CN113528482A - High-activity heparin framework synthase PmHS2 mutant and application thereof - Google Patents

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

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CN113528482A
CN113528482A CN202110881483.0A CN202110881483A CN113528482A CN 113528482 A CN113528482 A CN 113528482A CN 202110881483 A CN202110881483 A CN 202110881483A CN 113528482 A CN113528482 A CN 113528482A
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生举正
孙柳
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Abstract

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

Description

High-activity heparin framework synthase PmHS2 mutant and application thereof
Technical Field
The invention relates to a high-activity heparin framework synthase PmHS2 mutant and application thereof, belonging to the technical field of biology.
Background
Glycosaminoglycan (GAG) is a complex biomacromolecule consisting of repeating disaccharide units of hexuronic acid or hexose and hexosamine regularly arranged to form a linear unbranched acidic polysaccharide. Glycosaminoglycans are widely distributed and various, and classified into glycosaminoglycans without sulfuric acid modification and glycosaminoglycans with sulfuric acid modification according to the degree of sulfation, the glycosaminoglycans without sulfuric acid modification include only hyaluronic acid, and the glycosaminoglycans with sulfuric acid modification include dermatan sulfate/chondroitin sulfate, heparan sulfate/heparin, keratan sulfate, and the like.
Heparin (HP), an important glycosaminoglycan, is composed of repeating disaccharide units formed from D- β -glucuronic acid (or L- α -iduronic acid) and N-acetylglucosamine. It was first discovered from the liver and is named for its activity in enhancing the natural serine protease inhibitor antithrombin iii, catalyzing the inhibition of all serine proteases of the intrinsic coagulation pathway, thus having anticoagulant and antithrombotic effects, and also mediating important physiological processes including cell adhesion, lipid metabolism and growth factor regulation by binding to other proteins. The traditional Chinese medicine composition can be clinically used for treating angina, nephrotic syndrome, severe burns, rheumatoid arthritis and the like, and the demand of the traditional Chinese medicine composition is in the front of the biotechnology medicine field all year round internationally.
The sources of heparin are mainly divided into animal extraction, chemical synthesis and chemical enzymatic synthesis. The animal extracted heparin is mainly extracted from small intestinal mucosa of pigs and cows, but due to the difference of extraction methods and the structure and the number of basic units of the animal derived heparin are different, the structure, the configuration and the molecular weight of heparin products are different, and heparin pollution is possibly caused. In addition, heparin has a complex structure, and when a chemical synthesis method is used for extending a sugar chain, steps such as protection, deprotection, activation and coupling of an unstable group are required to be considered, and conditions such as regioselectivity and stereoselectivity are required to be considered, so that the difficulty is high and the yield is low. The chemical enzyme method synthesis of HP is a method combining a chemical method and an enzyme method, and the enzyme 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 heparin skeleton sugar chains 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-acetylglucosaminyl transferase activity and glucuronidase activity, the enzyme is widely applied to the synthesis by the chemical enzyme method, and the improvement of the enzyme activity is beneficial to the high-efficiency synthesis of the heparin skeleton sugar chains.
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 terms:
GlcA-pNP: the Chinese is called 4-nitrophenyl-beta-D-glucuronic acid, and the function of the Chinese is used as an initial receptor substrate for heparin oligosaccharide synthesis;
UDP-GlcNAc: the Chinese is fully called uridine diphosphate-N-acetylglucosamine, and the function of the Chinese is as a donor to provide acetylglucosamine for the synthesis of heparin oligosaccharide;
the technical scheme of the invention is as follows:
the mutant is any one site mutation of 130 th serine, 135 th serine, 197 th phenylalanine, 326 th threonine, 344 th threonine and 476 th isoleucine of 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 coding gene is shown as SEQ ID NO. 1; the 130 th serine of the amino acid sequence of 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 coding gene is shown as SEQ ID NO. 3; the 135 th serine of the amino acid sequence of 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 coding gene is shown as SEQ ID NO. 5; the phenylalanine at position 197 of an amino acid sequence of 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 coding gene is shown as SEQ ID NO. 7; the threonine at the 326 th site 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 coding gene is shown as SEQ ID NO. 9; the method is characterized in that threonine at the 344 th site of an amino acid sequence of 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 the mutant is shown as SEQ ID NO.12, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 11; isoleucine at the 476 th site of an amino acid sequence of 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.
