CN113583983A - Fusion protein or variant thereof and application thereof in preparation of calcifediol - Google Patents

Fusion protein or variant thereof and application thereof in preparation of calcifediol Download PDF

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CN113583983A
CN113583983A CN202010369514.XA CN202010369514A CN113583983A CN 113583983 A CN113583983 A CN 113583983A CN 202010369514 A CN202010369514 A CN 202010369514A CN 113583983 A CN113583983 A CN 113583983A
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吴燕
田振华
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Ecolab Biotechnology Shanghai Co Ltd
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Abstract

The invention provides a fusion protein or a variant thereof, which comprises K1 and RhFR, wherein the amino acid sequence of K1 is shown as SEQ ID NO. 1, and the amino acid sequence of RhFR is shown as amino acid 466-773 of SEQ ID NO. 5. The invention also provides a preparation method and application of the fusion protein or the variant thereof. When the fusion protein or the variant thereof is applied to catalyzing VD3 to synthesize 25-hydroxy vitamin D3 (calcifediol), no additional electron transfer related protein is needed to be added, the operation is simple and convenient, the electron transfer efficiency and the catalysis efficiency in the fusion protein are high, the yield of the obtained calcifediol is obviously improved, the production cost is reduced, and the method is suitable for industrial production.

Description

Fusion protein or variant thereof and application thereof in preparation of calcifediol
Technical Field
The invention relates to the technical field of biology, in particular to a fusion protein or a variant thereof, a gene thereof, a preparation method thereof and application of the fusion protein or the variant thereof in catalyzing VD3 to prepare calcifediol.
Background
25-hydroxy vitamin D3, also known as calcifediol, is an active metabolite of vitamin D3(VD3) and has strong physiological activity. The traditional method for synthesizing 25-hydroxyvitamin D3 is a chemical synthesis method, which requires multiple steps of group protection and deprotection, followed by light reaction, ring opening and isomerization to 25-hydroxyvitamin D3. In recent years, research on microbial transformation of VD3 is rapidly developed, but the transformation rate is low, and one of the keys for improving the yield of active VD3 is to screen and obtain a key metabolic enzyme cytochrome P450 enzyme with high transformation efficiency.
Cytochrome P450 enzyme widely exists in prokaryotes and eukaryotes, the single oxygenation reaction mediated by the cytochrome P450 enzyme usually needs the participation of electron transfer chain ferredoxin and ferredoxin reductase, and an efficient electron transfer system can effectively improve the reaction efficiency of P450. There are also reports of self-consistent type P450, and no protein related to electron transfer needs to be added in the reaction process. At present, 2 self-consistent types of P450, namely P450BM3 derived from Bacillus megaterium and P450 RhFREd derived from Rhodococcus rhodochrous, have been reported, and the protein structure of the self-consistent types of P450 comprises a P450 domain and an electron transfer domain, and no protein related to electron transfer needs to be added in the reaction process. However, no report is available on the use of self-consistent P450 for catalyzing VD3 to produce calcifediol.
Tamura et al (Biochemical and Biophysical Research Communications 2009,385, 170-175) engineered P450 enzymes from Pseudomonas autotropia to give 4 site-mutated mutants of Vdh-K1(T70R, V156L, E216M and E384R) and determined that Vdh-K1 is 21.6 times the activity of wild-type Vdhwt when used for the catalysis of VD 3. However, the catalysis for VD3 needs the participation of an electron transfer system, is complex to operate and high in cost, and still has low catalytic efficiency. Tamura et al (ChemBiochem 2013,14,2284-2291) modified P450 enzyme from Pseudomonas autotropica (mutation such as T107A) and heterologously expressed in Rhodococcus, by adding nisin to improve the permeability of cell membrane and reduce the mass transfer resistance of VD3 as substrate, finally catalyzing VD3 to obtain the ossification diol with the yield of 573 mug/mL for two hours, wherein the electron transfer systems AciFdx and AciFdxR are adopted in the method, but the yield is still to be improved.
Therefore, a method which has high catalytic efficiency, high yield of the obtained product, simple operation and low cost when used for catalyzing VD3 to prepare the calcifediol is urgently needed in the prior art.
