326 th threonine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into serine.
According to the invention, the mutant is a heparin skeleton synthase PmHS2 mutant (T344S), the amino acid sequence of the mutant is shown as SEQ ID NO.10, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 9; the 344 th threonine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into serine.
According to the invention, the mutant is a heparin skeleton synthase PmHS2 mutant (I476F), the amino acid sequence of which is shown as SEQ ID NO.12, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 11; the 476 th isoleucine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into phenylalanine.
A recombinant vector is obtained by inserting the coding gene of the heparin skeleton synthase PmHS2 mutant into a plasmid vector.
According to a preferred embodiment of the invention, the plasmid vector is pET-28a (+).
In the invention, the recombinant vector containing the PmHS2 mutant coding gene is synthesized by Nanjing Jinsrui company.
A recombinant strain is obtained by transforming the recombinant vector into a host cell.
Preferably, according to the invention, the host cell is E.coli; further preferably, the host cell is E.coli BL21 (DE 3).
The application of the heparin skeleton synthase mutant in the synthesis of heparin skeleton.
According to the invention, preferably, the application is to catalyze the reaction to generate a heparin skeleton with a structure of GlcNAc-GlcA-pNP by taking UDP-GlcNAc as a donor substrate and GlcA-pNP as an acceptor substrate; or the UDP-GlcA is used as a donor substrate, the GlcNAc-GlcA-pNP is used as an acceptor substrate, and the heparin skeleton with the structure of GlcA-GlcNAc-GlcA-pNP is generated by catalytic reaction.
Experimental procedures not described in detail in the present invention may be performed according to conventional experimental procedures in the art.
Advantageous effects
1. The invention is based on heparin skeleton synthase PmHS2, and selects 130 th, 135 th, 197 th, 326 th, 344 th and 476 th amino acids to carry out site-directed mutagenesis, mutates 130 th serine into threonine (S130T), mutates 135 th serine into alanine (S135A), mutates 197 th phenylalanine into lysine (F197Y), mutates 326 th threonine into serine (T326S), mutates 344 th threonine into serine (T344S), and mutates 476 th isoleucine into phenylalanine (I476F). Compared with the heparin skeleton synthase PmHS2, the heparin skeleton synthase PmHS2 mutant has very high GlcNAc transferase activity and GlcA transferase activity which are respectively 2-3.5 times and 1.5-3.5 times that of the heparin skeleton synthase PmHS2, and can effectively synthesize the GlcNAc-GlcA-pNP heparin skeleton by utilizing substrates UDP-GlcNAc and GlcA-pNP under the optimal condition; or the substrates UDP-GlcA and GlcNAc-GlcA-pNP are utilized to effectively synthesize the GlcA-GlcNAc-GlcA-pNP heparin skeleton, so that the GlcNAc transfer synthesis efficiency and GlcA transfer synthesis efficiency in heparin skeleton synthesis are improved, the application development of heparin bionic synthesis is greatly promoted, and a brand new page is opened for the research and development of glycosaminoglycan.
2. The heparin skeleton synthase PmHS2 mutant provided by the invention has good stability, and can still keep more than 40% of enzyme activity after being placed at 37 ℃ for 72 hours, which is more than 2 times of the existing heparin skeleton synthase PmHS 2. In particular to a heparin skeleton synthase mutant PmHS2 (F197Y), a heparin skeleton synthase mutant PmHS2 (S135A) and a protein expressed by the heparin skeleton synthase mutant PmHS2 (T344S), which can still keep more than 80 percent of enzyme activity after being placed for 72 hours at 37 ℃ and is 5 to 7 times of the existing heparin skeleton synthase PmHS 2.
Drawings
FIG. 1 is a diagram showing the SDS-PAGE detection result of a heparin skeleton synthase PmHS2 mutant for soluble expression of a target protein in recombinant escherichia coli;
FIG. 2 is a histogram of relative conversion of heparin skeleton synthase PmHS2 and its mutants;
FIG. 3 is a graph showing the stability of the heparin skeleton synthase PmHS2 mutant.
In the figure, the ordinate indicates the relative yield of the reaction product, and the abscissa indicates the time for which the protein was left at 37℃with the initial enzyme activity being 100%.
Detailed Description
The technical scheme of the present invention is further described below with reference to examples and drawings of the specification, but the scope of the present invention is not limited thereto. The technical means employed in the present invention are methods well known to those skilled in the art unless specifically stated.
The inventors obtained the amino acid sequence and nucleotide sequence information of heparin skeleton synthase PmHS2 (GenBank: AY 292200.1) from bioinformatics databases and then analyzed the highly conserved regions of the amino acid sequence using Jalview software. Simultaneously, a Swiss-Model tool is used for carrying out protein simulation modeling on the heparin skeleton synthase PmHS2, and a HotSpot Wizard 2.0 is used for predicting the active center of the enzyme. It was found that the 130, 135, 165, 197, 267, 296, 326, 344, 476 and 548 sites of the amino acid sequence of the heparin skeleton synthase PmHS2 are located near the active center and the highly conserved region, and that it is highly possible to increase the catalytic activity of PmHS2 by site-directed mutagenesis. Therefore, in combination with the dominant amino acids at these six positions for homology analysis, ten mutants of PmHS2 (S130T), pmHS2 (S135A), pmHS2 (D165Y), pmHS2 (F197Y), pmHS2 (F267L), pmHS2 (H296N), pmHS2 (T326S), pmHS2 (T344S), pmHS2 (I476F), and PmHS2 (S548A) were designed.
Wherein the coding gene of the mutant PmHS2 (S130T) is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the coding gene of the mutant PmHS2 (S135A) is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4; the coding gene of the mutant PmHS2 (F197Y) is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6; the coding gene of the mutant PmHS2 (T326S) is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO. 8; the coding gene of the mutant PmHS2 (T344S) is shown as SEQ ID NO.9, and the amino acid sequence is shown as SEQ ID NO. 10; the coding gene of the mutant PmHS2 (I476F) is shown as SEQ ID NO.11, and the amino acid sequence is shown as SEQ ID NO. 12. The mutant PmHS2 (D165Y) is characterized in that aspartic acid at 165 th position is mutated into tyrosine; the mutant PmHS2 (F267L) is characterized in that 267 th phenylalanine is mutated into leucine; the mutant PmHS2 (H296N) is characterized in that histidine 296 is mutated into asparagine; the mutant PmHS2 (S548A) was mutated from serine at position 548 to alanine. The unprotected mutants herein will not show their sequence, but their sequence acquisition method is the same.
The substrate carbohydrate reagents used in the present invention are available from sigma and from the manufacturer unless otherwise indicated. All plasmids for PmHS2 mutants were commissioned by the company nanjing gold sry. The adopted HPLC detection method uses an amino column of YMC, the liquid phase system is the liquid phase of Shimadzu, and the ultraviolet detection system is SPD-20A. Detecting ultraviolet absorption of each component at 310nm and 254nm after the heparin skeleton synthase catalytic product is separated by a chromatographic column, wherein the HPLC mobile phase condition is shown in table 1:
TABLE 1 HPLC analysis method for heparin oligosaccharides detection
Example 1 preparation of heparin skeleton synthase PmHS2 mutant
(1) Construction of expression strains
Taking mutant PmHS2 (F197Y) as an example, transforming a recombinant vector pET28a-His-PmHS2 (F197Y) -Stop codon (synthesized by Nanjing Style company) containing a heparin skeleton synthase PmHS2 (F197Y) coding gene (SEQ ID NO. 5) into competent cells of escherichia coli BL21 (DE 3), constructing a recombinant strain, culturing the recombinant strain on LB plates of kanamycin (100 mug/mL) for 12 hours, screening transformants (carrying out negative control experiments), and activating to obtain PmHS2 (F197Y) positive transformants; the amino acid sequence of the heparin skeleton synthase is shown as SEQ ID NO. 6.
The remaining nine positive transformants of mutant PmHS2 (S130T), pmHS2 (S135A), pmHS2 (D165Y), pmHS2 (F267L), pmHS2 (H296N), pmHS2 (T326S), pmHS2 (T344S), pmHS2 (I476F), and PmHS2 (S548A) were obtained in the same manner as described above.
(2) Expression and purification of proteins
Taking mutant PmHS2 (F197Y) as an example, single colonies of mutant PmHS2 (F197Y) positive transformants were subjected to activation culture in 50mL of sterile LB liquid medium for 12h (37 ℃ C., 225 r/min). Then 1% volume of the activated bacteria solution was added to LB liquid medium containing kanamycin (100. Mu.g/mL), and shake-cultured at 37℃and 225r/min for about 3 hours to OD 600 IPTG (final concentration 0.2 mM) was added at 0.6-0.8, and induction was performed at 22℃and 225r/min for 16-18h. Then, the cells were collected by centrifugation at 8000rpm for 10min and resuspended in equilibration buffer, sonicated on ice (15 s working, 45s intermittent, 33% amplitude, 1500KJ energy, 4 ℃) for 20min, and the cells after disruption were centrifuged at 12000rpm for 40min (4 ℃) to leave the supernatant which was filtered through a 0.22 μm filter. Purification by histidine tag with nickel column: firstly, using a balance buffer solution to balance 5-10 column volumes of a nickel column, filtering a supernatant fluid to flow through the nickel column to finish a sample loading step, using the balance buffer solution to balance 3-5 column volumes after sample loading, then using a washing buffer solution to wash out impurity proteins, and finally using an elution buffer solutionEluting to obtain the target protein. The purified protein was stored in 20% glycerol and each tube was stored in a-80℃refrigerator.