Preferably, according to the invention, the plasmid vector is pET-28a (+).
In the present invention, the recombinant vector containing the gene encoding the PmHS2 mutant was synthesized by Nanjing Kinsley.
A recombinant strain is obtained by transforming the above recombinant vector into a host cell.
Preferably according to the invention, the host cell is E.coli; further preferably, the host cell is escherichia coli BL21(DE 3).
The application of the heparin framework synthase mutant in synthesizing the heparin framework.
According to the invention, UDP-GlcNAc is used as a donor substrate and GlcA-pNP is used as an acceptor substrate, and the heparin framework with the structure of GlcNAc-GlcA-pNP is generated through catalytic reaction; or carrying out catalytic reaction by using UDP-GlcA as a donor substrate and GlcNAc-GlcA-pNP as an acceptor substrate to generate a heparin framework with the structure of GlcA-GlcNAc-GlcA-pNP.
The experimental procedures not described in detail in the present invention can be performed according to the routine experimental procedures in the technical field.
Advantageous effects
1. The invention selects 130 th, 135 th, 197 th, 326 th, 344 th and 476 th amino acids as the basis of heparin skeleton synthase PmHS2 to carry out site-directed mutagenesis, the 130 th serine is mutated into threonine (S130T), the 135 th serine is mutated into alanine (S135A), the 197 th phenylalanine is mutated into lysine (F197Y), the 326 th threonine is mutated into serine (T326S), the 344 th threonine is mutated into serine (T344S), and the 476 th isoleucine is mutated into phenylalanine (I476F). Compared with heparin framework synthase PmHS2, the heparin framework 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 of the heparin framework synthase PmHS2, and can effectively synthesize a GlcNAc-GlcA-pNP heparin framework by using substrates UDP-GlcNAc and GlcA-pNP under the optimal conditions; or the substrate UDP-GlcA and GlcNAc-GlcA-pNP are utilized to effectively synthesize the GlcA-GlcNAc-GlcA-pNP heparin framework, the GlcNAc transfer synthesis efficiency and the GlcA transfer synthesis efficiency in the synthesis of the heparin framework are improved, the application development of the heparin biomimetic synthesis is greatly promoted, and a brand new page is opened for the research and development of glycosaminoglycan.
2. The heparin framework synthase PmHS2 mutant provided by the invention has good stability, can still keep more than 40% of enzyme activity after being placed at 37 ℃ for 72 hours, and is more than 2 times of the activity of the existing heparin framework synthase PmHS 2. In particular to the protein expressed by the heparin framework synthase mutant PmHS2(F197Y), the heparin framework synthase mutant PmHS2(S135A) and the heparin framework synthase mutant PmHS2(T344S), which can still keep more than 80% of enzyme activity after being placed at 37 ℃ for 72 hours and is 5-7 times of the prior heparin framework synthase PmHS 2.
Drawings
FIG. 1 is a SDS-PAGE result of soluble expression of target protein in recombinant Escherichia coli by a heparin skeleton synthase PmHS2 mutant;
FIG. 2 is a bar graph of the relative conversion rates of heparin skeleton synthase PmHS2 and its mutants;
FIG. 3 is a stability profile of heparin scaffold synthase PmHS2 mutant.
In the figure, the ordinate is the relative yield of the reaction product, based on 100% of the initial enzyme activity, and the abscissa is the time the protein has been left at 37 ℃.
Detailed Description
The technical solution of the present invention is further described below with reference to the following embodiments and the accompanying drawings of the specification, but the scope of the present invention is not limited thereto. Unless otherwise specified, all technical means used in the present invention are well known to those skilled in the art.
The inventors obtained the amino acid sequence and nucleotide sequence information of heparin scaffold synthase PmHS2(GenBank: AY292200.1) from bioinformatics database, and then analyzed the highly conserved region of amino acid sequence using Jalview software. At the same time, protein simulation modeling was performed on heparin scaffold synthase PmHS2 using the Swiss-Model tool, and the active center of the enzyme was predicted using the HotSpot Wizard 2.0. It is found that the 130 th, 135 th, 165 th, 197 th, 267 th, 296 th, 326 th, 344 th, 476 th and 548 th of the amino acid sequence of the heparin skeleton synthase PmHS2 are positioned near an active center and a highly conserved region, and the catalytic activity of PmHS2 is probably improved by carrying out site-directed mutagenesis on the amino acid sequence. Thus, combining the dominant amino acids at these six positions of the homology analysis, ten mutants of PmHS2(S130T), PmHS2(S135A), PmHS2(D165Y), PmHS2(F197Y), PmHS2(F267L), PmHS2(H296N), PmHS2(T326S), PmHS2(T344S), PmHS2(I476F), 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 encoding 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 encoding 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 encoding 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 encoding 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 encoding 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) mutates aspartic acid at position 165 into tyrosine; the mutant PmHS2(F267L) is formed by mutating phenylalanine at position 267 to leucine; mutation of histidine at 296 of mutant PmHS2(H296N) into asparagine; the 548 th serine of the mutant PmHS2(S548A) is mutated into alanine. The unprotected mutants herein will not show their sequence, but their sequence is obtained in the same way.
