CN114591940A - Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof - Google Patents

Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof Download PDF

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CN114591940A
CN114591940A CN202210352448.4A CN202210352448A CN114591940A CN 114591940 A CN114591940 A CN 114591940A CN 202210352448 A CN202210352448 A CN 202210352448A CN 114591940 A CN114591940 A CN 114591940A
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许敬亮
吕永坤
贾箐
朱丽娟
张辉
赵安琪
熊文龙
阿拉牧
屈凌波
应汉杰
王诗元
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Abstract

The invention provides a fusion protein for catalyzing glucose to synthesize D-psicose and a construction method thereof, relating to the field of biochemical engineering. In order to avoid the problems of low conversion efficiency and the like caused by diffusion of intermediate products in a double-enzyme catalytic system, the invention constructs fusion proteins connected by connecting peptides with different lengths and improves the conversion efficiency by constructing a substrate channel. Specifically, the glucose isomerase gene and D-psicose 3-epimerase were linked using flexible linker peptides and rigid linker peptides of different lengths, and the results showed that the efficiency of D-psicose synthesis increased with the increase in the length of the linker peptide, and that the rigid linker peptide was superior to the flexible linker peptide. The fusion protein provided by the invention transfers the intermediate product to the next enzyme through the substrate channel, thereby reducing the diffusion of the intermediate product and improving the synthesis efficiency of D-psicose.

Description

Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to a fusion protein for catalyzing glucose to synthesize D-psicose and a construction method thereof.
Background
D-Psicose (D-Psicose or D-Allulose) is a six-carbon sugar with a very low content in nature, and is an epimer of the C-3 site of D-fructose. D-psicose is very difficult to digest and absorb and provides little energy for life activities, and thus is a very useful low-calorie sweetener. In the field of medical health, D-psicose can inhibit fatty liver enzyme and intestinal alpha-glycosidase, thereby reducing accumulation of fat in vivo and inhibiting increase of blood glucose concentration. In the field of food application, D-psicose has the advantages of high sweetness, good solubility, low calorie and low blood sugar reaction and the like, and is considered to be one of the most ideal sucrose substitutes. D-psicose is currently approved by the U.S. Food and Drug Administration (FDA) for safety. Therefore, D-psicose can be applied as a safe additive in the fields of medical care and food processing.
At first, D-psicose is synthesized by a chemical method, and with the progress of research, the chemical synthesis method has the defects of high cost, high process difficulty, complex purification, easy environmental pollution and the like, and is gradually replaced by a biotransformation method. The biotransformation of D-psicose was first proposed by professor Izumori, university of Xiangchuan, Japan, to accomplish the biotransformation between rare ketohexoses using hexitols as intermediates, and it includes epimerase, polyol dehydrogenase and aldoketose isomerase. Wherein the D-psicose 3-epimerase can realize the interconversion between D-fructose and D-psicose. The D-fructose can be generated by glucose isomerase to catalyze glucose to perform epimerization reaction. The method of producing D-psicose from glucose in one step using a two-enzyme system of glucose isomerase and D-psicose 3-epimerase has additional advantages in that the proximity of the two enzymes creates a substrate-rich microenvironment for the second enzyme, greatly reducing the time for the substrate to diffuse into the second enzyme. Therefore, the co-expression of glucose isomerase and D-psicose 3-epimerase provides an excellent opportunity for innovation in the one-step production of D-psicose.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for synthesizing D-psicose from glucose by using a fusion protein as a catalyst, thereby realizing direct synthesis of D-psicose from glucose which is an inexpensive substrate.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a fusion protein for catalyzing glucose to synthesize D-psicose and a construction method thereof.
Optionally, the escherichia coli is escherichia coli BL21(DE 3).
Optionally, the construction method of the fusion protein is as follows: the glucose isomerase-encoding gene derived from Acidothermus cellulolyticus 11B (AcceGI, SEQ ID NO:15) and the D-psicose 3-epimerase-encoding gene derived from Clostridium cellulolyticum H10 (CcDPEase, SEQ ID NO:16) were cloned between the BamHI and HindIII sites of the vector pET28a (PB) N or the BamHI site of the vector pET28a (PB) N-CdDPEase (see Table 1 for primers), respectively, using a one-step cloning kit (Nanjing nopraz Biotech, Inc.) to obtain recombinant plasmids pET28a (PB) N-GP, pET28a (PB) N-GS 6851, pET28a (PB) N-GS2P, pET28a (PB) N-GS3P, pET a (PB) N-GS1P, pET28a (PB) N-GS2 and GE 28 GE3 map (PB) GE a as shown in the figure). Wherein the expression of all pathway genes is controlled by a T7 promoter and a T7 terminator. The recombinant plasmid is transformed into escherichia coli BL21(DE3), and the recombinant escherichia coli BL21/GP, BL21/GS1P, BL21/GS2P, BL21/GS3P, BL21/GE1P, BL21/GE2P and BL21/GE3P are obtained. The recombinant strains can be used for catalyzing direct synthesis of D-psicose from glucose.
Optionally, the conditions for converting glucose into D-psicose by using the recombinant strains BL21/GP, BL21/GS1P, BL21/GS2P, BL21/GS3P, BL21/GE1P, BL21/GE2P and BL21/GE3P are as follows: culturing the recombinant E.coli to OD600When the concentration was 0.6-0.8, IPTG was added to the mixture at a final concentration of 0.5mM, and the mixture was induced at 25 ℃ and 200rpm for 8 hours. Centrifugally collecting thalli at 4 ℃, 8000rpm, washing the thalli by using 20mM phosphate buffer solution with pH of 4.5-8.5 to remove a culture medium, and then re-suspending the thalli by using the phosphate buffer solution with corresponding pH to obtain resting cells which can be used as a biocatalyst for subsequent reaction.
Optionally, the concentration of the substrate glucose is 100 g/L.
Optionally, the catalytic ion CoCl2At a concentration of 1mM, MgCl2Is 5 mM.
Optionally, the conversion condition is reaction for 2 hours at 45-85 ℃.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method uses cheap raw material glucose as a substrate, greatly reduces the production cost of the D-psicose, and is suitable for large-scale production.
(2) The product D-psicose is easy to separate and purify with fructose and glucose by an ion exchange resin method, the process is safe and environment-friendly, and pollution is not easy to cause.
(3) The co-expression of the two enzymes in the fusion protein creates a substrate-rich microenvironment for the second enzyme.
(4) The proximity of the two enzymes in the fusion protein reduces the time for the substrate to diffuse to the second enzyme, greatly increasing the rate of reaction.
Drawings
FIG. 1. reaction process for converting glucose into D-psicose. The reaction is to convert glucose into D-fructose by using glucose isomerase (AcceGI), and then convert the generated D-fructose into D-psicose by using D-psicose 3-epimerase (CcDPEase).
FIG. 2 functional verification of glucose isomerase and D-psicose 3-epimerase. (A) And (3) verifying and verifying the function of the glucose isomerase AcceGI by taking glucose as a substrate. (B) The function of D-psicose 3-epimerase CcDPEase was verified by using fructose as a substrate. Wherein BL21/AcceGI is Escherichia coli expressing glucose isomerase AcceGI; BL21/CcDPEase is Escherichia coli expressing D-psicose 3-epimerase CcDPEase; BL21/pET28a (PB) N is E.coli containing empty plasmid pET28a (PB) N as a blank control. D-Glu is D-glucose, D-Fru is D-fructose, and D-Psi is D-psicose.
FIG. 3 plasmid map of fusion protein. (A) pET28a (PB) N-GP, (B) pET28a (PB) N-GS1P, (C) pET28a (PB) N-GS2P, (D) pET28a (PB) N-GS3P, (E) pET28a (PB) N-GE1P, (F) pET28a (PB) N-GE2P, (G) pET28a (PB) N-GE 3P. The recombinant plasmid contains a glucose isomerase coding gene (AcceGI), a D-psicose 3-epimerase coding gene (CcDPEase) and a link peptide, wherein the expression of the genes is controlled by a T7 promoter and a T7 terminator and is connected in series in a monocistronic mode to form a fusion protein.
FIG. 4 protein expression of fusion proteins. Strain BL21/pET28a (PB) N containing empty plasmid pET28a (PB) N was a blank control.
FIG. 5 comparison of the activities of seven fusion proteins. (A) Comparison of conversion rates of seven fusion proteins to glucose, and (B) comparison of conversion rates of seven fusion proteins to fructose.
FIG. 6 optimal temperature and pH for the fusion protein to catalyze the production of D-psicose from glucose. (A) Temperature optimization of glucose to D-psicose conversion, (B) pH optimization of glucose to D-psicose conversion.
Detailed description of the invention
The invention provides a fusion protein for catalyzing glucose to synthesize D-psicose and a construction method thereof.
The following examples are provided to illustrate the method for producing D-psicose directly from glucose by using the fusion protein and the construction method of the fusion protein, but they should not be construed as limiting the scope of the present invention.