Wherein, the components of the balance buffer are: 0.3M NaCl,50mM NaH 2 PO 4 ，pH＝8；
The wash buffer had the following composition: 10mM imidazole, 0.3M NaCl,50mM NaH 2 PO 4 ，pH＝8；
The elution buffer had the following composition: 250mM imidazole, 0.3M NaCl,50mM NaH 2 PO 4 ，pH＝8。
The remaining nine mutant PmHS2 (S130T), pmHS2 (S135A), pmHS2 (D165Y), pmHS2 (F267L), pmHS2 (H296N), pmHS2 (T326S), pmHS2 (T344S), pmHS2 (I476F), pmHS2 (S548A) recombinant proteins were purified according to the same method as described above.
The expression condition of the purified recombinant protein is identified by polyacrylamide gel electrophoresis (SDS-PAGE) (shown in figure 1), and after the positive transformant is expressed in a recombinant mode, the protein is purified, the band is single, and the molecular weight of the target protein is consistent with the theoretical value (PmHS 2 is 75.39 kDa).
Example 2 Activity detection of heparin skeleton synthase PmHS2 mutant
The reaction was carried out using commercial GlcA-pNP (final concentration: 0.2 mM) as an acceptor substrate, UDP-GlcNAc (final concentration: 0.3 mM) as a donor substrate, and the heparin-skeleton-synthase PmHS2 mutant prepared in example 1 as a protease, with the reaction system shown in Table 2; the reaction was placed in a 37℃water bath for 30min, heated in boiling water for 5min to inactivate the protease and terminate the reaction, the reaction solution was filtered with a 0.22 μm filter membrane and subjected to HPLC detection as described in Table 1, the pNP group of the monosaccharide acceptor having a specific absorption at a detection wavelength of ultraviolet 310nm, and the mobile phase flow rate was 0.5mL/min.
TABLE 2 enzyme Activity verification reaction System for heparin skeleton synthase
The detection results are shown in fig. 2, wherein the conversion rate of PmHS2 is set to be 1, and the conversion rate of the mutant is reduced to be a multiple thereof.
As can be seen from FIG. 2, the heparin backbone synthase PmHS2 mutant has GlcNAc transferase activity and GlcA transferase activity, and can transfer GlcNAc groups to the non-reducing end of GlcA-pNP to produce heparin disaccharide GlcNAc-GlcA-pNP; the GlcA group can also be transferred to the non-reducing end of GlcNAc-GlcA-pNP to form the heparin-trisaccharide GlcA-GlcNAc-GlcA-pNP.
In addition, the GlcNAc transferase activity and GlcA transferase activity of each mutant were elevated to different extents.
Wherein, the activity of the GlcNAc transferase of the PmHS2 (S130T) is improved by 2.51 times of the activity of the original heparin skeleton synthase PmHS2, and the activity of the GlcA transferase is improved by 2.97 times of the activity of the original heparin skeleton synthase PmHS 2.
The GlcNAc transferase activity of PmHS2 (S135A) was increased by 3.31 times as much as that of the original heparin skeleton synthase PmHS2, and the GlcA transferase activity was increased by 1.63 times as much as that of the original heparin skeleton synthase PmHS 2.
The GlcNAc transferase activity of PmHS2 (F197Y) was increased by 2.81 times as much as that of the original heparin skeleton synthase PmHS2, and the GlcA transferase activity was increased by 2.76 times as much as that of the original heparin skeleton synthase PmHS 2.
The GlcNAc transferase activity of PmHS2 (T326S) was increased by 3.73 times as much as that of the original heparin skeleton synthase PmHS2, and the GlcA transferase activity was increased by 1.67 times as much as that of the original heparin skeleton synthase PmHS 2.
The GlcNAc transferase activity of PmHS2 (T344S) was increased by 2.08 times as much as that of the original heparin skeleton synthase PmHS2, and the GlcA transferase activity was increased by 3.35 times as much as that of the original heparin skeleton synthase PmHS 2.
The GlcNAc transferase activity of PmHS2 (I476F) was increased by 4.33 times as much as that of the original heparin skeleton synthase PmHS2, and the GlcA transferase activity was increased by 2.29 times as much as that of the original heparin skeleton synthase PmHS 2.
The changes in the activities of PmHS2 (D165Y), pmHS2 (F267L), pmHS2 (H297N), and PmHS2 (S548A) were not significant.
As shown by the data, compared with the heparin skeleton synthase PmHS2, the heparin skeleton synthase PmHS2 mutant provided by the invention has very high GlcNAc transferase activity and GlcA transferase activity which are respectively 2-3.5 times and 1.5-3.5 times that of the heparin skeleton synthase PmHS2, and can effectively synthesize a GlcNAc-GlcA-pNP heparin skeleton by utilizing substrates UDP-GlcNAc and GlcA-pNP under the optimal condition; or the substrates UDP-GlcA and GlcNAc-GlcA-pNP are utilized to effectively synthesize the GlcA-GlcNAc-GlcA-pNP heparin skeleton, so that the GlcNAc transfer synthesis efficiency and GlcA transfer synthesis efficiency in heparin skeleton synthesis are improved, the application development of heparin bionic synthesis is greatly promoted, and a brand new page is opened for the research and development of glycosaminoglycan.
EXAMPLE 3 stability study of heparin skeleton synthase PmHS2 mutant
The conventional PmHS2 mutant proteins obtained in example 1 and PmHS2 mutant proteins obtained in example 1 were left at 37℃for 0h, 24h, 48h and 72h, respectively, and then the conventional activities were measured in accordance with the reaction system shown in example 2 using commercially available GlcA-pNP (final concentration: 0.2 mM) as a acceptor substrate and UDP-GlcNAc (final concentration: 0.3 mM) as a donor substrate. The reaction is put into a water bath kettle at 37 ℃ for reaction for 30min, boiling water is heated for 5min to inactivate protease, thus stopping the reaction, the reaction liquid is filtered by a filter membrane of 0.22 mu m, HPLC detection is carried out according to the method shown in table 1, the pNP group of the monosaccharide receptor has specific absorption under the detection wavelength of ultraviolet 310nm, the flow rate of a mobile phase is 0.5mL/min, and the detection result is shown in figure 3.
As shown in FIG. 3, the heparin skeleton synthase PmHS2 mutant provided by the invention has good stability, can still maintain more than 55% of enzyme activity after being placed at 37 ℃ for 24 hours, and can still maintain more than 40% of enzyme activity after being placed at 37 ℃ for 72 hours. The enzyme activity of the heparin skeleton synthase PmHS2 is reduced to 40% after the heparin skeleton synthase PmHS2 is placed at 37 ℃ for 24 hours, and the enzyme activity is reduced to about 10% after the heparin skeleton synthase PmHS2 is placed at 37 ℃ for 72 hours. Wherein, the purified proteins of PmHS2 (F197Y), pmHS2 (S135A) and PmHS2 (T344S) can still keep more than 80 percent of enzyme activity after being placed for 72 hours at 37 ℃ and is 5 to 7 times of that of heparin skeleton synthase PmHS 2.