Unless otherwise indicated, the substrate saccharide reagents employed in the present invention were purchased from sigma and from Producer. All plasmids for the PmHS2 mutant were constructed by Nanjing Kinshire. The HPLC detection method adopts YMC amino column, liquid phase system is Shimadzu liquid phase, and 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 HPLC mobile phase conditions are shown in Table 1:
TABLE 1 HPLC analysis method used for detection of heparin oligosaccharides
Figure BDA0003192160760000041
Example 1 preparation of heparin scaffold synthase PmHS2 mutant
(1) Construction of expression Strain
Taking a mutant PmHS2(F197Y) as an example, a recombinant vector pET28a-His-PmHS2(F197Y) -Stop codon (synthesized by Nanjing Kingsler) containing a gene (SEQ ID NO.5) encoding heparin skeleton synthase PmHS2(F197Y) is transformed into escherichia coli BL21(DE3) competent cells to construct a recombinant strain, the recombinant strain is cultured on an LB plate of kanamycin (100 mu g/mL) for 12 hours, and transformants are screened (a negative control experiment is carried out) to activate to obtain a PmHS2(F197Y) positive transformant; the amino acid sequence of the heparin skeleton synthase is shown in SEQ ID NO. 6.
Positive transformants for the remaining nine mutants, 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
Using the mutant PmHS2(F197Y) as an example, a single colony of a transformant positive to the mutant PmHS2(F197Y) was activated and cultured in 50mL of sterile LB liquid medium for 12 hours (37 ℃, 225 r/min). Then, 1% volume of the activated bacterial suspension was added to LB liquid medium containing kanamycin (100. mu.g/mL), and the mixture was shake-cultured at 37 ℃ and 225r/min for about 3 hours to OD6000.6-0.8, adding IPTG (final concentration of 0.2mM), and inducing at 22 deg.C and 225r/min for 16-18 h. Then, the cells were collected by centrifugation at 8000rpm for 10min and resuspended in equilibration buffer, sonicated on ice (working 15s, pause 45s, amplitude 33%, energy 1500KJ, 4 ℃) for 20min, and the disrupted cells were centrifuged at 12000rpm for 40min (4 ℃), the supernatant was retained, and the supernatant was filtered through a 0.22 μm filter. Purification by histidine tag with nickel column: firstly, the nickel column uses an equilibrium buffer solution to balance 5-10 column volumes, filtered supernatant flows through the nickel column to finish the step of loading, the equilibrium buffer solution is used for balancing 3-5 column volumes after loading, then washing buffer solution is used for washing off impure protein, and finally elution buffer solution is used for eluting to obtain the target protein. The purified protein was stored in 20% glycerol and each tube was stored in a freezer at-80 ℃.
Wherein, the components of the equilibrium buffer solution are as follows: 0.3M NaCl, 50mM NaH2PO4，pH＝8；
The composition of the wash buffer was: 10mM imidazole, 0.3M NaCl, 50mM NaH2PO4，pH＝8；
The composition of the elution buffer was: 250mM imidazole, 0.3M NaCl, 50mM NaH2PO4，pH＝8。
The remaining nine mutants were purified according to the same method as described above to obtain recombinant proteins of PmHS2(S130T), PmHS2(S135A), PmHS2(D165Y), PmHS2(F267L), PmHS2(H296N), PmHS2(T326S), PmHS2(T344S), PmHS2(I476F), and PmHS2 (S548A).
The expression of the purified recombinant protein was identified by polyacrylamide gel electrophoresis (SDS-PAGE) (as shown in FIG. 1), and after the recombinant expression of the positive transformant, the protein was purified, the band was single, and the molecular weight of the target protein was consistent with the theoretical value (PmHS2 is 75.39 KDa).