Detailed Description
Example 1: functional verification of glucose isomerase and D-psicose isomerase
Coli BL21/AcceGI and BL21/CcDPEase each containing a glucose isomerase-encoding gene (AcceGI, SEQ ID NO:15) and a D-psicose 3-epimerase-encoding gene (CcDPEase, SEQ ID NO:16) were cultured overnight in LB medium at 37 ℃ and 200rpm, transferred to a 250mL Erlenmeyer flask containing 25mL of fresh LB medium with an inoculum size of 4% (v/v), and cultured at 37 ℃ and 200rpm to logarithmic phase. Then, IPTG was added to the cells at a final concentration of 500. mu.M, and the cells were further cultured at 25 ℃ and 200rpm for 8 hours to express the glucose isomerase gene and the D-psicose 3-epimerase gene. And respectively adding glucose and D-fructose to verify the functions of the glucose isomerase and the D-psicose isomerase. Coli BL21/pET28a (PB) N with empty plasmid pET28a (PB) N was used as a blank control, and the other operating conditions were the same.
To the system containing BL21/AcceGI was added glucose at a final concentration of 5g/L, 1mM CoCl25mM MgCl2Reacting for 1h at 80 ℃; to the system containing BL21/CcDPEase was added D-fructose at a final concentration of 5g/L, 1mM CoCl25mM MgCl2And reacting at 60 ℃ for 1 h. The reaction supernatant was detected by ion chromatography.
The method for analyzing D-fructose and D-psicose by ion chromatography is as follows: the ion chromatography model is a Saimeifei ICS-6000 high-pressure ion chromatograph, the detector is an electrochemical detector, the chromatographic column model is PA-20, the mobile phase A is ultrapure water, the mobile phase B is 200mM NaOH aqueous solution, and gradient elution is carried out to obtain (B%): 0min 10%, 15min 10%, 15.1min 100%, 25min 100%, 25.1min 10%, 35min 10%, constant flow rate 0.5mL/min, sample size 25 μ L, and column oven and detector temperature both 30 ℃.
The ion chromatographic analysis result shows that the peak-off time of the D-fructose and the D-psicose is 7.863min and 9.475min respectively.
The blank (E.coli BL21/pET28a (PB) N) synthesized neither D-fructose nor D-psicose; the experimental groups (E.coli BL21/AcceGI and BL21/CcDPEase) synthesized D-fructose and D-psicose, respectively (FIG. 2). The results show that glucose isomerase can convert D-glucose into D-fructose; d-psicose 3-epimerase can convert D-fructose into D-psicose.
Example 2: construction and functional analysis of fusion proteins
The glucose isomerase-encoding gene derived from Acidothermus cellulolyticus 11B (AcceGI, SEQ ID NO:15) and D-psicose 3-epimerase derived from Clostridium cellulolyticum H10 (CcDPEase, SEQ ID NO:16) were cloned between the BamHI and HindIII sites of vector pET28a (PB) N or the BamHI site of vector pET28a (PB) N-CcDPEase (primers see Table 1), respectively, using a one-step cloning kit (Nanjing Nozanza Biotech Co., Ltd.), to obtain recombinant plasmids pET28a (PB) N-GP, pET28a (PB) N-GS 6851, pET28a (PB) N-GS2P, pET28a (PB) N-pET 3P, T a (PB) N-GE P, pET28 (PB) N-GE3 PB a (PB) N-GE2 a, as shown in FIG. 3 map, with peptide linker 3626. Wherein the expression of all pathway genes is controlled by a T7 promoter and a T7 terminator. The recombinant plasmid is transformed into escherichia coli BL21(DE3), and the recombinant escherichia coli BL21/GP, BL21/GS1P, BL21/GS2P, BL21/GS3P, BL21/GE1P, BL21/GE2P and BL21/GE3P are obtained. The recombinant strains can be used for catalyzing the synthesis of D-psicose from glucose.
TABLE 1 primers used in the present invention
Figure BDA0003581242700000071
Figure BDA0003581242700000081
TABLE 2 linker peptides used in the construction of fusion proteins of the invention
Figure BDA0003581242700000082
Recombinant Escherichia coli BL21/GP, BL21/GS1P, BL21/GS2P, BL21/GS3P, BL21/GE1P, BL21/GE2P and BL21/GE3P are cultured overnight in LB medium at 37 ℃ and 200rpm, transferred to a 250mL triangular flask containing 25mL of fresh LB medium with an inoculum size of 4% (v/v), and cultured at 37 ℃ and 200rpm to logarithmic phase. Then, IPTG was added to the resulting mixture to a final concentration of 500. mu.M, and the mixture was further cultured at 25 ℃ and 200rpm for 8 hours to express glucose isomerase and D-psicose 3-epimerase genes in the fusion protein. Protein expression was examined by protein gel electrophoresis (SDS-PAGE).
The protein gel electrophoresis method for detecting the protein expression condition comprises the following steps: the protein gel electrophoresis model is a Saimer flying polyacrylamide protein gel electrophoresis apparatus, and the voltage of 150V is used for 30 min.
Protein gel electrophoresis analysis results show that the molecular weights corresponding to the fusion proteins GP, GS1P, GS2P, GS3P, GE1P, GE2P and GE3P are 81.475kDa, 81.79kDa, 82.105kDa, 82.420kDa, 81.945kDa, 82.416kDa and 82.886kDa respectively.
The blank control (E.coli BL21/pET28a (PB) N) had no obvious protein band; the experimental group (Escherichia coli BL21/GP, BL21/GS1P, BL21/GS2P, BL21/GS3P, BL21/GE1P, BL21/GE2P and BL21/GE3P) has obvious protein bands (FIG. 4). This result indicates that the genes of the fusion proteins are all expressed.
The induced system was divided into two groups, one of which was added with glucose at a final concentration of 50g/L, 1mM Co2+And 5mM Mg2+Reacting for 1h at 80 ℃; the other group was added with D-fructose to a final concentration of 50g/L, 1mM Co2+And 5mM Mg2+And reacting for 1h at 60 ℃. The conversion rates of the seven fusion proteins for converting glucose and converting D-fructose were examined by ion chromatography. Among the seven fusion proteins, GS3P and GE3P have higher conversion rate of converting D-glucose into D-fructose (FIG. 5A); at the same time, the conversion rate of D-fructose into D-psicose by GS3P and GE3P was also higher (FIG. 5B). This result indicates that the higher the number of linker peptides used, the higher the conversion rate of the fusion protein, in the case of the fusion protein formed by linking the same linker peptides.
Example 3: optimization of reaction conditions for catalyzing glucose to generate D-psicose by fusion proteins GE3P and GS3P
In order to improve the yield and the conversion rate of the D-psicose generated by one-step conversion of glucose catalyzed by the fusion protein, the invention further optimizes the reaction conditions for converting the D-psicose.
The invention optimizes the reaction conditions for converting the glucose into the D-psicose: the reaction temperature range is 45-85 ℃, the reaction pH range is 4.5-8.5, and the concentration of substrate glucose is 100 g/L. As a result, the optimum reaction temperature was 65 ℃ C (FIG. 6A), and the optimum reaction pH was 7.5 (FIG. 6B).
Under the optimal conditions, the yield of the D-psicose can reach 5.69 g/L. The result shows that the method for generating D-psicose by catalyzing glucose through the fusion protein can be smoothly carried out, and has great application potential.
The foregoing is only an alternative embodiment of the present invention, and it should be noted that modifications and embellishments could be made by those skilled in the art without departing from the principle of the present invention, and these should be considered as the protection scope of the present invention.