SEQUENCE LISTING
<110> university of Shandong
<120> high-activity heparin skeleton synthase PmHS2 mutant and application thereof
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1956
<212> DNA
<213> artificial sequence
<400> 1
atgaagggta agaaagagat gacccagatt caaatcgcga agaacccgcc gcaacacgag 60
aaagaaaacg agctgaacac ctttcagaac aaaatcgata gcctgaagac caccctgaac 120
aaagacatca ttagccagca aaccctgctg gcgaaacaag acagcaagca cccgctgagc 180
gcgagcctgg aaaacgagaa caaactgctg ctgaagcagc tgcaactggt gctgcaagaa 240
tttgagaaga tttacaccta taaccaggcg ctggaagcga aactggagaa ggataaacag 300
accaccagca tcaccgacct gtataacgaa gttgcgaaga gcgatctggg cctggtgaaa 360
gaaaccaaca gcgcgaaccc gctggttacc atcattatga ccagccacaa caccgcgcaa 420
ttcattgaag cgagcatcaa cagcctgctg ctgcagacct acaagaacat cgaaatcatt 480
atcgtggacg atgacagcag cgacaacacc tttgagattg cgagccgtat cgcgaacacc 540
accagcaaag tgcgtgtttt ccgtctgaac agcaacctgg gtacctattt tgcgaaaaac 600
accggtatcc tgaagagcaa aggcgacatt atcttctttc aggatagcga tgacgtttgc 660
caccacgaac gtattgagcg ttgcgtgaac atcctgctgg cgaacaaaga aaccatcgcg 720
gttcgttgcg cgtacagccg tctggcgccg gaaacccaac acattatcaa ggtgaacaac 780
atggactatc gtctgggttt cattaccctg ggcatgcacc gtaaagtttt tcaggagatc 840
ggcttcttta actgcaccac caagggtagc gatgacgagt tcttccaccg tattgcgaaa 900
tactatggca aggagaaaat caagaacctg ctgctgccgc tgtactataa caccatgcgt 960
gaaaacagcc tgttcaccga catggtggag tggatcgata accacaacat tatccagaag 1020
atgagcgaca cccgtcaaca ctacgcgacc ctgttccagg cgatgcacaa cgaaaccgcg 1080
agccacgatt ttaaaaacct gttccaattt ccgcgtattt acgacgcgct gccggttccg 1140
caggagatga gcaagctgag caacccgaaa atcccggtgt atattaacat ctgcagcatt 1200
ccgagccgta tcgcgcaact gcgtcgtatt atcggtattc tgaagaacca gtgcgaccac 1260
ttccacatct acctggatgg ctatgttgaa attccggact ttatcaagaa cctgggtaac 1320
aaagcgaccg tggttcactg caaagacaag gataacagca ttcgtgacaa cggcaagttc 1380
attctgctgg aggaactgat cgagaagaac caggatggtt actatatcac ctgcgatgac 1440
gatattatct acccgagcga ctatattaac accatgatca agaaactgaa cgagtacgac 1500
gataaagcgg ttattggtct gcacggcatc ctgttcccga gccgtatgac caagtatttt 1560
agcgcggatc gtctggtgta cagcttctat aaaccgctgg agaaagacaa ggcggtgaac 1620
gttctgggta ccggcaccgt tagctttcgt gtgagcctgt tcaaccaatt tagcctgagc 1680
gatttcaccc acagcggtat ggcggacatt tactttagcc tgctgtgcaa gaaaaacaac 1740
atcctgcaga tttgcatcag ccgtccggcg aactggctga ccgaagacaa ccgtgatagc 1800
gaaaccctgt accaccaata tcgtgacaac gatgaacagc aaacccagct gattatggag 1860
aacggtccgt ggggctacag cagcatctat ccgctggtta aaaaccatcc gaagttcacc 1920
gacctgattc cgtgcctgcc gttctatttc ctgtaa 1956
<210> 2
<211> 651
<212> PRT
<213> artificial sequence
<400> 2
Met Lys Gly Lys Lys Glu Met Thr Gln Ile Gln Ile Ala Lys Asn Pro
1 5 10 15
Pro Gln His Glu Lys Glu Asn Glu Leu Asn Thr Phe Gln Asn Lys Ile
20 25 30
Asp Ser Leu Lys Thr Thr Leu Asn Lys Asp Ile Ile Ser Gln Gln Thr
35 40 45
Leu Leu Ala Lys Gln Asp Ser Lys His Pro Leu Ser Ala Ser Leu Glu
50 55 60
Asn Glu Asn Lys Leu Leu Leu Lys Gln Leu Gln Leu Val Leu Gln Glu
65 70 75 80
Phe Glu Lys Ile Tyr Thr Tyr Asn Gln Ala Leu Glu Ala Lys Leu Glu
85 90 95
Lys Asp Lys Gln Thr Thr Ser Ile Thr Asp Leu Tyr Asn Glu Val Ala
100 105 110
Lys Ser Asp Leu Gly Leu Val Lys Glu Thr Asn Ser Ala Asn Pro Leu
115 120 125
Val Thr Ile Ile Met Thr Ser His Asn Thr Ala Gln Phe Ile Glu Ala
130 135 140
Ser Ile Asn Ser Leu Leu Leu Gln Thr Tyr Lys Asn Ile Glu Ile Ile
145 150 155 160
Ile Val Asp Asp Asp Ser Ser Asp Asn Thr Phe Glu Ile Ala Ser Arg
165 170 175
Ile Ala Asn Thr Thr Ser Lys Val Arg Val Phe Arg Leu Asn Ser Asn
180 185 190
Leu Gly Thr Tyr Phe Ala Lys Asn Thr Gly Ile Leu Lys Ser Lys Gly
195 200 205
Asp Ile Ile Phe Phe Gln Asp Ser Asp Asp Val Cys His His Glu Arg
210 215 220
Ile Glu Arg Cys Val Asn Ile Leu Leu Ala Asn Lys Glu Thr Ile Ala
225 230 235 240
Val Arg Cys Ala Tyr Ser Arg Leu Ala Pro Glu Thr Gln His Ile Ile
245 250 255
Lys Val Asn Asn Met Asp Tyr Arg Leu Gly Phe Ile Thr Leu Gly Met
260 265 270
His Arg Lys Val Phe Gln Glu Ile Gly Phe Phe Asn Cys Thr Thr Lys
275 280 285
Gly Ser Asp Asp Glu Phe Phe His Arg Ile Ala Lys Tyr Tyr Gly Lys
290 295 300
Glu Lys Ile Lys Asn Leu Leu Leu Pro Leu Tyr Tyr Asn Thr Met Arg
305 310 315 320
Glu Asn Ser Leu Phe Thr Asp Met Val Glu Trp Ile Asp Asn His Asn
325 330 335
Ile Ile Gln Lys Met Ser Asp Thr Arg Gln His Tyr Ala Thr Leu Phe
340 345 350
Gln Ala Met His Asn Glu Thr Ala Ser His Asp Phe Lys Asn Leu Phe
355 360 365
Gln Phe Pro Arg Ile Tyr Asp Ala Leu Pro Val Pro Gln Glu Met Ser
370 375 380
Lys Leu Ser Asn Pro Lys Ile Pro Val Tyr Ile Asn Ile Cys Ser Ile
385 390 395 400
Pro Ser Arg Ile Ala Gln Leu Arg Arg Ile Ile Gly Ile Leu Lys Asn
405 410 415
Gln Cys Asp His Phe His Ile Tyr Leu Asp Gly Tyr Val Glu Ile Pro
420 425 430
Asp Phe Ile Lys Asn Leu Gly Asn Lys Ala Thr Val Val His Cys Lys
435 440 445
Asp Lys Asp Asn Ser Ile Arg Asp Asn Gly Lys Phe Ile Leu Leu Glu
450 455 460
Glu Leu Ile Glu Lys Asn Gln Asp Gly Tyr Tyr Ile Thr Cys Asp Asp
465 470 475 480
Asp Ile Ile Tyr Pro Ser Asp Tyr Ile Asn Thr Met Ile Lys Lys Leu
485 490 495
Asn Glu Tyr Asp Asp Lys Ala Val Ile Gly Leu His Gly Ile Leu Phe
500 505 510
Pro Ser Arg Met Thr Lys Tyr Phe Ser Ala Asp Arg Leu Val Tyr Ser
515 520 525
Phe Tyr Lys Pro Leu Glu Lys Asp Lys Ala Val Asn Val Leu Gly Thr
530 535 540
Gly Thr Val Ser Phe Arg Val Ser Leu Phe Asn Gln Phe Ser Leu Ser
545 550 555 560
Asp Phe Thr His Ser Gly Met Ala Asp Ile Tyr Phe Ser Leu Leu Cys
565 570 575
Lys Lys Asn Asn Ile Leu Gln Ile Cys Ile Ser Arg Pro Ala Asn Trp
580 585 590
Leu Thr Glu Asp Asn Arg Asp Ser Glu Thr Leu Tyr His Gln Tyr Arg
595 600 605
Asp Asn Asp Glu Gln Gln Thr Gln Leu Ile Met Glu Asn Gly Pro Trp
610 615 620
Gly Tyr Ser Ser Ile Tyr Pro Leu Val Lys Asn His Pro Lys Phe Thr
625 630 635 640
Asp Leu Ile Pro Cys Leu Pro Phe Tyr Phe Leu
645 650
<210> 3
<211> 1956
<212> DNA
<213> artificial sequence
<400> 3
atgaagggta agaaagagat gacccagatt caaatcgcga agaacccgcc gcaacacgag 60
aaagaaaacg agctgaacac ctttcagaac aaaatcgata gcctgaagac caccctgaac 120
aaagacatca ttagccagca aaccctgctg gcgaaacaag acagcaagca cccgctgagc 180
gcgagcctgg aaaacgagaa caaactgctg ctgaagcagc tgcaactggt gctgcaagaa 240