Example 2 Activity assay of heparin scaffold synthase PmHS2 mutant
Using commercial GlcA-pNP (final concentration of 0.2mM) as an acceptor substrate and UDP-GlcNAc (final concentration of 0.3mM) as a donor substrate, a reaction was carried out using a heparin skeleton synthase PmHS2 and the heparin skeleton synthase PmHS2 mutant prepared in example 1 as proteases in the reaction system shown in Table 2; the reaction is placed in a water bath kettle at 37 ℃ for reaction for 30min, then boiling water is heated for 5min to inactivate protease so as to stop the reaction, the reaction solution is filtered by a filter membrane of 0.22 mu m and then 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, and the flow rate of a mobile phase is 0.5 mL/min.
TABLE 2 enzyme activity verification reaction System for heparin skeleton synthase
Figure BDA0003192160760000051
The results of the measurement are shown in FIG. 2, in which the conversion of PmHS2 was set to 1 and the conversion of the mutant was reduced to a multiple thereof.
As can be seen from FIG. 2, the heparin scaffold synthase PmHS2 mutant has GlcNAc transferase activity and GlcA transferase activity, and can transfer a GlcNAc group 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 produce heparin trisaccharide GlcA-GlcNAc-GlcA-pNP.
In addition, the GlcNAc transferase activity and the GlcA transferase activity of each mutant were improved to various degrees.
The GlcNAc transferase activity of PmHS2(S130T) is increased by 2.51 times of the activity of the PmHS2 of the pro-heparin skeleton synthase, and the GlcA transferase activity is increased by 2.97 times of the activity of the PmHS2 of the pro-heparin skeleton synthase.
The GlcNAc transferase activity of PmHS2(S135A) was increased to 3.31 times that of the pro-heparin skeleton synthase PmHS2, and the GlcA transferase activity was increased to 1.63 times that of the pro-heparin skeleton synthase PmHS 2.
The GlcNAc transferase activity of PmHS2(F197Y) was increased to 2.81 times of the activity of PmHS2, and the GlcA transferase activity was increased to 2.76 times of the activity of PmHS 2.
The GlcNAc transferase activity of PmHS2(T326S) is increased to 3.73 times of the activity of PmHS2, and the GlcA transferase activity is increased to 1.67 times of the activity of PmHS 2.
The GlcNAc transferase activity of PmHS2(T344S) is improved by 2.08 times of the activity of the PmHS2 of the protoparin skeleton synthase, and the GlcA transferase activity is improved by 3.35 times of the activity of the PmHS2 of the protoparin skeleton synthase.
The GlcNAc transferase activity of PmHS2(I476F) is increased to 4.33 times of the activity of PmHS2, and the GlcA transferase activity is increased to 2.29 times of the activity of PmHS 2.
The activity of PmHS2(D165Y), PmHS2(F267L), PmHS2(H297N) and PmHS2(S548A) is not obviously changed.
From the data, compared with the heparin framework synthase PmHS2, the heparin framework 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 of the heparin framework synthase PmHS2, and can effectively synthesize a GlcNAc-GlcA-pNP heparin framework by using UDP-GlcNAc and GlcA-pNP as substrates under the optimal condition; or the substrate UDP-GlcA and GlcNAc-GlcA-pNP are utilized to effectively synthesize the GlcA-GlcNAc-GlcA-pNP heparin framework, the GlcNAc transfer synthesis efficiency and the GlcA transfer synthesis efficiency in the synthesis of the heparin framework are improved, the application development of the heparin biomimetic synthesis is greatly promoted, and a brand new page is opened for the research and development of glycosaminoglycan.
Example 3 stability study of heparin scaffold synthase PmHS2 mutant
The current activities of the available PmHS2 and the PmHS2 mutant protein obtained in example 1 were determined by subjecting them to a reaction system shown in example 2, in which commercial GlcA-pNP (final concentration of 0.2mM) was used as an acceptor substrate and UDP-GlcNAc (final concentration of 0.3mM) was used as a donor substrate after 0h, 24h, 48h, and 72h, respectively, at 37 ℃. After the above reaction was placed in a 37 ℃ water bath for 30min, boiling water was heated for 5min to inactivate protease and terminate the reaction, the reaction solution was filtered through a 0.22 μm filter membrane and subjected to HPLC detection as described in Table 1, the pNP group of the monosaccharide acceptor was specifically absorbed at a detection wavelength of ultraviolet 310nm, the mobile phase flow rate was 0.5mL/min, and the detection results are shown in FIG. 3.