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725 730
<210> 2
<211> 738
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Leu Thr Thr Ala Ser Ser Lys Thr Ile Glu Val Ala
35 40 45
Thr Pro Ser Lys Glu Asp Arg Phe Ser Phe Gly Leu Trp Thr Val Gly
50 55 60
Trp Gln Ala Arg Asp Pro Phe Gly Glu Ala Thr Arg Pro Pro Leu Asp
65 70 75 80
Pro Val Glu Ala Val His Lys Leu Ala Glu Leu Gly Ala Tyr Gly Val
85 90 95
Thr Phe His Asp Asp Asp Leu Val Pro Phe Gly Ser Ser Asp Ala Glu
100 105 110
Arg Ala Arg Leu Ile Asp Arg Phe Lys Lys Ala Leu Ala Asp Thr Gly
115 120 125
Leu Val Val Pro Met Met Thr Thr Asn Leu Phe Thr His Pro Ile Phe
130 135 140
Lys Asp Gly Ala Phe Thr Ala Asn Asp Arg Ser Ile Arg Arg Tyr Ala
145 150 155 160
Ile Arg Lys Val Met Arg Asn Leu Asp Leu Ala Ala Glu Leu Gly Ala
165 170 175
Arg Thr Tyr Val Phe Trp Gly Gly Arg Glu Gly Ser Glu Ile Asp Ala
180 185 190
Ala Lys Asp Ile Arg Ala Ala Leu Asp Arg Tyr Arg Glu Ala Ile Asp
195 200 205
Thr Leu Ala Gln Tyr Val Lys Asp Gln Gly Tyr Gly Ile Arg Phe Ala
210 215 220
Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Phe Leu Pro Thr
225 230 235 240
Ile Gly His Ala Leu Ala Phe Ile Asn Ser Leu Glu His Ser Asp Ile
245 250 255
Val Gly Leu Asn Pro Glu Val Gly His Glu Gln Met Ser Asn Leu Asn
260 265 270
Phe Val His Gly Ile Ala Gln Ala Leu Trp His Gly Lys Leu Phe His
275 280 285
Ile Asp Leu Asn Gly Gln His Gly Pro Lys Tyr Asp Gln Asp Leu Val
290 295 300
Phe Gly His Gly Asp Leu Leu Ser Ala Phe Phe Leu Val Asp Leu Leu
305 310 315 320
Glu Asn Gly Phe Pro Gly Gly Gly Pro Val Tyr Asp Gly Pro Arg His
325 330 335
Phe Asp Tyr Lys Pro Met Arg Thr Glu Asp Ile Asp Gly Val Trp Ala
340 345 350
Ser Ala Ala Ala Asn Met Arg Thr Tyr Leu Leu Leu Lys Gln Arg Ala
355 360 365
Lys Ala Phe Arg Ala Asp Pro Glu Val Gln Ala Ala Leu Thr Ala Ser
370 375 380
Arg Val Pro Glu Leu Ala Val Pro Thr Leu Gly Glu Gly Glu Ser Tyr
385 390 395 400
Ala Asp Leu Leu Ala Asp Arg Ser Ala Trp Glu Glu Phe Asp Val Asp
405 410 415
Arg Ala Ala Asn Gln Gly Tyr Gly Tyr Ala Arg Leu Asp Gln Leu Ala
420 425 430
Ile Glu His Leu Leu Gly Ala Arg Gly Gly Gly Gly Gly Ser Lys His
435 440 445
Gly Ile Tyr Tyr Ala Tyr Trp Glu Gln Glu Trp Glu Ala Asp Tyr Lys
450 455 460
Tyr Tyr Ile Glu Lys Val Ala Lys Leu Gly Phe Asp Ile Leu Glu Ile
465 470 475 480
Ala Ala Ser Pro Leu Pro Phe Tyr Ser Asp Ile Gln Ile Asn Glu Leu
485 490 495
Lys Ala Cys Ala His Gly Asn Gly Ile Thr Leu Thr Val Gly His Gly
500 505 510
Pro Ser Ala Glu Gln Asn Leu Ser Ser Pro Asp Pro Asp Ile Arg Lys
515 520 525
Asn Ala Lys Ala Phe Tyr Thr Asp Leu Leu Lys Arg Leu Tyr Lys Leu
530 535 540
Asp Val His Leu Ile Gly Gly Ala Leu Tyr Ser Tyr Trp Pro Ile Asp
545 550 555 560
Tyr Thr Lys Thr Ile Asp Lys Lys Gly Asp Trp Glu Arg Ser Val Glu
565 570 575
Ser Val Arg Glu Val Ala Lys Val Ala Glu Ala Cys Gly Val Asp Phe
580 585 590
Cys Leu Glu Val Leu Asn Arg Phe Glu Asn Tyr Leu Ile Asn Thr Ala
595 600 605
Gln Glu Gly Val Asp Phe Val Lys Gln Val Asp His Asn Asn Val Lys
610 615 620
Val Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Asp Ser Ile Gly
625 630 635 640
Gly Ala Ile Arg Thr Ala Gly Ser Tyr Leu Gly His Leu His Thr Gly
645 650 655
Glu Cys Asn Arg Lys Val Pro Gly Arg Gly Arg Ile Pro Trp Val Glu
660 665 670
Ile Gly Glu Ala Leu Ala Asp Ile Gly Tyr Asn Gly Ser Val Val Met
675 680 685
Glu Pro Phe Val Arg Met Gly Gly Thr Val Gly Ser Asn Ile Lys Val
690 695 700
Trp Arg Asp Ile Ser Asn Gly Ala Asp Glu Lys Met Leu Asp Arg Glu
705 710 715 720
Ala Gln Ala Ala Leu Asp Phe Ser Arg Tyr Val Leu Glu Cys His Lys
725 730 735
His Ser
<210> 3
<211> 743
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 3
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Leu Thr Thr Ala Ser Ser Lys Thr Ile Glu Val Ala
35 40 45
Thr Pro Ser Lys Glu Asp Arg Phe Ser Phe Gly Leu Trp Thr Val Gly
50 55 60
Trp Gln Ala Arg Asp Pro Phe Gly Glu Ala Thr Arg Pro Pro Leu Asp
65 70 75 80
Pro Val Glu Ala Val His Lys Leu Ala Glu Leu Gly Ala Tyr Gly Val
85 90 95
Thr Phe His Asp Asp Asp Leu Val Pro Phe Gly Ser Ser Asp Ala Glu
100 105 110
Arg Ala Arg Leu Ile Asp Arg Phe Lys Lys Ala Leu Ala Asp Thr Gly
115 120 125
Leu Val Val Pro Met Met Thr Thr Asn Leu Phe Thr His Pro Ile Phe
130 135 140
Lys Asp Gly Ala Phe Thr Ala Asn Asp Arg Ser Ile Arg Arg Tyr Ala
145 150 155 160
Ile Arg Lys Val Met Arg Asn Leu Asp Leu Ala Ala Glu Leu Gly Ala
165 170 175
Arg Thr Tyr Val Phe Trp Gly Gly Arg Glu Gly Ser Glu Ile Asp Ala
180 185 190
Ala Lys Asp Ile Arg Ala Ala Leu Asp Arg Tyr Arg Glu Ala Ile Asp
195 200 205
Thr Leu Ala Gln Tyr Val Lys Asp Gln Gly Tyr Gly Ile Arg Phe Ala
210 215 220
Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Phe Leu Pro Thr
225 230 235 240
Ile Gly His Ala Leu Ala Phe Ile Asn Ser Leu Glu His Ser Asp Ile
245 250 255
Val Gly Leu Asn Pro Glu Val Gly His Glu Gln Met Ser Asn Leu Asn
260 265 270
Phe Val His Gly Ile Ala Gln Ala Leu Trp His Gly Lys Leu Phe His
275 280 285
Ile Asp Leu Asn Gly Gln His Gly Pro Lys Tyr Asp Gln Asp Leu Val
290 295 300
Phe Gly His Gly Asp Leu Leu Ser Ala Phe Phe Leu Val Asp Leu Leu
305 310 315 320
Glu Asn Gly Phe Pro Gly Gly Gly Pro Val Tyr Asp Gly Pro Arg His
325 330 335
Phe Asp Tyr Lys Pro Met Arg Thr Glu Asp Ile Asp Gly Val Trp Ala
340 345 350
Ser Ala Ala Ala Asn Met Arg Thr Tyr Leu Leu Leu Lys Gln Arg Ala
355 360 365
Lys Ala Phe Arg Ala Asp Pro Glu Val Gln Ala Ala Leu Thr Ala Ser
370 375 380
Arg Val Pro Glu Leu Ala Val Pro Thr Leu Gly Glu Gly Glu Ser Tyr
385 390 395 400
Ala Asp Leu Leu Ala Asp Arg Ser Ala Trp Glu Glu Phe Asp Val Asp
405 410 415
Arg Ala Ala Asn Gln Gly Tyr Gly Tyr Ala Arg Leu Asp Gln Leu Ala
420 425 430
Ile Glu His Leu Leu Gly Ala Arg Gly Gly Gly Gly Gly Ser Gly Gly
435 440 445
Gly Gly Ser Lys His Gly Ile Tyr Tyr Ala Tyr Trp Glu Gln Glu Trp
450 455 460
Glu Ala Asp Tyr Lys Tyr Tyr Ile Glu Lys Val Ala Lys Leu Gly Phe
465 470 475 480
Asp Ile Leu Glu Ile Ala Ala Ser Pro Leu Pro Phe Tyr Ser Asp Ile
485 490 495
Gln Ile Asn Glu Leu Lys Ala Cys Ala His Gly Asn Gly Ile Thr Leu
500 505 510
Thr Val Gly His Gly Pro Ser Ala Glu Gln Asn Leu Ser Ser Pro Asp
515 520 525
Pro Asp Ile Arg Lys Asn Ala Lys Ala Phe Tyr Thr Asp Leu Leu Lys
530 535 540
Arg Leu Tyr Lys Leu Asp Val His Leu Ile Gly Gly Ala Leu Tyr Ser
545 550 555 560
Tyr Trp Pro Ile Asp Tyr Thr Lys Thr Ile Asp Lys Lys Gly Asp Trp
565 570 575
Glu Arg Ser Val Glu Ser Val Arg Glu Val Ala Lys Val Ala Glu Ala
580 585 590
Cys Gly Val Asp Phe Cys Leu Glu Val Leu Asn Arg Phe Glu Asn Tyr
595 600 605