tttgagaaga tttacaccta taaccaggcg ctggaagcga aactggagaa ggataaacag 300
accaccagca tcaccgacct gtataacgaa gttgcgaaga gcgatctggg cctggtgaaa 360
gaaaccaaca gcgcgaaccc gctggttagc atcattatga ccgcgcacaa caccgcgcaa 420
ttcattgaag cgagcatcaa cagcctgctg ctgcagacct acaagaacat cgaaatcatt 480
atcgtggacg atgacagcag cgacaacacc tttgagattg cgagccgtat cgcgaacacc 540
accagcaaag tgcgtgtttt ccgtctgaac agcaacctgg gtacctattt tgcgaaaaac 600
accggtatcc tgaagagcaa aggcgacatt atcttctttc aggatagcga tgacgtttgc 660
caccacgaac gtattgagcg ttgcgtgaac atcctgctgg cgaacaaaga aaccatcgcg 720
gttcgttgcg cgtacagccg tctggcgccg gaaacccaac acattatcaa ggtgaacaac 780
atggactatc gtctgggttt cattaccctg ggcatgcacc gtaaagtttt tcaggagatc 840
ggcttcttta actgcaccac caagggtagc gatgacgagt tcttccaccg tattgcgaaa 900
tactatggca aggagaaaat caagaacctg ctgctgccgc tgtactataa caccatgcgt 960
gaaaacagcc tgttcaccga catggtggag tggatcgata accacaacat tatccagaag 1020
atgagcgaca cccgtcaaca ctacgcgacc ctgttccagg cgatgcacaa cgaaaccgcg 1080
agccacgatt ttaaaaacct gttccaattt ccgcgtattt acgacgcgct gccggttccg 1140
caggagatga gcaagctgag caacccgaaa atcccggtgt atattaacat ctgcagcatt 1200
ccgagccgta tcgcgcaact gcgtcgtatt atcggtattc tgaagaacca gtgcgaccac 1260
ttccacatct acctggatgg ctatgttgaa attccggact ttatcaagaa cctgggtaac 1320
aaagcgaccg tggttcactg caaagacaag gataacagca ttcgtgacaa cggcaagttc 1380
attctgctgg aggaactgat cgagaagaac caggatggtt actatatcac ctgcgatgac 1440
gatattatct acccgagcga ctatattaac accatgatca agaaactgaa cgagtacgac 1500
gataaagcgg ttattggtct gcacggcatc ctgttcccga gccgtatgac caagtatttt 1560
agcgcggatc gtctggtgta cagcttctat aaaccgctgg agaaagacaa ggcggtgaac 1620
gttctgggta ccggcaccgt tagctttcgt gtgagcctgt tcaaccaatt tagcctgagc 1680
gatttcaccc acagcggtat ggcggacatt tactttagcc tgctgtgcaa gaaaaacaac 1740
atcctgcaga tttgcatcag ccgtccggcg aactggctga ccgaagacaa ccgtgatagc 1800
gaaaccctgt accaccaata tcgtgacaac gatgaacagc aaacccagct gattatggag 1860
aacggtccgt ggggctacag cagcatctat ccgctggtta aaaaccatcc gaagttcacc 1920
gacctgattc cgtgcctgcc gttctatttc ctgtaa 1956
<210> 4
<211> 651
<212> PRT
<213> artificial sequence
<400> 4
Met Lys Gly Lys Lys Glu Met Thr Gln Ile Gln Ile Ala Lys Asn Pro
1 5 10 15
Pro Gln His Glu Lys Glu Asn Glu Leu Asn Thr Phe Gln Asn Lys Ile
20 25 30
Asp Ser Leu Lys Thr Thr Leu Asn Lys Asp Ile Ile Ser Gln Gln Thr
35 40 45
Leu Leu Ala Lys Gln Asp Ser Lys His Pro Leu Ser Ala Ser Leu Glu
50 55 60
Asn Glu Asn Lys Leu Leu Leu Lys Gln Leu Gln Leu Val Leu Gln Glu
65 70 75 80
Phe Glu Lys Ile Tyr Thr Tyr Asn Gln Ala Leu Glu Ala Lys Leu Glu
85 90 95
Lys Asp Lys Gln Thr Thr Ser Ile Thr Asp Leu Tyr Asn Glu Val Ala
100 105 110
Lys Ser Asp Leu Gly Leu Val Lys Glu Thr Asn Ser Ala Asn Pro Leu
115 120 125
Val Ser Ile Ile Met Thr Ala His Asn Thr Ala Gln Phe Ile Glu Ala
130 135 140
Ser Ile Asn Ser Leu Leu Leu Gln Thr Tyr Lys Asn Ile Glu Ile Ile
145 150 155 160
Ile Val Asp Asp Asp Ser Ser Asp Asn Thr Phe Glu Ile Ala Ser Arg
165 170 175
Ile Ala Asn Thr Thr Ser Lys Val Arg Val Phe Arg Leu Asn Ser Asn
180 185 190
Leu Gly Thr Tyr Phe Ala Lys Asn Thr Gly Ile Leu Lys Ser Lys Gly
195 200 205
Asp Ile Ile Phe Phe Gln Asp Ser Asp Asp Val Cys His His Glu Arg
210 215 220
Ile Glu Arg Cys Val Asn Ile Leu Leu Ala Asn Lys Glu Thr Ile Ala
225 230 235 240
Val Arg Cys Ala Tyr Ser Arg Leu Ala Pro Glu Thr Gln His Ile Ile
245 250 255
Lys Val Asn Asn Met Asp Tyr Arg Leu Gly Phe Ile Thr Leu Gly Met
260 265 270
His Arg Lys Val Phe Gln Glu Ile Gly Phe Phe Asn Cys Thr Thr Lys
275 280 285
Gly Ser Asp Asp Glu Phe Phe His Arg Ile Ala Lys Tyr Tyr Gly Lys
290 295 300
Glu Lys Ile Lys Asn Leu Leu Leu Pro Leu Tyr Tyr Asn Thr Met Arg
305 310 315 320
Glu Asn Ser Leu Phe Thr Asp Met Val Glu Trp Ile Asp Asn His Asn
325 330 335
Ile Ile Gln Lys Met Ser Asp Thr Arg Gln His Tyr Ala Thr Leu Phe
340 345 350
Gln Ala Met His Asn Glu Thr Ala Ser His Asp Phe Lys Asn Leu Phe
355 360 365
Gln Phe Pro Arg Ile Tyr Asp Ala Leu Pro Val Pro Gln Glu Met Ser
370 375 380
Lys Leu Ser Asn Pro Lys Ile Pro Val Tyr Ile Asn Ile Cys Ser Ile
385 390 395 400
Pro Ser Arg Ile Ala Gln Leu Arg Arg Ile Ile Gly Ile Leu Lys Asn
405 410 415
Gln Cys Asp His Phe His Ile Tyr Leu Asp Gly Tyr Val Glu Ile Pro
420 425 430
Asp Phe Ile Lys Asn Leu Gly Asn Lys Ala Thr Val Val His Cys Lys
435 440 445
Asp Lys Asp Asn Ser Ile Arg Asp Asn Gly Lys Phe Ile Leu Leu Glu
450 455 460
Glu Leu Ile Glu Lys Asn Gln Asp Gly Tyr Tyr Ile Thr Cys Asp Asp
465 470 475 480
Asp Ile Ile Tyr Pro Ser Asp Tyr Ile Asn Thr Met Ile Lys Lys Leu
485 490 495
Asn Glu Tyr Asp Asp Lys Ala Val Ile Gly Leu His Gly Ile Leu Phe
500 505 510
Pro Ser Arg Met Thr Lys Tyr Phe Ser Ala Asp Arg Leu Val Tyr Ser
515 520 525
Phe Tyr Lys Pro Leu Glu Lys Asp Lys Ala Val Asn Val Leu Gly Thr
530 535 540
Gly Thr Val Ser Phe Arg Val Ser Leu Phe Asn Gln Phe Ser Leu Ser
545 550 555 560
Asp Phe Thr His Ser Gly Met Ala Asp Ile Tyr Phe Ser Leu Leu Cys
565 570 575
Lys Lys Asn Asn Ile Leu Gln Ile Cys Ile Ser Arg Pro Ala Asn Trp
580 585 590
Leu Thr Glu Asp Asn Arg Asp Ser Glu Thr Leu Tyr His Gln Tyr Arg
595 600 605
Asp Asn Asp Glu Gln Gln Thr Gln Leu Ile Met Glu Asn Gly Pro Trp
610 615 620
Gly Tyr Ser Ser Ile Tyr Pro Leu Val Lys Asn His Pro Lys Phe Thr
625 630 635 640
Asp Leu Ile Pro Cys Leu Pro Phe Tyr Phe Leu
645 650
<210> 5
<211> 1956
<212> DNA
<213> artificial sequence
<400> 5
atgaagggta agaaagagat gacccagatt caaatcgcga agaacccgcc gcaacacgag 60
aaagaaaacg agctgaacac ctttcagaac aaaatcgata gcctgaagac caccctgaac 120
aaagacatca ttagccagca aaccctgctg gcgaaacaag acagcaagca cccgctgagc 180
gcgagcctgg aaaacgagaa caaactgctg ctgaagcagc tgcaactggt gctgcaagaa 240
tttgagaaga tttacaccta taaccaggcg ctggaagcga aactggagaa ggataaacag 300
accaccagca tcaccgacct gtataacgaa gttgcgaaga gcgatctggg cctggtgaaa 360
gaaaccaaca gcgcgaaccc gctggttagc atcattatga ccagccacaa caccgcgcaa 420
ttcattgaag cgagcatcaa cagcctgctg ctgcagacct acaagaacat cgaaatcatt 480
atcgtggacg atgacagcag cgacaacacc tttgagattg cgagccgtat cgcgaacacc 540
accagcaaag tgcgtgtttt ccgtctgaac agcaacctgg gtacctatta tgcgaaaaac 600
accggtatcc tgaagagcaa aggcgacatt atcttctttc aggatagcga tgacgtttgc 660