Disclosure of Invention
The invention aims to solve the technical problems of low yield, high cost, complicated operation and the like in the preparation of the calcifediol by catalyzing VD3 with cytochrome P450 enzyme in the prior art, and provides a fusion protein or a variant thereof, a gene thereof, a preparation method thereof and application of the fusion protein or the variant thereof in catalyzing VD3 to prepare the calcifediol. When the fusion protein or the variant thereof is applied to catalyzing VD3 to synthesize 25-hydroxy vitamin D3 (calcifediol), no additional electron transfer related protein is needed to be added, the operation is simple and convenient, the electron transfer efficiency and the catalysis efficiency in the fusion protein are high, the yield of the obtained calcifediol is obviously improved, the production cost is reduced, and the method is suitable for industrial production.
The invention provides a plurality of methods for improving the yield of VD3 catalyzed by P450 enzyme, including searching for high-efficiency P450 enzyme, optimizing reaction conditions and the like, and through a great deal of research, the inventors unexpectedly find that when a specific type of P450 is modified into self-consistent type P450 enzyme in a specific mode, the catalytic efficiency of catalyzing VD3 to prepare the calcifediol can be obviously improved, and the yield of the calcifediol can be obviously improved.
In order to solve the above technical problems, the present invention provides a fusion protein or a variant thereof, which comprises K1 and RhFR, wherein the amino acid sequence of K1 is shown as SEQ ID NO. 1, and the amino acid sequence of RhFR is shown as amino acid 466-773 of SEQ ID NO. 5.
Preferably, the fusion protein is K1 and RhFR in sequence from N-terminus to C-terminus.
Preferably, the K1 and RhFR are connected by a linker (linker), and the amino acid sequence of the linker is preferably shown as the amino acids 445-465 of SEQ ID NO. 5.
Preferably, the fusion protein or variant thereof is co-expressed with a chaperone, preferably Gro 7. In a preferred embodiment of the invention, a chaperone such as Gro7 chaperone may be added to express the fusion protein, allowing the fusion protein to be co-expressed with the chaperone. In the present invention, the Gro7 chaperone protein may be of commercial origin, for example Gro7 available from Biovector Science Lab, Inc.
Preferably, the variant is an insertion or deletion of an amino acid at the N-terminus of the RhFR; more preferably, the variant is an insertion or deletion of 1-14 amino acids at the N-terminus of the RhFR; even more preferably, the variant is an insertion or deletion of 3-6 amino acids at the N-terminus of the RhFR.
Preferably, the amino acid sequence of the fusion protein or the variant thereof is shown as SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17 or SEQ ID NO 19.
More preferably, the nucleotide sequence encoding the fusion protein or the variant thereof is shown as SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18 or SEQ ID NO 20.
In order to solve the above technical problem, the second aspect of the present invention provides a fusion gene encoding the fusion protein according to the first aspect of the present invention or a variant thereof.
In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector containing the fusion gene according to the second aspect of the present invention.
Preferably, the backbone vector of the recombinant expression vector is pET28 a.
In order to solve the above technical problems, the fourth aspect of the present invention provides a transformant comprising the fusion gene according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention.
Preferably, the transformant is obtained by introducing the fusion gene according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention into a host.
More preferably, the host is escherichia coli, preferably escherichia coli e.coli BL21(DE3) cells.
In order to solve the above technical problems, a fifth aspect of the present invention provides a method for preparing a fusion protein or a variant thereof, comprising the steps of:
(1) obtaining a transformant according to the fourth aspect of the present invention;
(2) screening said transformants, expressing and purifying said fusion protein or variant thereof.
In order to solve the above technical problems, a sixth aspect of the present invention provides a method for producing a calcifediol, the method comprising the steps of: the fusion protein or the variant thereof according to the first aspect of the present invention catalyzes the hydroxylation reaction of vitamin D3 in the presence of a reaction solvent and reduced coenzyme NADH/NADPH.
Preferably, the vitamin D3 is cosolvent pre-dissolved vitamin D3; the co-solvent preferably comprises one or more of DMSO, tween 80, Triton X100, methanol, ethanol, isopropanol and DMF, for example ethanol.
Preferably, the method further comprises the step of adding cyclodextrin, which may be, for example, hydroxypropyl- β -cyclodextrin, to the reaction solvent before the hydroxylation reaction is performed; the hydroxypropyl-beta-cyclodextrin accounts for 0.05-0.4% of the reaction system by mass volume, and is 0.25% for example.
Preferably, the reaction temperature is 20-33 ℃, for example, 22 ℃, 25 ℃, 28 ℃ or 30 ℃. The inventors found during their experiments that the yield of the product obtained is reduced when the temperature is not in this range.
Preferably, the pH of the reaction is 6.0 to 8.0, for example 7.4. The inventors have found during the course of experiments that the yield of the product obtained is reduced when the pH is outside the range specified in the present invention.