Leu Ile Asn Thr Ala Gln Glu Gly Val Asp Phe Val Lys Gln Val Asp
610 615 620
His Asn Asn Val Lys Val Met Leu Asp Thr Phe His Met Asn Ile Glu
625 630 635 640
Glu Asp Ser Ile Gly Gly Ala Ile Arg Thr Ala Gly Ser Tyr Leu Gly
645 650 655
His Leu His Thr Gly Glu Cys Asn Arg Lys Val Pro Gly Arg Gly Arg
660 665 670
Ile Pro Trp Val Glu Ile Gly Glu Ala Leu Ala Asp Ile Gly Tyr Asn
675 680 685
Gly Ser Val Val Met Glu Pro Phe Val Arg Met Gly Gly Thr Val Gly
690 695 700
Ser Asn Ile Lys Val Trp Arg Asp Ile Ser Asn Gly Ala Asp Glu Lys
705 710 715 720
Met Leu Asp Arg Glu Ala Gln Ala Ala Leu Asp Phe Ser Arg Tyr Val
725 730 735
Leu Glu Cys His Lys His Ser
740
<210> 4
<211> 748
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Leu Thr Thr Ala Ser Ser Lys Thr Ile Glu Val Ala
35 40 45
Thr Pro Ser Lys Glu Asp Arg Phe Ser Phe Gly Leu Trp Thr Val Gly
50 55 60
Trp Gln Ala Arg Asp Pro Phe Gly Glu Ala Thr Arg Pro Pro Leu Asp
65 70 75 80
Pro Val Glu Ala Val His Lys Leu Ala Glu Leu Gly Ala Tyr Gly Val
85 90 95
Thr Phe His Asp Asp Asp Leu Val Pro Phe Gly Ser Ser Asp Ala Glu
100 105 110
Arg Ala Arg Leu Ile Asp Arg Phe Lys Lys Ala Leu Ala Asp Thr Gly
115 120 125
Leu Val Val Pro Met Met Thr Thr Asn Leu Phe Thr His Pro Ile Phe
130 135 140
Lys Asp Gly Ala Phe Thr Ala Asn Asp Arg Ser Ile Arg Arg Tyr Ala
145 150 155 160
Ile Arg Lys Val Met Arg Asn Leu Asp Leu Ala Ala Glu Leu Gly Ala
165 170 175
Arg Thr Tyr Val Phe Trp Gly Gly Arg Glu Gly Ser Glu Ile Asp Ala
180 185 190
Ala Lys Asp Ile Arg Ala Ala Leu Asp Arg Tyr Arg Glu Ala Ile Asp
195 200 205
Thr Leu Ala Gln Tyr Val Lys Asp Gln Gly Tyr Gly Ile Arg Phe Ala
210 215 220
Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Phe Leu Pro Thr
225 230 235 240
Ile Gly His Ala Leu Ala Phe Ile Asn Ser Leu Glu His Ser Asp Ile
245 250 255
Val Gly Leu Asn Pro Glu Val Gly His Glu Gln Met Ser Asn Leu Asn
260 265 270
Phe Val His Gly Ile Ala Gln Ala Leu Trp His Gly Lys Leu Phe His
275 280 285
Ile Asp Leu Asn Gly Gln His Gly Pro Lys Tyr Asp Gln Asp Leu Val
290 295 300
Phe Gly His Gly Asp Leu Leu Ser Ala Phe Phe Leu Val Asp Leu Leu
305 310 315 320
Glu Asn Gly Phe Pro Gly Gly Gly Pro Val Tyr Asp Gly Pro Arg His
325 330 335
Phe Asp Tyr Lys Pro Met Arg Thr Glu Asp Ile Asp Gly Val Trp Ala
340 345 350
Ser Ala Ala Ala Asn Met Arg Thr Tyr Leu Leu Leu Lys Gln Arg Ala
355 360 365
Lys Ala Phe Arg Ala Asp Pro Glu Val Gln Ala Ala Leu Thr Ala Ser
370 375 380
Arg Val Pro Glu Leu Ala Val Pro Thr Leu Gly Glu Gly Glu Ser Tyr
385 390 395 400
Ala Asp Leu Leu Ala Asp Arg Ser Ala Trp Glu Glu Phe Asp Val Asp
405 410 415
Arg Ala Ala Asn Gln Gly Tyr Gly Tyr Ala Arg Leu Asp Gln Leu Ala
420 425 430
Ile Glu His Leu Leu Gly Ala Arg Gly Gly Gly Gly Gly Ser Gly Gly
435 440 445
Gly Gly Ser Gly Gly Gly Gly Ser Lys His Gly Ile Tyr Tyr Ala Tyr
450 455 460
Trp Glu Gln Glu Trp Glu Ala Asp Tyr Lys Tyr Tyr Ile Glu Lys Val
465 470 475 480
Ala Lys Leu Gly Phe Asp Ile Leu Glu Ile Ala Ala Ser Pro Leu Pro
485 490 495
Phe Tyr Ser Asp Ile Gln Ile Asn Glu Leu Lys Ala Cys Ala His Gly
500 505 510
Asn Gly Ile Thr Leu Thr Val Gly His Gly Pro Ser Ala Glu Gln Asn
515 520 525
Leu Ser Ser Pro Asp Pro Asp Ile Arg Lys Asn Ala Lys Ala Phe Tyr
530 535 540
Thr Asp Leu Leu Lys Arg Leu Tyr Lys Leu Asp Val His Leu Ile Gly
545 550 555 560
Gly Ala Leu Tyr Ser Tyr Trp Pro Ile Asp Tyr Thr Lys Thr Ile Asp
565 570 575
Lys Lys Gly Asp Trp Glu Arg Ser Val Glu Ser Val Arg Glu Val Ala
580 585 590
Lys Val Ala Glu Ala Cys Gly Val Asp Phe Cys Leu Glu Val Leu Asn
595 600 605
Arg Phe Glu Asn Tyr Leu Ile Asn Thr Ala Gln Glu Gly Val Asp Phe
610 615 620
Val Lys Gln Val Asp His Asn Asn Val Lys Val Met Leu Asp Thr Phe
625 630 635 640
His Met Asn Ile Glu Glu Asp Ser Ile Gly Gly Ala Ile Arg Thr Ala
645 650 655
Gly Ser Tyr Leu Gly His Leu His Thr Gly Glu Cys Asn Arg Lys Val
660 665 670
Pro Gly Arg Gly Arg Ile Pro Trp Val Glu Ile Gly Glu Ala Leu Ala
675 680 685
Asp Ile Gly Tyr Asn Gly Ser Val Val Met Glu Pro Phe Val Arg Met
690 695 700
Gly Gly Thr Val Gly Ser Asn Ile Lys Val Trp Arg Asp Ile Ser Asn
705 710 715 720
Gly Ala Asp Glu Lys Met Leu Asp Arg Glu Ala Gln Ala Ala Leu Asp
725 730 735
Phe Ser Arg Tyr Val Leu Glu Cys His Lys His Ser
740 745
<210> 5
<211> 738
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 5
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Leu Thr Thr Ala Ser Ser Lys Thr Ile Glu Val Ala
35 40 45
Thr Pro Ser Lys Glu Asp Arg Phe Ser Phe Gly Leu Trp Thr Val Gly
50 55 60
Trp Gln Ala Arg Asp Pro Phe Gly Glu Ala Thr Arg Pro Pro Leu Asp
65 70 75 80
Pro Val Glu Ala Val His Lys Leu Ala Glu Leu Gly Ala Tyr Gly Val
85 90 95
Thr Phe His Asp Asp Asp Leu Val Pro Phe Gly Ser Ser Asp Ala Glu
100 105 110
Arg Ala Arg Leu Ile Asp Arg Phe Lys Lys Ala Leu Ala Asp Thr Gly
115 120 125
Leu Val Val Pro Met Met Thr Thr Asn Leu Phe Thr His Pro Ile Phe
130 135 140
Lys Asp Gly Ala Phe Thr Ala Asn Asp Arg Ser Ile Arg Arg Tyr Ala
145 150 155 160
Ile Arg Lys Val Met Arg Asn Leu Asp Leu Ala Ala Glu Leu Gly Ala
165 170 175
Arg Thr Tyr Val Phe Trp Gly Gly Arg Glu Gly Ser Glu Ile Asp Ala
180 185 190
Ala Lys Asp Ile Arg Ala Ala Leu Asp Arg Tyr Arg Glu Ala Ile Asp
195 200 205
Thr Leu Ala Gln Tyr Val Lys Asp Gln Gly Tyr Gly Ile Arg Phe Ala
210 215 220
Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Phe Leu Pro Thr
225 230 235 240
Ile Gly His Ala Leu Ala Phe Ile Asn Ser Leu Glu His Ser Asp Ile
245 250 255
Val Gly Leu Asn Pro Glu Val Gly His Glu Gln Met Ser Asn Leu Asn
260 265 270
Phe Val His Gly Ile Ala Gln Ala Leu Trp His Gly Lys Leu Phe His
275 280 285
Ile Asp Leu Asn Gly Gln His Gly Pro Lys Tyr Asp Gln Asp Leu Val
290 295 300
Phe Gly His Gly Asp Leu Leu Ser Ala Phe Phe Leu Val Asp Leu Leu
305 310 315 320
Glu Asn Gly Phe Pro Gly Gly Gly Pro Val Tyr Asp Gly Pro Arg His
325 330 335
Phe Asp Tyr Lys Pro Met Arg Thr Glu Asp Ile Asp Gly Val Trp Ala
340 345 350
Ser Ala Ala Ala Asn Met Arg Thr Tyr Leu Leu Leu Lys Gln Arg Ala
355 360 365
Lys Ala Phe Arg Ala Asp Pro Glu Val Gln Ala Ala Leu Thr Ala Ser
370 375 380
Arg Val Pro Glu Leu Ala Val Pro Thr Leu Gly Glu Gly Glu Ser Tyr
385 390 395 400
Ala Asp Leu Leu Ala Asp Arg Ser Ala Trp Glu Glu Phe Asp Val Asp
405 410 415
Arg Ala Ala Asn Gln Gly Tyr Gly Tyr Ala Arg Leu Asp Gln Leu Ala
420 425 430
Ile Glu His Leu Leu Gly Ala Arg Gly Glu Ala Ala Ala Lys Lys His
435 440 445
Gly Ile Tyr Tyr Ala Tyr Trp Glu Gln Glu Trp Glu Ala Asp Tyr Lys
450 455 460
Tyr Tyr Ile Glu Lys Val Ala Lys Leu Gly Phe Asp Ile Leu Glu Ile
465 470 475 480