caccacgaac gtattgagcg ttgcgtgaac atcctgctgg cgaacaaaga aaccatcgcg 720
gttcgttgcg cgtacagccg tctggcgccg gaaacccaac acattatcaa ggtgaacaac 780
atggactatc gtctgggttt cattaccctg ggcatgcacc gtaaagtttt tcaggagatc 840
ggcttcttta actgcaccac caagggtagc gatgacgagt tcttccaccg tattgcgaaa 900
tactatggca aggagaaaat caagaacctg ctgctgccgc tgtactataa caccatgcgt 960
gaaaacagcc tgttcaccga catggtggag tggatcgata accacaacat tatccagaag 1020
atgagcgaca cccgtcaaca ctacgcgacc ctgttccagg cgatgcacaa cgaaaccgcg 1080
agccacgatt ttaaaaacct gttccaattt ccgcgtattt acgacgcgct gccggttccg 1140
caggagatga gcaagctgag caacccgaaa atcccggtgt atattaacat ctgcagcatt 1200
ccgagccgta tcgcgcaact gcgtcgtatt atcggtattc tgaagaacca gtgcgaccac 1260
ttccacatct acctggatgg ctatgttgaa attccggact ttatcaagaa cctgggtaac 1320
aaagcgaccg tggttcactg caaagacaag gataacagca ttcgtgacaa cggcaagttc 1380
attctgctgg aggaactgat cgagaagaac caggatggtt actatatcac ctgcgatgac 1440
gatattatct acccgagcga ctatattaac accatgatca agaaactgaa cgagtacgac 1500
gataaagcgg ttattggtct gcacggcatc ctgttcccga gccgtatgac caagtatttt 1560
agcgcggatc gtctggtgta cagcttctat aaaccgctgg agaaagacaa ggcggtgaac 1620
gttctgggta ccggcaccgt tagctttcgt gtgagcctgt tcaaccaatt tagcctgagc 1680
gatttcaccc acagcggtat ggcggacatt tactttagcc tgctgtgcaa gaaaaacaac 1740
atcctgcaga tttgcatcag ccgtccggcg aactggctga ccgaagacaa ccgtgatagc 1800
gaaaccctgt accaccaata tcgtgacaac gatgaacagc aaacccagct gattatggag 1860
aacggtccgt ggggctacag cagcatctat ccgctggtta aaaaccatcc gaagttcacc 1920
gacctgattc cgtgcctgcc gttctatttc ctgtaa 1956
<210> 6
<211> 651
<212> PRT
<213> artificial sequence
<400> 6
Met Lys Gly Lys Lys Glu Met Thr Gln Ile Gln Ile Ala Lys Asn Pro
1 5 10 15
Pro Gln His Glu Lys Glu Asn Glu Leu Asn Thr Phe Gln Asn Lys Ile
20 25 30
Asp Ser Leu Lys Thr Thr Leu Asn Lys Asp Ile Ile Ser Gln Gln Thr
35 40 45
Leu Leu Ala Lys Gln Asp Ser Lys His Pro Leu Ser Ala Ser Leu Glu
50 55 60
Asn Glu Asn Lys Leu Leu Leu Lys Gln Leu Gln Leu Val Leu Gln Glu
65 70 75 80
Phe Glu Lys Ile Tyr Thr Tyr Asn Gln Ala Leu Glu Ala Lys Leu Glu
85 90 95
Lys Asp Lys Gln Thr Thr Ser Ile Thr Asp Leu Tyr Asn Glu Val Ala
100 105 110
Lys Ser Asp Leu Gly Leu Val Lys Glu Thr Asn Ser Ala Asn Pro Leu
115 120 125
Val Ser Ile Ile Met Thr Ser His Asn Thr Ala Gln Phe Ile Glu Ala
130 135 140
Ser Ile Asn Ser Leu Leu Leu Gln Thr Tyr Lys Asn Ile Glu Ile Ile
145 150 155 160
Ile Val Asp Asp Asp Ser Ser Asp Asn Thr Phe Glu Ile Ala Ser Arg
165 170 175
Ile Ala Asn Thr Thr Ser Lys Val Arg Val Phe Arg Leu Asn Ser Asn
180 185 190
Leu Gly Thr Tyr Tyr Ala Lys Asn Thr Gly Ile Leu Lys Ser Lys Gly
195 200 205
Asp Ile Ile Phe Phe Gln Asp Ser Asp Asp Val Cys His His Glu Arg
210 215 220
Ile Glu Arg Cys Val Asn Ile Leu Leu Ala Asn Lys Glu Thr Ile Ala
225 230 235 240
Val Arg Cys Ala Tyr Ser Arg Leu Ala Pro Glu Thr Gln His Ile Ile
245 250 255
Lys Val Asn Asn Met Asp Tyr Arg Leu Gly Phe Ile Thr Leu Gly Met
260 265 270
His Arg Lys Val Phe Gln Glu Ile Gly Phe Phe Asn Cys Thr Thr Lys
275 280 285
Gly Ser Asp Asp Glu Phe Phe His Arg Ile Ala Lys Tyr Tyr Gly Lys
290 295 300
Glu Lys Ile Lys Asn Leu Leu Leu Pro Leu Tyr Tyr Asn Thr Met Arg
305 310 315 320
Glu Asn Ser Leu Phe Thr Asp Met Val Glu Trp Ile Asp Asn His Asn
325 330 335
Ile Ile Gln Lys Met Ser Asp Thr Arg Gln His Tyr Ala Thr Leu Phe
340 345 350
Gln Ala Met His Asn Glu Thr Ala Ser His Asp Phe Lys Asn Leu Phe
355 360 365
Gln Phe Pro Arg Ile Tyr Asp Ala Leu Pro Val Pro Gln Glu Met Ser
370 375 380
Lys Leu Ser Asn Pro Lys Ile Pro Val Tyr Ile Asn Ile Cys Ser Ile
385 390 395 400
Pro Ser Arg Ile Ala Gln Leu Arg Arg Ile Ile Gly Ile Leu Lys Asn
405 410 415
Gln Cys Asp His Phe His Ile Tyr Leu Asp Gly Tyr Val Glu Ile Pro
420 425 430
Asp Phe Ile Lys Asn Leu Gly Asn Lys Ala Thr Val Val His Cys Lys
435 440 445
Asp Lys Asp Asn Ser Ile Arg Asp Asn Gly Lys Phe Ile Leu Leu Glu
450 455 460
Glu Leu Ile Glu Lys Asn Gln Asp Gly Tyr Tyr Ile Thr Cys Asp Asp
465 470 475 480
Asp Ile Ile Tyr Pro Ser Asp Tyr Ile Asn Thr Met Ile Lys Lys Leu
485 490 495
Asn Glu Tyr Asp Asp Lys Ala Val Ile Gly Leu His Gly Ile Leu Phe
500 505 510
Pro Ser Arg Met Thr Lys Tyr Phe Ser Ala Asp Arg Leu Val Tyr Ser
515 520 525
Phe Tyr Lys Pro Leu Glu Lys Asp Lys Ala Val Asn Val Leu Gly Thr
530 535 540
Gly Thr Val Ser Phe Arg Val Ser Leu Phe Asn Gln Phe Ser Leu Ser
545 550 555 560
Asp Phe Thr His Ser Gly Met Ala Asp Ile Tyr Phe Ser Leu Leu Cys
565 570 575
Lys Lys Asn Asn Ile Leu Gln Ile Cys Ile Ser Arg Pro Ala Asn Trp
580 585 590
Leu Thr Glu Asp Asn Arg Asp Ser Glu Thr Leu Tyr His Gln Tyr Arg
595 600 605
Asp Asn Asp Glu Gln Gln Thr Gln Leu Ile Met Glu Asn Gly Pro Trp
610 615 620
Gly Tyr Ser Ser Ile Tyr Pro Leu Val Lys Asn His Pro Lys Phe Thr
625 630 635 640
Asp Leu Ile Pro Cys Leu Pro Phe Tyr Phe Leu
645 650
<210> 7
<211> 1956
<212> DNA
<213> artificial sequence
<400> 7
atgaagggta agaaagagat gacccagatt caaatcgcga agaacccgcc gcaacacgag 60
aaagaaaacg agctgaacac ctttcagaac aaaatcgata gcctgaagac caccctgaac 120
aaagacatca ttagccagca aaccctgctg gcgaaacaag acagcaagca cccgctgagc 180
gcgagcctgg aaaacgagaa caaactgctg ctgaagcagc tgcaactggt gctgcaagaa 240
tttgagaaga tttacaccta taaccaggcg ctggaagcga aactggagaa ggataaacag 300
accaccagca tcaccgacct gtataacgaa gttgcgaaga gcgatctggg cctggtgaaa 360
gaaaccaaca gcgcgaaccc gctggttagc atcattatga ccagccacaa caccgcgcaa 420
ttcattgaag cgagcatcaa cagcctgctg ctgcagacct acaagaacat cgaaatcatt 480
atcgtggacg atgacagcag cgacaacacc tttgagattg cgagccgtat cgcgaacacc 540
accagcaaag tgcgtgtttt ccgtctgaac agcaacctgg gtacctattt tgcgaaaaac 600
accggtatcc tgaagagcaa aggcgacatt atcttctttc aggatagcga tgacgtttgc 660
caccacgaac gtattgagcg ttgcgtgaac atcctgctgg cgaacaaaga aaccatcgcg 720
gttcgttgcg cgtacagccg tctggcgccg gaaacccaac acattatcaa ggtgaacaac 780
atggactatc gtctgggttt cattaccctg ggcatgcacc gtaaagtttt tcaggagatc 840
ggcttcttta actgcaccac caagggtagc gatgacgagt tcttccaccg tattgcgaaa 900
tactatggca aggagaaaat caagaacctg ctgctgccgc tgtactataa caccatgcgt 960
gaaaacagcc tgttcagcga catggtggag tggatcgata accacaacat tatccagaag 1020
atgagcgaca cccgtcaaca ctacgcgacc ctgttccagg cgatgcacaa cgaaaccgcg 1080