Preferably, the concentration of vitamin D3 is 1g/L to 10g/L, such as 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10 g/L. In the experimental process of the inventor, the VD3 is too high in concentration, cannot be dissolved or possibly inhibits enzyme activity by a substrate, the reaction is incomplete, the concentration is too low, and the yield is also low.
Preferably, the molar ratio of said NADH/NADPH to said vitamin D3 is 0.001:1 to 2:1, such as 0.2: 1.
Preferably, the preparation method further comprises the following steps: in the presence of dehydrogenase and a hydrogen donor, NAD (oxidized coenzyme)+/NADP+Carrying out reduction reaction to obtain the reduced coenzyme NADH/NADPH;
more preferably, the dehydrogenase is glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase; and/or the hydrogen donor is glucose, isopropanol or formate;
more preferably, when the dehydrogenase is alcohol dehydrogenase, the hydrogen donor is isopropanol; when the dehydrogenase is glucose dehydrogenase, the hydrogen donor is glucose; when the dehydrogenase is formate dehydrogenase, the hydrogen donor is formate.
In order to solve the above technical problems, the seventh aspect of the present invention provides a fusion protein or a variant thereof according to the first aspect of the present invention, a fusion gene according to the second aspect of the present invention, a recombinant expression vector according to the third aspect of the present invention, or a transformant according to the fourth aspect of the present invention for use in the preparation of calcifediol.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: when the fusion protein or the variant thereof is applied to catalyzing VD3 to synthesize 25-hydroxy vitamin D3 (calcifediol), no additional electron transfer related protein is needed to be added, the operation is simple and convenient, the electron transfer efficiency and the catalysis efficiency in the fusion protein are high, the yield of the obtained calcifediol is obviously improved, the production cost is reduced, and the method is suitable for industrial production. In a preferred embodiment of the invention, the fusion protein or the variant thereof catalyzes VD3, and the yield of the obtained ossifying glycol is up to 4.427 g/L.
Drawings
FIG. 1 is a detection map of K1-RhFR-I6-Gro7 in example 6.
FIG. 2 is a graph of the VD3 substrate control.
FIG. 3 is a graph of the results of a ossifying glycol control.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
HPLC analysis method of product
Chromatographic conditions are as follows: a chromatographic column: poroshell EC-C18(4.0 μm, 4.6X 150 mm); detection wavelength: 265 nm; flow rate: 1 mL/min; column temperature: 35 ℃; sample introduction volume: 10 μ L. The gradient elution procedure was as follows: 0-8min, H2O: acetonitrile 85%: 15 percent; 8-20min, H2O: acetonitrile 0: 100 percent; 20-21min, H2O: acetonitrile 85%: 15 percent; 21-27min, H2O: acetonitrile 85%: 15 percent.
pET28a was purchased from Novagen; DpnI, NdeI, HindIII enzymes were purchased from Thermo Fisher; exnase II enzyme purchased from Nanjing Novozam Biotech Ltd; coli BL21(DE3) competent cells were purchased from Changsheng biotechnology, LLC of Beijing ancient cooking; NAD + was purchased from Shenzhen Bangtai bioengineering, Inc.; vitamin D3 and the plasmid extraction kit were purchased from Biotechnology engineering (Shanghai) Ltd.
EXAMPLE 1 construction of the fusion protease Strain
1.1 protein sequence analysis
In this example, a P450 enzyme Vdh-K1 capable of catalyzing VD3 is fused with the reduction regions of P450BM3 and P450 RhFRed to construct a self-consistent P450 fusion protein, and the specific steps are as follows:
each gene was synthesized based on the gene sequences SEQ ID NO:2, 4, 6 of cytochrome P450 enzymes SEQ ID NO:1, 3, 5 reported on NCBI. The company for synthesizing genes is Suzhou Jinweizhi Biotechnology GmbH (Suzhou Industrial park Star lake street 218 Bionanotechnology park, C3). The information of each gene is shown in Table 1.
TABLE 1
Figure BDA0002477627290000051
Analyzing the amino acid sequence of the BM3 protein by using Discovery studio software, and finding that the amino acids at the 1 st to 459 th positions of the N end are the P450 structural domain of the protein; linker with amino acids 460 and 479 as P450 domain and electron transfer domain; the 480 rd and 1048 th amino acids are the electron transfer structural domain of the protein.
Analyzing the amino acid sequence of the RhFRed protein by using Discovery studio software, and finding that the 24 th to 444 th amino acids of the N end are the P450 structural domain of the protein; the 445-465 th amino acid is a Linker of the P450 structural domain and the electron transfer structural domain; the 466-773 amino acid is the electron transfer structure domain of the protein.