Ala Ala Ser Pro Leu Pro Phe Tyr Ser Asp Ile Gln Ile Asn Glu Leu
485 490 495
Lys Ala Cys Ala His Gly Asn Gly Ile Thr Leu Thr Val Gly His Gly
500 505 510
Pro Ser Ala Glu Gln Asn Leu Ser Ser Pro Asp Pro Asp Ile Arg Lys
515 520 525
Asn Ala Lys Ala Phe Tyr Thr Asp Leu Leu Lys Arg Leu Tyr Lys Leu
530 535 540
Asp Val His Leu Ile Gly Gly Ala Leu Tyr Ser Tyr Trp Pro Ile Asp
545 550 555 560
Tyr Thr Lys Thr Ile Asp Lys Lys Gly Asp Trp Glu Arg Ser Val Glu
565 570 575
Ser Val Arg Glu Val Ala Lys Val Ala Glu Ala Cys Gly Val Asp Phe
580 585 590
Cys Leu Glu Val Leu Asn Arg Phe Glu Asn Tyr Leu Ile Asn Thr Ala
595 600 605
Gln Glu Gly Val Asp Phe Val Lys Gln Val Asp His Asn Asn Val Lys
610 615 620
Val Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Asp Ser Ile Gly
625 630 635 640
Gly Ala Ile Arg Thr Ala Gly Ser Tyr Leu Gly His Leu His Thr Gly
645 650 655
Glu Cys Asn Arg Lys Val Pro Gly Arg Gly Arg Ile Pro Trp Val Glu
660 665 670
Ile Gly Glu Ala Leu Ala Asp Ile Gly Tyr Asn Gly Ser Val Val Met
675 680 685
Glu Pro Phe Val Arg Met Gly Gly Thr Val Gly Ser Asn Ile Lys Val
690 695 700
Trp Arg Asp Ile Ser Asn Gly Ala Asp Glu Lys Met Leu Asp Arg Glu
705 710 715 720
Ala Gln Ala Ala Leu Asp Phe Ser Arg Tyr Val Leu Glu Cys His Lys
725 730 735
His Ser
<210> 6
<211> 743
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Leu Thr Thr Ala Ser Ser Lys Thr Ile Glu Val Ala
35 40 45
Thr Pro Ser Lys Glu Asp Arg Phe Ser Phe Gly Leu Trp Thr Val Gly
50 55 60
Trp Gln Ala Arg Asp Pro Phe Gly Glu Ala Thr Arg Pro Pro Leu Asp
65 70 75 80
Pro Val Glu Ala Val His Lys Leu Ala Glu Leu Gly Ala Tyr Gly Val
85 90 95
Thr Phe His Asp Asp Asp Leu Val Pro Phe Gly Ser Ser Asp Ala Glu
100 105 110
Arg Ala Arg Leu Ile Asp Arg Phe Lys Lys Ala Leu Ala Asp Thr Gly
115 120 125
Leu Val Val Pro Met Met Thr Thr Asn Leu Phe Thr His Pro Ile Phe
130 135 140
Lys Asp Gly Ala Phe Thr Ala Asn Asp Arg Ser Ile Arg Arg Tyr Ala
145 150 155 160
Ile Arg Lys Val Met Arg Asn Leu Asp Leu Ala Ala Glu Leu Gly Ala
165 170 175
Arg Thr Tyr Val Phe Trp Gly Gly Arg Glu Gly Ser Glu Ile Asp Ala
180 185 190
Ala Lys Asp Ile Arg Ala Ala Leu Asp Arg Tyr Arg Glu Ala Ile Asp
195 200 205
Thr Leu Ala Gln Tyr Val Lys Asp Gln Gly Tyr Gly Ile Arg Phe Ala
210 215 220
Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Phe Leu Pro Thr
225 230 235 240
Ile Gly His Ala Leu Ala Phe Ile Asn Ser Leu Glu His Ser Asp Ile
245 250 255
Val Gly Leu Asn Pro Glu Val Gly His Glu Gln Met Ser Asn Leu Asn
260 265 270
Phe Val His Gly Ile Ala Gln Ala Leu Trp His Gly Lys Leu Phe His
275 280 285
Ile Asp Leu Asn Gly Gln His Gly Pro Lys Tyr Asp Gln Asp Leu Val
290 295 300
Phe Gly His Gly Asp Leu Leu Ser Ala Phe Phe Leu Val Asp Leu Leu
305 310 315 320
Glu Asn Gly Phe Pro Gly Gly Gly Pro Val Tyr Asp Gly Pro Arg His
325 330 335
Phe Asp Tyr Lys Pro Met Arg Thr Glu Asp Ile Asp Gly Val Trp Ala
340 345 350
Ser Ala Ala Ala Asn Met Arg Thr Tyr Leu Leu Leu Lys Gln Arg Ala
355 360 365
Lys Ala Phe Arg Ala Asp Pro Glu Val Gln Ala Ala Leu Thr Ala Ser
370 375 380
Arg Val Pro Glu Leu Ala Val Pro Thr Leu Gly Glu Gly Glu Ser Tyr
385 390 395 400
Ala Asp Leu Leu Ala Asp Arg Ser Ala Trp Glu Glu Phe Asp Val Asp
405 410 415
Arg Ala Ala Asn Gln Gly Tyr Gly Tyr Ala Arg Leu Asp Gln Leu Ala
420 425 430
Ile Glu His Leu Leu Gly Ala Arg Gly Glu Ala Ala Ala Lys Glu Ala
435 440 445
Ala Ala Lys Lys His Gly Ile Tyr Tyr Ala Tyr Trp Glu Gln Glu Trp
450 455 460
Glu Ala Asp Tyr Lys Tyr Tyr Ile Glu Lys Val Ala Lys Leu Gly Phe
465 470 475 480
Asp Ile Leu Glu Ile Ala Ala Ser Pro Leu Pro Phe Tyr Ser Asp Ile
485 490 495
Gln Ile Asn Glu Leu Lys Ala Cys Ala His Gly Asn Gly Ile Thr Leu
500 505 510
Thr Val Gly His Gly Pro Ser Ala Glu Gln Asn Leu Ser Ser Pro Asp
515 520 525
Pro Asp Ile Arg Lys Asn Ala Lys Ala Phe Tyr Thr Asp Leu Leu Lys
530 535 540
Arg Leu Tyr Lys Leu Asp Val His Leu Ile Gly Gly Ala Leu Tyr Ser
545 550 555 560
Tyr Trp Pro Ile Asp Tyr Thr Lys Thr Ile Asp Lys Lys Gly Asp Trp
565 570 575
Glu Arg Ser Val Glu Ser Val Arg Glu Val Ala Lys Val Ala Glu Ala
580 585 590
Cys Gly Val Asp Phe Cys Leu Glu Val Leu Asn Arg Phe Glu Asn Tyr
595 600 605
Leu Ile Asn Thr Ala Gln Glu Gly Val Asp Phe Val Lys Gln Val Asp
610 615 620
His Asn Asn Val Lys Val Met Leu Asp Thr Phe His Met Asn Ile Glu
625 630 635 640
Glu Asp Ser Ile Gly Gly Ala Ile Arg Thr Ala Gly Ser Tyr Leu Gly
645 650 655
His Leu His Thr Gly Glu Cys Asn Arg Lys Val Pro Gly Arg Gly Arg
660 665 670
Ile Pro Trp Val Glu Ile Gly Glu Ala Leu Ala Asp Ile Gly Tyr Asn
675 680 685
Gly Ser Val Val Met Glu Pro Phe Val Arg Met Gly Gly Thr Val Gly
690 695 700
Ser Asn Ile Lys Val Trp Arg Asp Ile Ser Asn Gly Ala Asp Glu Lys
705 710 715 720
Met Leu Asp Arg Glu Ala Gln Ala Ala Leu Asp Phe Ser Arg Tyr Val
725 730 735
Leu Glu Cys His Lys His Ser
740
<210> 7
<211> 748
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 7
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Ser Leu Thr Thr Ala Ser Ser Lys Thr Ile Glu Val Ala
35 40 45
Thr Pro Ser Lys Glu Asp Arg Phe Ser Phe Gly Leu Trp Thr Val Gly
50 55 60
Trp Gln Ala Arg Asp Pro Phe Gly Glu Ala Thr Arg Pro Pro Leu Asp
65 70 75 80
Pro Val Glu Ala Val His Lys Leu Ala Glu Leu Gly Ala Tyr Gly Val
85 90 95
Thr Phe His Asp Asp Asp Leu Val Pro Phe Gly Ser Ser Asp Ala Glu
100 105 110
Arg Ala Arg Leu Ile Asp Arg Phe Lys Lys Ala Leu Ala Asp Thr Gly
115 120 125
Leu Val Val Pro Met Met Thr Thr Asn Leu Phe Thr His Pro Ile Phe
130 135 140
Lys Asp Gly Ala Phe Thr Ala Asn Asp Arg Ser Ile Arg Arg Tyr Ala
145 150 155 160
Ile Arg Lys Val Met Arg Asn Leu Asp Leu Ala Ala Glu Leu Gly Ala
165 170 175
Arg Thr Tyr Val Phe Trp Gly Gly Arg Glu Gly Ser Glu Ile Asp Ala
180 185 190
Ala Lys Asp Ile Arg Ala Ala Leu Asp Arg Tyr Arg Glu Ala Ile Asp
195 200 205
Thr Leu Ala Gln Tyr Val Lys Asp Gln Gly Tyr Gly Ile Arg Phe Ala
210 215 220
Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Phe Leu Pro Thr
225 230 235 240
Ile Gly His Ala Leu Ala Phe Ile Asn Ser Leu Glu His Ser Asp Ile
245 250 255
Val Gly Leu Asn Pro Glu Val Gly His Glu Gln Met Ser Asn Leu Asn
260 265 270
Phe Val His Gly Ile Ala Gln Ala Leu Trp His Gly Lys Leu Phe His
275 280 285
Ile Asp Leu Asn Gly Gln His Gly Pro Lys Tyr Asp Gln Asp Leu Val
290 295 300
Phe Gly His Gly Asp Leu Leu Ser Ala Phe Phe Leu Val Asp Leu Leu
305 310 315 320
Glu Asn Gly Phe Pro Gly Gly Gly Pro Val Tyr Asp Gly Pro Arg His
325 330 335
Phe Asp Tyr Lys Pro Met Arg Thr Glu Asp Ile Asp Gly Val Trp Ala
340 345 350
Ser Ala Ala Ala Asn Met Arg Thr Tyr Leu Leu Leu Lys Gln Arg Ala