agccacgatt ttaaaaacct gttccaattt ccgcgtattt acgacgcgct gccggttccg 1140
caggagatga gcaagctgag caacccgaaa atcccggtgt atattaacat ctgcagcatt 1200
ccgagccgta tcgcgcaact gcgtcgtatt atcggtattc tgaagaacca gtgcgaccac 1260
ttccacatct acctggatgg ctatgttgaa attccggact ttatcaagaa cctgggtaac 1320
aaagcgaccg tggttcactg caaagacaag gataacagca ttcgtgacaa cggcaagttc 1380
attctgctgg aggaactgat cgagaagaac caggatggtt actatatcac ctgcgatgac 1440
gatattatct acccgagcga ctatattaac accatgatca agaaactgaa cgagtacgac 1500
gataaagcgg ttattggtct gcacggcatc ctgttcccga gccgtatgac caagtatttt 1560
agcgcggatc gtctggtgta cagcttctat aaaccgctgg agaaagacaa ggcggtgaac 1620
gttctgggta ccggcaccgt tagctttcgt gtgagcctgt tcaaccaatt tagcctgagc 1680
gatttcaccc acagcggtat ggcggacatt tactttagcc tgctgtgcaa gaaaaacaac 1740
atcctgcaga tttgcatcag ccgtccggcg aactggctga ccgaagacaa ccgtgatagc 1800
gaaaccctgt accaccaata tcgtgacaac gatgaacagc aaacccagct gattatggag 1860
aacggtccgt ggggctacag cagcatctat ccgctggtta aaaaccatcc gaagttcacc 1920
gacctgattc cgtgcctgcc gttctatttc ctgtaa 1956
<210> 8
<211> 651
<212> PRT
<213> artificial sequence
<400> 8
Met Lys Gly Lys Lys Glu Met Thr Gln Ile Gln Ile Ala Lys Asn Pro
1 5 10 15
Pro Gln His Glu Lys Glu Asn Glu Leu Asn Thr Phe Gln Asn Lys Ile
20 25 30
Asp Ser Leu Lys Thr Thr Leu Asn Lys Asp Ile Ile Ser Gln Gln Thr
35 40 45
Leu Leu Ala Lys Gln Asp Ser Lys His Pro Leu Ser Ala Ser Leu Glu
50 55 60
Asn Glu Asn Lys Leu Leu Leu Lys Gln Leu Gln Leu Val Leu Gln Glu
65 70 75 80
Phe Glu Lys Ile Tyr Thr Tyr Asn Gln Ala Leu Glu Ala Lys Leu Glu
85 90 95
Lys Asp Lys Gln Thr Thr Ser Ile Thr Asp Leu Tyr Asn Glu Val Ala
100 105 110
Lys Ser Asp Leu Gly Leu Val Lys Glu Thr Asn Ser Ala Asn Pro Leu
115 120 125
Val Ser Ile Ile Met Thr Ser His Asn Thr Ala Gln Phe Ile Glu Ala
130 135 140
Ser Ile Asn Ser Leu Leu Leu Gln Thr Tyr Lys Asn Ile Glu Ile Ile
145 150 155 160
Ile Val Asp Asp Asp Ser Ser Asp Asn Thr Phe Glu Ile Ala Ser Arg
165 170 175
Ile Ala Asn Thr Thr Ser Lys Val Arg Val Phe Arg Leu Asn Ser Asn
180 185 190
Leu Gly Thr Tyr Phe Ala Lys Asn Thr Gly Ile Leu Lys Ser Lys Gly
195 200 205
Asp Ile Ile Phe Phe Gln Asp Ser Asp Asp Val Cys His His Glu Arg
210 215 220
Ile Glu Arg Cys Val Asn Ile Leu Leu Ala Asn Lys Glu Thr Ile Ala
225 230 235 240
Val Arg Cys Ala Tyr Ser Arg Leu Ala Pro Glu Thr Gln His Ile Ile
245 250 255
Lys Val Asn Asn Met Asp Tyr Arg Leu Gly Phe Ile Thr Leu Gly Met
260 265 270
His Arg Lys Val Phe Gln Glu Ile Gly Phe Phe Asn Cys Thr Thr Lys
275 280 285
Gly Ser Asp Asp Glu Phe Phe His Arg Ile Ala Lys Tyr Tyr Gly Lys
290 295 300
Glu Lys Ile Lys Asn Leu Leu Leu Pro Leu Tyr Tyr Asn Thr Met Arg
305 310 315 320
Glu Asn Ser Leu Phe Ser Asp Met Val Glu Trp Ile Asp Asn His Asn
325 330 335
Ile Ile Gln Lys Met Ser Asp Thr Arg Gln His Tyr Ala Thr Leu Phe
340 345 350
Gln Ala Met His Asn Glu Thr Ala Ser His Asp Phe Lys Asn Leu Phe
355 360 365
Gln Phe Pro Arg Ile Tyr Asp Ala Leu Pro Val Pro Gln Glu Met Ser
370 375 380
Lys Leu Ser Asn Pro Lys Ile Pro Val Tyr Ile Asn Ile Cys Ser Ile
385 390 395 400
Pro Ser Arg Ile Ala Gln Leu Arg Arg Ile Ile Gly Ile Leu Lys Asn
405 410 415
Gln Cys Asp His Phe His Ile Tyr Leu Asp Gly Tyr Val Glu Ile Pro
420 425 430
Asp Phe Ile Lys Asn Leu Gly Asn Lys Ala Thr Val Val His Cys Lys
435 440 445
Asp Lys Asp Asn Ser Ile Arg Asp Asn Gly Lys Phe Ile Leu Leu Glu
450 455 460
Glu Leu Ile Glu Lys Asn Gln Asp Gly Tyr Tyr Ile Thr Cys Asp Asp
465 470 475 480
Asp Ile Ile Tyr Pro Ser Asp Tyr Ile Asn Thr Met Ile Lys Lys Leu
485 490 495
Asn Glu Tyr Asp Asp Lys Ala Val Ile Gly Leu His Gly Ile Leu Phe
500 505 510
Pro Ser Arg Met Thr Lys Tyr Phe Ser Ala Asp Arg Leu Val Tyr Ser
515 520 525
Phe Tyr Lys Pro Leu Glu Lys Asp Lys Ala Val Asn Val Leu Gly Thr
530 535 540
Gly Thr Val Ser Phe Arg Val Ser Leu Phe Asn Gln Phe Ser Leu Ser
545 550 555 560
Asp Phe Thr His Ser Gly Met Ala Asp Ile Tyr Phe Ser Leu Leu Cys
565 570 575
Lys Lys Asn Asn Ile Leu Gln Ile Cys Ile Ser Arg Pro Ala Asn Trp
580 585 590
Leu Thr Glu Asp Asn Arg Asp Ser Glu Thr Leu Tyr His Gln Tyr Arg
595 600 605
Asp Asn Asp Glu Gln Gln Thr Gln Leu Ile Met Glu Asn Gly Pro Trp
610 615 620
Gly Tyr Ser Ser Ile Tyr Pro Leu Val Lys Asn His Pro Lys Phe Thr
625 630 635 640
Asp Leu Ile Pro Cys Leu Pro Phe Tyr Phe Leu
645 650
<210> 9
<211> 1956
<212> DNA
<213> artificial sequence
<400> 9
atgaagggta agaaagagat gacccagatt caaatcgcga agaacccgcc gcaacacgag 60
aaagaaaacg agctgaacac ctttcagaac aaaatcgata gcctgaagac caccctgaac 120
aaagacatca ttagccagca aaccctgctg gcgaaacaag acagcaagca cccgctgagc 180
gcgagcctgg aaaacgagaa caaactgctg ctgaagcagc tgcaactggt gctgcaagaa 240
tttgagaaga tttacaccta taaccaggcg ctggaagcga aactggagaa ggataaacag 300
accaccagca tcaccgacct gtataacgaa gttgcgaaga gcgatctggg cctggtgaaa 360
gaaaccaaca gcgcgaaccc gctggttagc atcattatga ccagccacaa caccgcgcaa 420
ttcattgaag cgagcatcaa cagcctgctg ctgcagacct acaagaacat cgaaatcatt 480
atcgtggacg atgacagcag cgacaacacc tttgagattg cgagccgtat cgcgaacacc 540
accagcaaag tgcgtgtttt ccgtctgaac agcaacctgg gtacctattt tgcgaaaaac 600
accggtatcc tgaagagcaa aggcgacatt atcttctttc aggatagcga tgacgtttgc 660
caccacgaac gtattgagcg ttgcgtgaac atcctgctgg cgaacaaaga aaccatcgcg 720
gttcgttgcg cgtacagccg tctggcgccg gaaacccaac acattatcaa ggtgaacaac 780
atggactatc gtctgggttt cattaccctg ggcatgcacc gtaaagtttt tcaggagatc 840
ggcttcttta actgcaccac caagggtagc gatgacgagt tcttccaccg tattgcgaaa 900
tactatggca aggagaaaat caagaacctg ctgctgccgc tgtactataa caccatgcgt 960
gaaaacagcc tgttcaccga catggtggag tggatcgata accacaacat tatccagaag 1020
atgagcgaca gccgtcaaca ctacgcgacc ctgttccagg cgatgcacaa cgaaaccgcg 1080
agccacgatt ttaaaaacct gttccaattt ccgcgtattt acgacgcgct gccggttccg 1140
caggagatga gcaagctgag caacccgaaa atcccggtgt atattaacat ctgcagcatt 1200
ccgagccgta tcgcgcaact gcgtcgtatt atcggtattc tgaagaacca gtgcgaccac 1260
ttccacatct acctggatgg ctatgttgaa attccggact ttatcaagaa cctgggtaac 1320
aaagcgaccg tggttcactg caaagacaag gataacagca ttcgtgacaa cggcaagttc 1380
attctgctgg aggaactgat cgagaagaac caggatggtt actatatcac ctgcgatgac 1440
gatattatct acccgagcga ctatattaac accatgatca agaaactgaa cgagtacgac 1500
gataaagcgg ttattggtct gcacggcatc ctgttcccga gccgtatgac caagtatttt 1560