1.2 construction of engineered strains of K1, FdR, Fdx proteins and engineered strains of fusion proteins
The K1, BM3, RhFRed and synthesized electron transport system AciFdR, AciFdX genes (shown in Table 2 below) synthesized in example 1.1 were linked to pET28a and restriction sites NdeI & HindIII, respectively, and the enzymatically linked vector was transformed into host E.coli BL21(DE3) competent cells to obtain engineered strains containing K1, BM3, RhFRed, AciFdR, AciFdX, respectively. Plasmids were extracted using the plasmid extraction kit of Shanghai, to obtain plasmids pET28a-K1, pET28a-BM3, pET28a-RhFRed, pET28a-FdR, and pET28a-Fdx, respectively.
And (3) co-transforming the obtained pET28a-K1 and pET28a-FdR plasmids and Gro7 companion plasmids into E.coli BL21 respectively to obtain BL21-pET28a-K1-Gro7 and BL21-pET28a-FdR-Gro7 engineering bacteria. BL21 is transformed by pET28a-Fdx plasmid to obtain BL21-pET28a-Fdx engineering bacteria.
TABLE 2
Enzyme numbering GeneSource NCBI accession number
AciFdx Acinetobacter sp.OC4 BAE78451.1
AciFdR Acinetobacter sp.OC4 BAE78453.1
The target fragment delta K1-1 is amplified by taking a plasmid pET28a-K1 as a template and K1-Rh-F1 and K1-Rh-R1 as primers. And (3) amplifying a vector fragment RhFR of which the 5 'end and the 3' end of delta K1-1 have 15bP homologous arms and the 3 'end and the 5' end of delta K1-1 have 15bP homologous arms by using a pET28a-RhFRed plasmid as a template and RhFR-F1 and RhFR-R1 as primers. The PCR product was digested with Dpn1 at 37 ℃ for 2 hours. After the reaction is completed, the delta K1-1 and the RhFR are recombined by recombinase Exnase II at 37 ℃ for 0.5 hour, the recombined product is transformed into BL21 competent cells, the cells are spread on LB culture medium containing 50 mu g/mL kanamycin and cultured overnight at 37 ℃ to obtain BL21-pET28a-K1-RhFR transformant, namely BL21-pET28a-K1-RhFR engineering bacteria. BL21-pET28a-K1-RhFR transformant is selected and inoculated into 5mL LB liquid culture medium containing 50 mu g/mL kanamycin, shaking culture is carried out for 6h at 37 ℃, pET28a-K1-RhFR plasmid is extracted, pET28a-K1-RhFR plasmid and Gro7 mate plasmid are co-transformed into BL21, and BL21-pET28a-K1-RhFR-Gro7 engineering bacteria are obtained. The primers and their sequences are shown in Table 2.
The target fragment delta K1-2 is amplified by taking a protoplasmid pET28a-K1 as a template and K1-BM3-F1 and K1-BM3-R1 as primers. And (3) amplifying a vector fragment BM3R of which the 5 'end and the 3' end of delta K1-2 have 15bP homology arms and the 3 'end and the 5' end of K1-2 have 15bP homology arms by using a pET28a-BM3 plasmid as a template and BM3R-F1 and BM3R-R1 as primers. The PCR product was digested with Dpn1 at 37 ℃ for 2 hours. After the reaction is completed, the delta K1-2 and BM3R are recombined by recombinase Exnase II at 37 ℃ for 0.5 hour, and the recombined products are transformed into BL21 competent cells, spread on LB culture medium containing 50 mu g/mL kanamycin and cultured overnight at 37 ℃ to obtain BL21-pET28a-K1-BM3R transformants, namely BL21-pET28a-K1-BM3R engineering bacteria. BL21-pET28a-K1-BM3R transformants were selected and inoculated into 5mL LB liquid medium containing 50. mu.g/mL kanamycin, shake-cultured at 37 ℃ for 6h to extract pET28a-K1-BM3R plasmids, and BL21 was co-transformed with pET28a-K1-BM3R plasmids and Gro7 companion plasmids (purchased from Biovector Science Lab, Inc) to obtain BL21-pET28a-K1-BM3R-Gro7 engineering bacteria. The primers and their sequences are shown in Table 3.