355 360 365
Lys Ala Phe Arg Ala Asp Pro Glu Val Gln Ala Ala Leu Thr Ala Ser
370 375 380
Arg Val Pro Glu Leu Ala Val Pro Thr Leu Gly Glu Gly Glu Ser Tyr
385 390 395 400
Ala Asp Leu Leu Ala Asp Arg Ser Ala Trp Glu Glu Phe Asp Val Asp
405 410 415
Arg Ala Ala Asn Gln Gly Tyr Gly Tyr Ala Arg Leu Asp Gln Leu Ala
420 425 430
Ile Glu His Leu Leu Gly Ala Arg Gly Glu Ala Ala Ala Lys Glu Ala
435 440 445
Ala Ala Lys Glu Ala Ala Ala Lys Lys His Gly Ile Tyr Tyr Ala Tyr
450 455 460
Trp Glu Gln Glu Trp Glu Ala Asp Tyr Lys Tyr Tyr Ile Glu Lys Val
465 470 475 480
Ala Lys Leu Gly Phe Asp Ile Leu Glu Ile Ala Ala Ser Pro Leu Pro
485 490 495
Phe Tyr Ser Asp Ile Gln Ile Asn Glu Leu Lys Ala Cys Ala His Gly
500 505 510
Asn Gly Ile Thr Leu Thr Val Gly His Gly Pro Ser Ala Glu Gln Asn
515 520 525
Leu Ser Ser Pro Asp Pro Asp Ile Arg Lys Asn Ala Lys Ala Phe Tyr
530 535 540
Thr Asp Leu Leu Lys Arg Leu Tyr Lys Leu Asp Val His Leu Ile Gly
545 550 555 560
Gly Ala Leu Tyr Ser Tyr Trp Pro Ile Asp Tyr Thr Lys Thr Ile Asp
565 570 575
Lys Lys Gly Asp Trp Glu Arg Ser Val Glu Ser Val Arg Glu Val Ala
580 585 590
Lys Val Ala Glu Ala Cys Gly Val Asp Phe Cys Leu Glu Val Leu Asn
595 600 605
Arg Phe Glu Asn Tyr Leu Ile Asn Thr Ala Gln Glu Gly Val Asp Phe
610 615 620
Val Lys Gln Val Asp His Asn Asn Val Lys Val Met Leu Asp Thr Phe
625 630 635 640
His Met Asn Ile Glu Glu Asp Ser Ile Gly Gly Ala Ile Arg Thr Ala
645 650 655
Gly Ser Tyr Leu Gly His Leu His Thr Gly Glu Cys Asn Arg Lys Val
660 665 670
Pro Gly Arg Gly Arg Ile Pro Trp Val Glu Ile Gly Glu Ala Leu Ala
675 680 685
Asp Ile Gly Tyr Asn Gly Ser Val Val Met Glu Pro Phe Val Arg Met
690 695 700
Gly Gly Thr Val Gly Ser Asn Ile Lys Val Trp Arg Asp Ile Ser Asn
705 710 715 720
Gly Ala Asp Glu Lys Met Leu Asp Arg Glu Ala Gln Ala Ala Leu Asp
725 730 735
Phe Ser Arg Tyr Val Leu Glu Cys His Lys His Ser
740 745
<210> 8
<211> 2202
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 8
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtaaacacg gtatctacta cgcttactgg gaacaagaat gggaagctga ttacaaatac 1380
tacatcgaaa aagtagctaa attaggtttc gacatcttag aaatcgctgc ttctccactt 1440
ccattctact ctgatatcca aattaacgaa ttaaaagcat gcgctcacgg taacggtatt 1500
acacttactg ttggtcacgg cccatctgct gaacaaaact tatcttctcc tgacccagac 1560
attcgtaaaa acgctaaagc attctacact gatcttttaa aacgtttata caaattagat 1620
gtacacttaa ttggtggtgc tctttactct tactggccaa tcgactacac taaaactatc 1680
gataaaaaag gtgactggga acgttctgtt gaatcagtac gtgaagttgc taaagttgct 1740
gaggcttgtg gtgttgattt ctgtttagaa gtattaaacc gtttcgaaaa ctacttaatc 1800
aacactgctc aagaaggtgt tgatttcgtt aaacaagttg atcacaataa cgttaaagta 1860
atgttagata cattccatat gaacatcgaa gaagattcta tcggtggtgc aatccgtact 1920
gctggttctt acttaggtca ccttcacact ggtgaatgta accgtaaagt acctggtcgt 1980
ggtcgtatcc catgggttga gatcggtgaa gcattagctg acatcggtta caacggttct 2040
gtagtaatgg aaccattcgt acgtatgggt ggtactgttg gttctaacat taaagtttgg 2100
cgtgacatct ctaacggtgc tgatgaaaaa atgttagatc gtgaagctca agctgcatta 2160
gatttctctc gttacgttct tgaatgtcac aaacactctt aa 2202
<210> 9
<211> 2217
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 9
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtggtggag gcggaagtaa acacggtatc tactacgctt actgggaaca agaatgggaa 1380
gctgattaca aatactacat cgaaaaagta gctaaattag gtttcgacat cttagaaatc 1440
gctgcttctc cacttccatt ctactctgat atccaaatta acgaattaaa agcatgcgct 1500
cacggtaacg gtattacact tactgttggt cacggcccat ctgctgaaca aaacttatct 1560
tctcctgacc cagacattcg taaaaacgct aaagcattct acactgatct tttaaaacgt 1620
ttatacaaat tagatgtaca cttaattggt ggtgctcttt actcttactg gccaatcgac 1680
tacactaaaa ctatcgataa aaaaggtgac tgggaacgtt ctgttgaatc agtacgtgaa 1740
gttgctaaag ttgctgaggc ttgtggtgtt gatttctgtt tagaagtatt aaaccgtttc 1800
gaaaactact taatcaacac tgctcaagaa ggtgttgatt tcgttaaaca agttgatcac 1860
aataacgtta aagtaatgtt agatacattc catatgaaca tcgaagaaga ttctatcggt 1920
ggtgcaatcc gtactgctgg ttcttactta ggtcaccttc acactggtga atgtaaccgt 1980
aaagtacctg gtcgtggtcg tatcccatgg gttgagatcg gtgaagcatt agctgacatc 2040
ggttacaacg gttctgtagt aatggaacca ttcgtacgta tgggtggtac tgttggttct 2100
aacattaaag tttggcgtga catctctaac ggtgctgatg aaaaaatgtt agatcgtgaa 2160
gctcaagctg cattagattt ctctcgttac gttcttgaat gtcacaaaca ctcttaa 2217
<210> 10
<211> 2232
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 10
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtggtggag gcggaagtgg cggtggtggc agcaaacacg gtatctacta cgcttactgg 1380
gaacaagaat gggaagctga ttacaaatac tacatcgaaa aagtagctaa attaggtttc 1440
gacatcttag aaatcgctgc ttctccactt ccattctact ctgatatcca aattaacgaa 1500
ttaaaagcat gcgctcacgg taacggtatt acacttactg ttggtcacgg cccatctgct 1560
gaacaaaact tatcttctcc tgacccagac attcgtaaaa acgctaaagc attctacact 1620
gatcttttaa aacgtttata caaattagat gtacacttaa ttggtggtgc tctttactct 1680
tactggccaa tcgactacac taaaactatc gataaaaaag gtgactggga acgttctgtt 1740
gaatcagtac gtgaagttgc taaagttgct gaggcttgtg gtgttgattt ctgtttagaa 1800
gtattaaacc gtttcgaaaa ctacttaatc aacactgctc aagaaggtgt tgatttcgtt 1860
aaacaagttg atcacaataa cgttaaagta atgttagata cattccatat gaacatcgaa 1920
gaagattcta tcggtggtgc aatccgtact gctggttctt acttaggtca ccttcacact 1980
ggtgaatgta accgtaaagt acctggtcgt ggtcgtatcc catgggttga gatcggtgaa 2040
gcattagctg acatcggtta caacggttct gtagtaatgg aaccattcgt acgtatgggt 2100
ggtactgttg gttctaacat taaagtttgg cgtgacatct ctaacggtgc tgatgaaaaa 2160
atgttagatc gtgaagctca agctgcatta gatttctctc gttacgttct tgaatgtcac 2220
aaacactctt aa 2232
<210> 11
<211> 2247
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 11
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtggggggg gaggttcagg tggaggcgga agtggcggtg gtggcagcaa acacggtatc 1380
tactacgctt actgggaaca agaatgggaa gctgattaca aatactacat cgaaaaagta 1440
gctaaattag gtttcgacat cttagaaatc gctgcttctc cacttccatt ctactctgat 1500
atccaaatta acgaattaaa agcatgcgct cacggtaacg gtattacact tactgttggt 1560
cacggcccat ctgctgaaca aaacttatct tctcctgacc cagacattcg taaaaacgct 1620
aaagcattct acactgatct tttaaaacgt ttatacaaat tagatgtaca cttaattggt 1680
ggtgctcttt actcttactg gccaatcgac tacactaaaa ctatcgataa aaaaggtgac 1740
tgggaacgtt ctgttgaatc agtacgtgaa gttgctaaag ttgctgaggc ttgtggtgtt 1800
gatttctgtt tagaagtatt aaaccgtttc gaaaactact taatcaacac tgctcaagaa 1860
ggtgttgatt tcgttaaaca agttgatcac aataacgtta aagtaatgtt agatacattc 1920
catatgaaca tcgaagaaga ttctatcggt ggtgcaatcc gtactgctgg ttcttactta 1980
ggtcaccttc acactggtga atgtaaccgt aaagtacctg gtcgtggtcg tatcccatgg 2040