agcgcggatc gtctggtgta cagcttctat aaaccgctgg agaaagacaa ggcggtgaac 1620
gttctgggta ccggcaccgt tagctttcgt gtgagcctgt tcaaccaatt tagcctgagc 1680
gatttcaccc acagcggtat ggcggacatt tactttagcc tgctgtgcaa gaaaaacaac 1740
atcctgcaga tttgcatcag ccgtccggcg aactggctga ccgaagacaa ccgtgatagc 1800
gaaaccctgt accaccaata tcgtgacaac gatgaacagc aaacccagct gattatggag 1860
aacggtccgt ggggctacag cagcatctat ccgctggtta aaaaccatcc gaagttcacc 1920
gacctgattc cgtgcctgcc gttctatttc ctgtaa 1956
<210> 10
<211> 651
<212> PRT
<213> artificial sequence
<400> 10
Met Lys Gly Lys Lys Glu Met Thr Gln Ile Gln Ile Ala Lys Asn Pro
1 5 10 15
Pro Gln His Glu Lys Glu Asn Glu Leu Asn Thr Phe Gln Asn Lys Ile
20 25 30
Asp Ser Leu Lys Thr Thr Leu Asn Lys Asp Ile Ile Ser Gln Gln Thr
35 40 45
Leu Leu Ala Lys Gln Asp Ser Lys His Pro Leu Ser Ala Ser Leu Glu
50 55 60
Asn Glu Asn Lys Leu Leu Leu Lys Gln Leu Gln Leu Val Leu Gln Glu
65 70 75 80
Phe Glu Lys Ile Tyr Thr Tyr Asn Gln Ala Leu Glu Ala Lys Leu Glu
85 90 95
Lys Asp Lys Gln Thr Thr Ser Ile Thr Asp Leu Tyr Asn Glu Val Ala
100 105 110
Lys Ser Asp Leu Gly Leu Val Lys Glu Thr Asn Ser Ala Asn Pro Leu
115 120 125
Val Ser Ile Ile Met Thr Ser His Asn Thr Ala Gln Phe Ile Glu Ala
130 135 140
Ser Ile Asn Ser Leu Leu Leu Gln Thr Tyr Lys Asn Ile Glu Ile Ile
145 150 155 160
Ile Val Asp Asp Asp Ser Ser Asp Asn Thr Phe Glu Ile Ala Ser Arg
165 170 175
Ile Ala Asn Thr Thr Ser Lys Val Arg Val Phe Arg Leu Asn Ser Asn
180 185 190
Leu Gly Thr Tyr Phe Ala Lys Asn Thr Gly Ile Leu Lys Ser Lys Gly
195 200 205
Asp Ile Ile Phe Phe Gln Asp Ser Asp Asp Val Cys His His Glu Arg
210 215 220
Ile Glu Arg Cys Val Asn Ile Leu Leu Ala Asn Lys Glu Thr Ile Ala
225 230 235 240
Val Arg Cys Ala Tyr Ser Arg Leu Ala Pro Glu Thr Gln His Ile Ile
245 250 255
Lys Val Asn Asn Met Asp Tyr Arg Leu Gly Phe Ile Thr Leu Gly Met
260 265 270
His Arg Lys Val Phe Gln Glu Ile Gly Phe Phe Asn Cys Thr Thr Lys
275 280 285
Gly Ser Asp Asp Glu Phe Phe His Arg Ile Ala Lys Tyr Tyr Gly Lys
290 295 300
Glu Lys Ile Lys Asn Leu Leu Leu Pro Leu Tyr Tyr Asn Thr Met Arg
305 310 315 320
Glu Asn Ser Leu Phe Thr Asp Met Val Glu Trp Ile Asp Asn His Asn
325 330 335
Ile Ile Gln Lys Met Ser Asp Ser Arg Gln His Tyr Ala Thr Leu Phe
340 345 350
Gln Ala Met His Asn Glu Thr Ala Ser His Asp Phe Lys Asn Leu Phe
355 360 365
Gln Phe Pro Arg Ile Tyr Asp Ala Leu Pro Val Pro Gln Glu Met Ser
370 375 380
Lys Leu Ser Asn Pro Lys Ile Pro Val Tyr Ile Asn Ile Cys Ser Ile
385 390 395 400
Pro Ser Arg Ile Ala Gln Leu Arg Arg Ile Ile Gly Ile Leu Lys Asn
405 410 415
Gln Cys Asp His Phe His Ile Tyr Leu Asp Gly Tyr Val Glu Ile Pro
420 425 430
Asp Phe Ile Lys Asn Leu Gly Asn Lys Ala Thr Val Val His Cys Lys
435 440 445
Asp Lys Asp Asn Ser Ile Arg Asp Asn Gly Lys Phe Ile Leu Leu Glu
450 455 460
Glu Leu Ile Glu Lys Asn Gln Asp Gly Tyr Tyr Ile Thr Cys Asp Asp
465 470 475 480
Asp Ile Ile Tyr Pro Ser Asp Tyr Ile Asn Thr Met Ile Lys Lys Leu
485 490 495
Asn Glu Tyr Asp Asp Lys Ala Val Ile Gly Leu His Gly Ile Leu Phe
500 505 510
Pro Ser Arg Met Thr Lys Tyr Phe Ser Ala Asp Arg Leu Val Tyr Ser
515 520 525
Phe Tyr Lys Pro Leu Glu Lys Asp Lys Ala Val Asn Val Leu Gly Thr
530 535 540
Gly Thr Val Ser Phe Arg Val Ser Leu Phe Asn Gln Phe Ser Leu Ser
545 550 555 560
Asp Phe Thr His Ser Gly Met Ala Asp Ile Tyr Phe Ser Leu Leu Cys
565 570 575
Lys Lys Asn Asn Ile Leu Gln Ile Cys Ile Ser Arg Pro Ala Asn Trp
580 585 590
Leu Thr Glu Asp Asn Arg Asp Ser Glu Thr Leu Tyr His Gln Tyr Arg
595 600 605
Asp Asn Asp Glu Gln Gln Thr Gln Leu Ile Met Glu Asn Gly Pro Trp
610 615 620
Gly Tyr Ser Ser Ile Tyr Pro Leu Val Lys Asn His Pro Lys Phe Thr
625 630 635 640
Asp Leu Ile Pro Cys Leu Pro Phe Tyr Phe Leu
645 650
<210> 11
<211> 1956
<212> DNA
<213> artificial sequence
<400> 11
atgaagggta agaaagagat gacccagatt caaatcgcga agaacccgcc gcaacacgag 60
aaagaaaacg agctgaacac ctttcagaac aaaatcgata gcctgaagac caccctgaac 120
aaagacatca ttagccagca aaccctgctg gcgaaacaag acagcaagca cccgctgagc 180
gcgagcctgg aaaacgagaa caaactgctg ctgaagcagc tgcaactggt gctgcaagaa 240
tttgagaaga tttacaccta taaccaggcg ctggaagcga aactggagaa ggataaacag 300
accaccagca tcaccgacct gtataacgaa gttgcgaaga gcgatctggg cctggtgaaa 360
gaaaccaaca gcgcgaaccc gctggttagc atcattatga ccagccacaa caccgcgcaa 420
ttcattgaag cgagcatcaa cagcctgctg ctgcagacct acaagaacat cgaaatcatt 480
atcgtggacg atgacagcag cgacaacacc tttgagattg cgagccgtat cgcgaacacc 540
accagcaaag tgcgtgtttt ccgtctgaac agcaacctgg gtacctattt tgcgaaaaac 600
accggtatcc tgaagagcaa aggcgacatt atcttctttc aggatagcga tgacgtttgc 660
caccacgaac gtattgagcg ttgcgtgaac atcctgctgg cgaacaaaga aaccatcgcg 720
gttcgttgcg cgtacagccg tctggcgccg gaaacccaac acattatcaa ggtgaacaac 780
atggactatc gtctgggttt cattaccctg ggcatgcacc gtaaagtttt tcaggagatc 840
ggcttcttta actgcaccac caagggtagc gatgacgagt tcttccaccg tattgcgaaa 900
tactatggca aggagaaaat caagaacctg ctgctgccgc tgtactataa caccatgcgt 960
gaaaacagcc tgttcaccga catggtggag tggatcgata accacaacat tatccagaag 1020
atgagcgaca cccgtcaaca ctacgcgacc ctgttccagg cgatgcacaa cgaaaccgcg 1080
agccacgatt ttaaaaacct gttccaattt ccgcgtattt acgacgcgct gccggttccg 1140
caggagatga gcaagctgag caacccgaaa atcccggtgt atattaacat ctgcagcatt 1200
ccgagccgta tcgcgcaact gcgtcgtatt atcggtattc tgaagaacca gtgcgaccac 1260
ttccacatct acctggatgg ctatgttgaa attccggact ttatcaagaa cctgggtaac 1320
aaagcgaccg tggttcactg caaagacaag gataacagca ttcgtgacaa cggcaagttc 1380
attctgctgg aggaactgat cgagaagaac caggatggtt actattttac ctgcgatgac 1440
gatattatct acccgagcga ctatattaac accatgatca agaaactgaa cgagtacgac 1500
gataaagcgg ttattggtct gcacggcatc ctgttcccga gccgtatgac caagtatttt 1560
agcgcggatc gtctggtgta cagcttctat aaaccgctgg agaaagacaa ggcggtgaac 1620
gttctgggta ccggcaccgt tagctttcgt gtgagcctgt tcaaccaatt tagcctgagc 1680
gatttcaccc acagcggtat ggcggacatt tactttagcc tgctgtgcaa gaaaaacaac 1740
atcctgcaga tttgcatcag ccgtccggcg aactggctga ccgaagacaa ccgtgatagc 1800
gaaaccctgt accaccaata tcgtgacaac gatgaacagc aaacccagct gattatggag 1860
aacggtccgt ggggctacag cagcatctat ccgctggtta aaaaccatcc gaagttcacc 1920
gacctgattc cgtgcctgcc gttctatttc ctgtaa 1956