As can be seen from FIG. 3, the PmHS2 mutant of heparin framework synthase provided by the invention has good stability, and can still maintain more than 55% of enzyme activity after being placed at 37 ℃ for 24h, and can still maintain more than 40% of enzyme activity after being placed at 37 ℃ for 72 h. The enzyme activity of the heparin skeleton synthase PmHS2 is reduced to 40 percent after being placed at 37 ℃ for 24 hours, and the enzyme activity is reduced to about 10 percent after being placed at 37 ℃ for 72 hours. Wherein the purified proteins of PmHS2(F197Y), PmHS2(S135A) and PmHS2(T344S) can still keep more than 80% of enzyme activity after being placed at 37 ℃ for 72 hours, and are 5-7 times of the enzyme activity of the PmHS2 of heparin skeleton synthase.
SEQUENCE LISTING
<110> Shandong university
<120> high-activity heparin framework 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 (10)

1. The mutant is characterized in that the mutant is any one site mutation of 130 th serine, 135 th serine, 197 th phenylalanine, 326 th threonine, 344 th threonine and 476 th isoleucine of heparin framework synthase PmHS 2.
2. The high-activity heparin framework synthase PmHS2 mutant according to claim 1, wherein the mutant is a heparin framework synthase PmHS2 mutant (S130T), the amino acid sequence of which is shown as SEQ ID No.2, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 1; the 130 th serine of the amino acid sequence of heparin skeleton synthase PmHS2 is mutated into threonine.
3. The high-activity heparin framework synthase PmHS2 mutant according to claim 1, wherein the mutant is a heparin framework synthase PmHS2 mutant (S135A), the amino acid sequence of which is shown as SEQ ID No.4, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 3; the 135 th serine of the amino acid sequence of heparin skeleton synthase PmHS2 is mutated into alanine.
4. The high-activity heparin framework synthase PmHS2 mutant according to claim 1, wherein the mutant is a heparin framework synthase PmHS2 mutant (F197Y), the amino acid sequence of which is shown as SEQ ID No.6, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 5; the phenylalanine at position 197 of an amino acid sequence of heparin skeleton synthase PmHS2 is mutated into lysine.
5. The high-activity heparin framework synthase PmHS2 mutant according to claim 1, wherein the mutant is a heparin framework synthase PmHS2 mutant (T326S), the amino acid sequence of which is shown as SEQ ID No.8, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 7; the threonine at the 326 th site of the amino acid sequence of the heparin skeleton synthase PmHS2 is mutated into serine.
6. The high-activity heparin framework synthase PmHS2 mutant according to claim 1, wherein the mutant is a heparin framework synthase PmHS2 mutant (T344S), the amino acid sequence of which is shown as SEQ ID No.10, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 9; the method is characterized in that threonine at the 344 th site of an amino acid sequence of heparin skeleton synthase PmHS2 is mutated into serine.
7. The high-activity heparin framework synthase PmHS2 mutant according to claim 1, wherein the mutant is a heparin framework synthase PmHS2 mutant (I476F), the amino acid sequence of which is shown as SEQ ID No.12, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 11; isoleucine at the 476 th site of an amino acid sequence of heparin skeleton synthase PmHS2 is mutated into phenylalanine.
8. A recombinant vector, which is characterized in that a gene encoding the heparin skeleton synthase PmHS2 mutant according to any one of claims 2 to 7 is inserted into a plasmid vector;
preferably, the plasmid vector is pET-28a (+).
9. A recombinant strain obtained by transforming the recombinant vector according to claim 8 into a host cell;
preferably, the host cell is E.coli.
10. Use of the heparin scaffold synthase mutant according to any one of claims 1 to 7 for synthesizing a heparin scaffold;
preferably, the application takes UDP-GlcNAc as a donor substrate and GlcA-pNP as an acceptor substrate, and the heparin framework with the structure of GlcNAc-GlcA-pNP is generated by catalytic reaction; or carrying out catalytic reaction by using UDP-GlcA as a donor substrate and GlcNAc-GlcA-pNP as an acceptor substrate to generate a heparin framework with the structure of GlcA-GlcNAc-GlcA-pNP.
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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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