gttgagatcg gtgaagcatt agctgacatc ggttacaacg gttctgtagt aatggaacca 2100
ttcgtacgta tgggtggtac tgttggttct aacattaaag tttggcgtga catctctaac 2160
ggtgctgatg aaaaaatgtt agatcgtgaa gctcaagctg cattagattt ctctcgttac 2220
gttcttgaat gtcacaaaca ctcttaa 2247
<210> 12
<211> 2217
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 12
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtgaagcag ctgccaagaa acacggtatc tactacgctt actgggaaca agaatgggaa 1380
gctgattaca aatactacat cgaaaaagta gctaaattag gtttcgacat cttagaaatc 1440
gctgcttctc cacttccatt ctactctgat atccaaatta acgaattaaa agcatgcgct 1500
cacggtaacg gtattacact tactgttggt cacggcccat ctgctgaaca aaacttatct 1560
tctcctgacc cagacattcg taaaaacgct aaagcattct acactgatct tttaaaacgt 1620
ttatacaaat tagatgtaca cttaattggt ggtgctcttt actcttactg gccaatcgac 1680
tacactaaaa ctatcgataa aaaaggtgac tgggaacgtt ctgttgaatc agtacgtgaa 1740
gttgctaaag ttgctgaggc ttgtggtgtt gatttctgtt tagaagtatt aaaccgtttc 1800
gaaaactact taatcaacac tgctcaagaa ggtgttgatt tcgttaaaca agttgatcac 1860
aataacgtta aagtaatgtt agatacattc catatgaaca tcgaagaaga ttctatcggt 1920
ggtgcaatcc gtactgctgg ttcttactta ggtcaccttc acactggtga atgtaaccgt 1980
aaagtacctg gtcgtggtcg tatcccatgg gttgagatcg gtgaagcatt agctgacatc 2040
ggttacaacg gttctgtagt aatggaacca ttcgtacgta tgggtggtac tgttggttct 2100
aacattaaag tttggcgtga catctctaac ggtgctgatg aaaaaatgtt agatcgtgaa 2160
gctcaagctg cattagattt ctctcgttac gttcttgaat gtcacaaaca ctcttaa 2217
<210> 13
<211> 2232
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 13
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtgaagcag ctgccaagga ggcagctgcg aagaaacacg gtatctacta cgcttactgg 1380
gaacaagaat gggaagctga ttacaaatac tacatcgaaa aagtagctaa attaggtttc 1440
gacatcttag aaatcgctgc ttctccactt ccattctact ctgatatcca aattaacgaa 1500
ttaaaagcat gcgctcacgg taacggtatt acacttactg ttggtcacgg cccatctgct 1560
gaacaaaact tatcttctcc tgacccagac attcgtaaaa acgctaaagc attctacact 1620
gatcttttaa aacgtttata caaattagat gtacacttaa ttggtggtgc tctttactct 1680
tactggccaa tcgactacac taaaactatc gataaaaaag gtgactggga acgttctgtt 1740
gaatcagtac gtgaagttgc taaagttgct gaggcttgtg gtgttgattt ctgtttagaa 1800
gtattaaacc gtttcgaaaa ctacttaatc aacactgctc aagaaggtgt tgatttcgtt 1860
aaacaagttg atcacaataa cgttaaagta atgttagata cattccatat gaacatcgaa 1920
gaagattcta tcggtggtgc aatccgtact gctggttctt acttaggtca ccttcacact 1980
ggtgaatgta accgtaaagt acctggtcgt ggtcgtatcc catgggttga gatcggtgaa 2040
gcattagctg acatcggtta caacggttct gtagtaatgg aaccattcgt acgtatgggt 2100
ggtactgttg gttctaacat taaagtttgg cgtgacatct ctaacggtgc tgatgaaaaa 2160
atgttagatc gtgaagctca agctgcatta gatttctctc gttacgttct tgaatgtcac 2220
aaacactctt aa 2232
<210> 14
<211> 2247
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 14
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggtgaggcag ctgcgaaaga agcagctgcc aaggaggcag ctgcgaagaa acacggtatc 1380
tactacgctt actgggaaca agaatgggaa gctgattaca aatactacat cgaaaaagta 1440
gctaaattag gtttcgacat cttagaaatc gctgcttctc cacttccatt ctactctgat 1500
atccaaatta acgaattaaa agcatgcgct cacggtaacg gtattacact tactgttggt 1560
cacggcccat ctgctgaaca aaacttatct tctcctgacc cagacattcg taaaaacgct 1620
aaagcattct acactgatct tttaaaacgt ttatacaaat tagatgtaca cttaattggt 1680
ggtgctcttt actcttactg gccaatcgac tacactaaaa ctatcgataa aaaaggtgac 1740
tgggaacgtt ctgttgaatc agtacgtgaa gttgctaaag ttgctgaggc ttgtggtgtt 1800
gatttctgtt tagaagtatt aaaccgtttc gaaaactact taatcaacac tgctcaagaa 1860
ggtgttgatt tcgttaaaca agttgatcac aataacgtta aagtaatgtt agatacattc 1920
catatgaaca tcgaagaaga ttctatcggt ggtgcaatcc gtactgctgg ttcttactta 1980
ggtcaccttc acactggtga atgtaaccgt aaagtacctg gtcgtggtcg tatcccatgg 2040
gttgagatcg gtgaagcatt agctgacatc ggttacaacg gttctgtagt aatggaacca 2100
ttcgtacgta tgggtggtac tgttggttct aacattaaag tttggcgtga catctctaac 2160
ggtgctgatg aaaaaatgtt agatcgtgaa gctcaagctg cattagattt ctctcgttac 2220
gttcttgaat gtcacaaaca ctcttaa 2247
<210> 15
<211> 1326
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 15
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgtctct tactactgct 120
tcttctaaaa ctatcgaagt tgcaactcca tcaaaagaag atcgtttctc tttcggtctt 180
tggactgttg gttggcaagc tcgtgaccca ttcggtgaag ctactcgccc accacttgac 240
ccagttgaag ctgtacacaa acttgctgaa ttaggtgcat acggtgttac tttccacgat 300
gatgaccttg ttccattcgg ttcttctgat gctgaacgcg ctcgcttaat cgatcgtttc 360
aaaaaagcac tagctgatac tggtttagtt gtaccaatga tgacaacaaa cttattcact 420
catcctatct tcaaagatgg tgctttcact gctaacgatc gttctatccg tcgttatgct 480
atccgcaaag taatgcgtaa cttagattta gcggctgaat taggtgctcg tacatacgta 540
ttctggggtg gtcgtgaagg ttctgaaatc gatgctgcaa aagacatccg tgctgcttta 600
gatcgttacc gtgaagctat tgacactctt gctcaatacg ttaaagatca aggttacggt 660
attcgtttcg cattagaacc aaaaccaaac gaaccacgcg gtgacatctt cttaccaact 720
atcggtcacg ctttagcttt catcaactct ttagaacact ctgatattgt tggtcttaac 780
cctgaagtag gtcacgaaca aatgtctaac ttaaacttcg tgcacggtat cgctcaagct 840
ctttggcacg gtaaattatt ccacattgat cttaacggtc aacacggtcc taaatacgac 900
caagacttag ttttcggtca cggtgattta ttatctgctt tcttccttgt agacttactt 960
gaaaacggtt tcccaggtgg aggaccagta tacgatggac ctcgtcactt cgattacaaa 1020
cctatgcgta ctgaagatat tgatggtgtt tgggcatcag cagcagctaa catgcgtact 1080
tacttacttt taaaacaacg tgcaaaagca ttccgtgctg atcctgaagt tcaagcagct 1140
ttaacagctt cacgcgttcc tgaattagct gttccaacat taggtgaagg tgaatcttac 1200
gctgatttat tagctgatcg ttcagcttgg gaagaatttg atgttgatcg tgctgctaac 1260
caaggttacg gttacgctcg tcttgatcaa ttagctatcg aacacttatt aggtgctcgt 1320
ggttaa 1326
<210> 16
<211> 984
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 16
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaagca tgactggtgg acagcaaatg ggtcgcggat ccatgaaaca cggtatctac 120
tacgcttact gggaacaaga atgggaagct gattacaaat actacatcga aaaagtagct 180
aaattaggtt tcgacatctt agaaatcgct gcttctccac ttccattcta ctctgatatc 240
caaattaacg aattaaaagc atgcgctcac ggtaacggta ttacacttac tgttggtcac 300
ggcccatctg ctgaacaaaa cttatcttct cctgacccag acattcgtaa aaacgctaaa 360
gcattctaca ctgatctttt aaaacgttta tacaaattag atgtacactt aattggtggt 420
gctctttact cttactggcc aatcgactac actaaaacta tcgataaaaa aggtgactgg 480
gaacgttctg ttgaatcagt acgtgaagtt gctaaagttg ctgaggcttg tggtgttgat 540
ttctgtttag aagtattaaa ccgtttcgaa aactacttaa tcaacactgc tcaagaaggt 600
gttgatttcg ttaaacaagt tgatcacaat aacgttaaag taatgttaga tacattccat 660
atgaacatcg aagaagattc tatcggtggt gcaatccgta ctgctggttc ttacttaggt 720