<210> 12
<211> 651
<212> PRT
<213> artificial sequence
<400> 12
Met Lys Gly Lys Lys Glu Met Thr Gln Ile Gln Ile Ala Lys Asn Pro
1 5 10 15
Pro Gln His Glu Lys Glu Asn Glu Leu Asn Thr Phe Gln Asn Lys Ile
20 25 30
Asp Ser Leu Lys Thr Thr Leu Asn Lys Asp Ile Ile Ser Gln Gln Thr
35 40 45
Leu Leu Ala Lys Gln Asp Ser Lys His Pro Leu Ser Ala Ser Leu Glu
50 55 60
Asn Glu Asn Lys Leu Leu Leu Lys Gln Leu Gln Leu Val Leu Gln Glu
65 70 75 80
Phe Glu Lys Ile Tyr Thr Tyr Asn Gln Ala Leu Glu Ala Lys Leu Glu
85 90 95
Lys Asp Lys Gln Thr Thr Ser Ile Thr Asp Leu Tyr Asn Glu Val Ala
100 105 110
Lys Ser Asp Leu Gly Leu Val Lys Glu Thr Asn Ser Ala Asn Pro Leu
115 120 125
Val Ser Ile Ile Met Thr Ser His Asn Thr Ala Gln Phe Ile Glu Ala
130 135 140
Ser Ile Asn Ser Leu Leu Leu Gln Thr Tyr Lys Asn Ile Glu Ile Ile
145 150 155 160
Ile Val Asp Asp Asp Ser Ser Asp Asn Thr Phe Glu Ile Ala Ser Arg
165 170 175
Ile Ala Asn Thr Thr Ser Lys Val Arg Val Phe Arg Leu Asn Ser Asn
180 185 190
Leu Gly Thr Tyr Phe Ala Lys Asn Thr Gly Ile Leu Lys Ser Lys Gly
195 200 205
Asp Ile Ile Phe Phe Gln Asp Ser Asp Asp Val Cys His His Glu Arg
210 215 220
Ile Glu Arg Cys Val Asn Ile Leu Leu Ala Asn Lys Glu Thr Ile Ala
225 230 235 240
Val Arg Cys Ala Tyr Ser Arg Leu Ala Pro Glu Thr Gln His Ile Ile
245 250 255
Lys Val Asn Asn Met Asp Tyr Arg Leu Gly Phe Ile Thr Leu Gly Met
260 265 270
His Arg Lys Val Phe Gln Glu Ile Gly Phe Phe Asn Cys Thr Thr Lys
275 280 285
Gly Ser Asp Asp Glu Phe Phe His Arg Ile Ala Lys Tyr Tyr Gly Lys
290 295 300
Glu Lys Ile Lys Asn Leu Leu Leu Pro Leu Tyr Tyr Asn Thr Met Arg
305 310 315 320
Glu Asn Ser Leu Phe Thr Asp Met Val Glu Trp Ile Asp Asn His Asn
325 330 335
Ile Ile Gln Lys Met Ser Asp Thr Arg Gln His Tyr Ala Thr Leu Phe
340 345 350
Gln Ala Met His Asn Glu Thr Ala Ser His Asp Phe Lys Asn Leu Phe
355 360 365
Gln Phe Pro Arg Ile Tyr Asp Ala Leu Pro Val Pro Gln Glu Met Ser
370 375 380
Lys Leu Ser Asn Pro Lys Ile Pro Val Tyr Ile Asn Ile Cys Ser Ile
385 390 395 400
Pro Ser Arg Ile Ala Gln Leu Arg Arg Ile Ile Gly Ile Leu Lys Asn
405 410 415
Gln Cys Asp His Phe His Ile Tyr Leu Asp Gly Tyr Val Glu Ile Pro
420 425 430
Asp Phe Ile Lys Asn Leu Gly Asn Lys Ala Thr Val Val His Cys Lys
435 440 445
Asp Lys Asp Asn Ser Ile Arg Asp Asn Gly Lys Phe Ile Leu Leu Glu
450 455 460
Glu Leu Ile Glu Lys Asn Gln Asp Gly Tyr Tyr Phe Thr Cys Asp Asp
465 470 475 480
Asp Ile Ile Tyr Pro Ser Asp Tyr Ile Asn Thr Met Ile Lys Lys Leu
485 490 495
Asn Glu Tyr Asp Asp Lys Ala Val Ile Gly Leu His Gly Ile Leu Phe
500 505 510
Pro Ser Arg Met Thr Lys Tyr Phe Ser Ala Asp Arg Leu Val Tyr Ser
515 520 525
Phe Tyr Lys Pro Leu Glu Lys Asp Lys Ala Val Asn Val Leu Gly Thr
530 535 540
Gly Thr Val Ser Phe Arg Val Ser Leu Phe Asn Gln Phe Ser Leu Ser
545 550 555 560
Asp Phe Thr His Ser Gly Met Ala Asp Ile Tyr Phe Ser Leu Leu Cys
565 570 575
Lys Lys Asn Asn Ile Leu Gln Ile Cys Ile Ser Arg Pro Ala Asn Trp
580 585 590
Leu Thr Glu Asp Asn Arg Asp Ser Glu Thr Leu Tyr His Gln Tyr Arg
595 600 605
Asp Asn Asp Glu Gln Gln Thr Gln Leu Ile Met Glu Asn Gly Pro Trp
610 615 620
Gly Tyr Ser Ser Ile Tyr Pro Leu Val Lys Asn His Pro Lys Phe Thr
625 630 635 640
Asp Leu Ile Pro Cys Leu Pro Phe Tyr Phe Leu
645 650

Claims (8)

1. The high-activity heparin skeleton synthase PmHS2 mutant T326S is characterized in that the amino acid sequence is shown as SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 7; the 326 th threonine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into serine; genBank of the amino acid sequence of the heparin skeleton synthase PmHS2 is AY292200.1.
2. A high-activity heparin skeleton synthase PmHS2 mutant I476F is characterized in that the amino acid sequence is shown as SEQ ID NO.12, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 11; the 476 th isoleucine of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into phenylalanine; genBank of the amino acid sequence of the heparin skeleton synthase PmHS2 is AY292200.1.
3. A recombinant vector comprising a plasmid vector and a gene encoding the heparin skeleton synthase PmHS2 mutant according to claim 1 or claim 2 inserted into the plasmid vector.
4. The recombinant vector of claim 3, wherein the plasmid vector is pET-28a (+).
5. A recombinant strain obtained by transforming the recombinant vector of claim 3 into a host cell.
6. The recombinant strain of claim 5, wherein the host cell is e.
7. Use of the heparin scaffold synthase mutant according to claim 1 or claim 2, for the synthesis of heparin scaffolds.
8. The use according to claim 7 wherein the use is of UDP-GlcNAc as the donor substrate and GlcA-pNP as the acceptor substrate to catalyze a reaction to produce a heparin backbone having the structure GlcNAc-GlcA-pNP; or the UDP-GlcA is used as a donor substrate, the GlcNAc-GlcA-pNP is used as an acceptor substrate, and the heparin skeleton with the structure of GlcA-GlcNAc-GlcA-pNP is generated by catalytic reaction.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002256501B2 (en) * 2001-05-08 2008-05-29 The Board Of Regents Of The University Of Oklahoma Heparin/heparosan synthase and methods of making and using same
CN101855354A (en) * 2007-09-12 2010-10-06 拜尔作物科学股份公司 Plants which synthesize increased amounts of glucosaminglycans
WO2014132468A1 (en) * 2013-03-01 2014-09-04 独立行政法人理化学研究所 Sugar chain compound and method for producing sugar chain compound
CN107189992A (en) * 2017-06-29 2017-09-22 江南大学 A kind of heparosan synthase and its application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002256501B2 (en) * 2001-05-08 2008-05-29 The Board Of Regents Of The University Of Oklahoma Heparin/heparosan synthase and methods of making and using same
CN101855354A (en) * 2007-09-12 2010-10-06 拜尔作物科学股份公司 Plants which synthesize increased amounts of glucosaminglycans
WO2014132468A1 (en) * 2013-03-01 2014-09-04 独立行政法人理化学研究所 Sugar chain compound and method for producing sugar chain compound
CN107189992A (en) * 2017-06-29 2017-09-22 江南大学 A kind of heparosan synthase and its application

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