caccttcaca ctggtgaatg taaccgtaaa gtacctggtc gtggtcgtat cccatgggtt 780
gagatcggtg aagcattagc tgacatcggt tacaacggtt ctgtagtaat ggaaccattc 840
gtacgtatgg gtggtactgt tggttctaac attaaagttt ggcgtgacat ctctaacggt 900
gctgatgaaa aaatgttaga tcgtgaagct caagctgcat tagatttctc tcgttacgtt 960
cttgaatgtc acaaacactc ttaa 984

Claims (8)

1. A fusion protein for catalyzing glucose to synthesize D-psicose and a construction thereof are characterized in that glucose is used as a substrate, and the fusion protein (GP, SEQ ID NO:1, GS1P, SEQ ID NO:2, GS2P, SEQ ID NO:3, GS3P, SEQ ID NO:4, GE1P, SEQ ID NO:5, GE2P, SEQ ID NO:6, GE3P and SEQ ID NO:7) is used for directly converting the glucose into the D-psicose (the reaction process is shown in figure 1).
2. The fusion protein according to claim 1, wherein the fusion protein-encoding gene (GP, SEQ ID NO:8, GS1P, SEQ ID NO:9, GS2P, SEQ ID NO:10, GS3P, SEQ ID NO:11, GE1P, SEQ ID NO:12, GE2P, SEQ ID NO:13 and GE3P, SEQ ID NO:14) is a fusion of a glucose isomerase-encoding gene (AcceGI, SEQ ID NO:15), a D-psicose 3-epimerase-encoding gene (CcDPEase, SEQ ID NO:16) and a linker peptide-encoding gene.
3. The method of claim 1 and claim 2, wherein S is flexible linker peptide (GGGGS) and E is rigid alpha helical linker peptide (EAAAK), and wherein the number indicates the number of tandem copies of the linker peptide used, e.g., GS2P indicates that a glucose isomerase-encoding gene and a D-psicose 3-epimerase-encoding gene are fused by linking the two copies of the flexible linker peptide. The glucose isomerase coding gene is derived from Acidothermus cellulolyticus 11B. The coding gene of the D-psicose 3-epimerase is derived from Clostridium cellulolyticum H10.
4. The method for synthesizing D-psicose according to claim 1, wherein plasmid pET28a (PB) N (SEQ ID NO:17) is used as the expression vector of the transformation pathway, and recombinant plasmids pET28a (PB) N-GP, pET28a (PB) N-GS1P, pET28a (PB) N-GS2P, pET28a (PB) N-GS3P, pET28a (PB) N-GE1P, pET28a (PB) N-GE2P and pET28a (PB) N-GE3P containing all pathway genes are constructed (the plasmid structure is shown in FIG. 3).
5. According to the claim 1, the claim 2, the claim 3 and the claim 4, the Escherichia coli BL21(DE3) is used as an expression host, the recombinant plasmid is transformed into Escherichia coli BL21(DE3), and the engineering strains BL21/GP, BL21/GS1P, BL21/GS2P, BL21/GS3P, BL21/GE1P, BL21/GE2P and BL21/GE3P containing all fusion genes are obtained.
6. The method for synthesizing D-psicose using fusion protein with glucose as substrate according to claim 1 and the recombinant engineered strain of claim 5, wherein the transformation conditions used are as follows: (1) in LB culture medium, culturing Escherichia coli recombinant engineering bacteria to OD6000.6-0.8, isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.5mM, and gene expression was induced at 25 ℃ and 200rpm for 8 h; (2) centrifuging at 4 deg.C and 8000rpm to collect thallus, removing culture medium with 20mM Phosphate Buffer Solution (PBS) with pH of 4.5-8.5, and resuspending thallus with phosphate buffer solution with corresponding pH to obtain resting cell; (3) glucose was added as a substrate to resting cells at a final concentration of 100g/L and 1mM CoCl2And 5mM MgCl2Reacting for 2 hours at 45-85 ℃ as catalytic ions; (4) the yield of D-psicose was analyzed using ion chromatography.
7. According to claim 1 and claim 6, the optimal temperature for catalyzing the direct conversion of glucose into D-psicose by using the fusion protein in the present invention is: at 65 ℃, the optimal pH is: 7.5.
8. according to claim 1 and claim 7, the method for directly producing D-psicose from glucose by using fusion protein catalysis has the advantage that the yield of D-psicose is not less than 5.69g/L under the optimal temperature and pH conditions.
CN202210352448.4A 2022-04-04 2022-04-04 Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof Pending CN114591940A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023242877A1 (en) * 2022-06-16 2023-12-21 Fertis India Pvt Ltd Enzymatic synthesis of d-allulose and its derivatives from fusion enzymes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103710329A (en) * 2013-12-24 2014-04-09 山西天骄食业有限公司 Method for preparation of co-expressed recombinase with genetic engineering technology
CN107723307A (en) * 2017-10-09 2018-02-23 中国科学院天津工业生物技术研究所 A kind of method and its application for efficiently preparing the epimerase of D psicoses 3
CN113801240A (en) * 2021-08-26 2021-12-17 华南理工大学 D-psicose-3-epimerase activity aggregate and preparation method and application thereof
CN113980880A (en) * 2021-09-24 2022-01-28 吉林中粮生化有限公司 Genetically engineered bacterium, application thereof and method for producing psicose by taking glucose as raw material

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103710329A (en) * 2013-12-24 2014-04-09 山西天骄食业有限公司 Method for preparation of co-expressed recombinase with genetic engineering technology
CN107723307A (en) * 2017-10-09 2018-02-23 中国科学院天津工业生物技术研究所 A kind of method and its application for efficiently preparing the epimerase of D psicoses 3
CN113801240A (en) * 2021-08-26 2021-12-17 华南理工大学 D-psicose-3-epimerase activity aggregate and preparation method and application thereof
CN113980880A (en) * 2021-09-24 2022-01-28 吉林中粮生化有限公司 Genetically engineered bacterium, application thereof and method for producing psicose by taking glucose as raw material

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
WANMENG MU ET AL.: ""Characterization of a thermostable glucose isomerase with an acidic pH optimum from Acidothermus cellulolyticus"", 《FOOD RESEARCH INTERNATIONAL》, vol. 47, pages 364 - 367, XP028926041, DOI: 10.1016/j.foodres.2011.09.006 *
WANMENG MU ET AL.: ""Cloning, expression, and characterization of a D-Psicose 3-Epimerase from Clostridium cellulolyticum H10"", 《J. AGRIC. FOOD CHEM. 》, vol. 59, pages 7785 - 7792 *
YAN MEN ET AL.: ""Co-expression of d-glucose isomerase and d-psicose 3-epimerase: Development of an efficient one-step production of d-psicose"", 《ENZYME AND MICROBIAL TECHNOLOGY》, vol. 64, pages 212 - 127 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023242877A1 (en) * 2022-06-16 2023-12-21 Fertis India Pvt Ltd Enzymatic synthesis of d-allulose and its derivatives from fusion enzymes

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