TABLE 3
Figure BDA0002477627290000061
Figure BDA0002477627290000071
EXAMPLE 2 preparation of fusion protease
Single colonies of BL21-pET28a-K1-Gro7, BL21-pET28a-FdR-Gro7, BL21-pET28a-Fdx engineering bacteria and fusion protein engineering bacteria BL21-pET28a-K1-RhFR, BL21-pET28a-K1-RhFR-Gro7, BL21-pET28a-K1-BM3R, BL21-pET28a-K1-BM3R-Gro7 constructed in example 1 were inoculated into 5ml LB liquid medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol, and shake-cultured at 37 ℃ for 12h, respectively. Was inoculated into 50ml of a fresh LB liquid medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol at an inoculum size of 2 v/v%, shaken at 37 ℃ until the OD600 reached about 0.8, added with IPTG to a final concentration of 0.5mM, and induced at 22 ℃ for 22 hours. And after the culture is finished, centrifuging the culture solution at 10000rpm for 10min, removing the supernatant, collecting thalli (namely bacterial sludge), and storing the thalli in a refrigerator at the temperature of-20 ℃ for later use.
The bacterial sludge of each fusion protein was homogenized with 100mM PBS7.4 at a ratio of 1:4(W/V), the homogenate was centrifuged at 4000rpm for 20min, and the precipitate was discarded. And flocculating the supernatant by using 2 per mill of PEI, and centrifuging for 20min at 4000rpm, wherein the supernatant is crude enzyme liquid of K1-Gro7, AciFdR-Gro7, AciFdx protein and fusion protein K1-BM3R, K1-BM3R-Gro7, K1-RhFR and K1-RhFR-Gro 7.
Example 3 fusion protein P450 Activity assay
The concentration of K1 fusion protein P450 was determined by CO difference spectroscopy.
The determination method comprises the following steps: 1mL of a sample to be tested (i.e., the crude enzyme solution of the fusion protein in example 2) was placed in 2 10mL centrifuge tubes, which were labeled as a control tube and a sample tube. The sample is taken to a fume hood, a centrifuge tube is firstly taken to be filled with a proper amount of water, a CO pipeline is inserted into the water, and a three-way valve is adjusted until the CO gas outlet speed is about 1 second and one bubble is formed. 1mg of sodium hydrosulfite powder is added into the control tube and the sample tube respectively, and the solution is inverted repeatedly to dissolve the sodium hydrosulfite completely and mixed evenly. Respectively transferring the liquid of the control tube and the liquid of the sample tube into a cuvette, and scanning an absorbance value of 400-500nm on an ultraviolet spectrophotometer.
Calculating the enzyme concentration:
CP450＝(ΔA450-ΔA490)/(ε450·L)
wherein:
CP450the concentration of the P450 enzyme in the sample to be tested, in nmol/mL;
ΔA450，A450sample (I)-A450ControlA difference of (d);
ΔA490，A490sample (I)-A490ControlA difference of (d);
ε450the molar absorptivity of P450 is 0.091mL/nmol-1·cm-1。
L, optical path length, 1 cm.
The results of the measurement are shown in table 4 below:
TABLE 4
Figure BDA0002477627290000081
The obtained fusion proteins all have P450 activity, which indicates that the construction of the fusion proteins is successful.
Example 4 acquisition and expression of Glucose Dehydrogenase (GDH) Gene
The glucose dehydrogenase gene was synthesized from the glucose dehydrogenase gene sequence derived from Bacillus subtilis (Bacillus. mu. s s. mu. btilis)168(NCBI accession NP-388275.1).
Glucose dehydrogenase gene pET21a, enzyme cutting site NdeI&HindIII, and transforming the enzyme-linked vector into host E.coli BL21(DE3) competent cells to obtain an engineering strain containing glucose dehydrogenase gene. After the engineering bacteria containing glucose dehydrogenase gene is activated by plate streak, a single colony is selected and inoculated into 5ml LB liquid culture medium containing 100 mug/ml ampicillin, and shake culture is carried out for 12h at 37 ℃. Transferred into 50ml of fresh LB liquid medium containing 100. mu.g/ml ampicillin in an inoculum size of 2% (v/v), and shaken to OD at 37 ℃600When the concentration reached about 0.8, IPTG was added to a final concentration of 0.5mM, and induced culture was carried out at 18 ℃ for 16 hours. And after the culture is finished, centrifuging the culture solution at 10000rpm for 10min, removing the supernatant, collecting thalli (namely glucose dehydrogenase bacterial sludge), and storing in a refrigerator at the temperature of-20 ℃ for later use.
Example 5 fusion protease in vitro catalysis of VD3
Substrate VD3 (available from Shanghai Demer medical science and technology, Inc.) was made up with ethanol into a mother liquor with a concentration of 50g/L and solubilized by adding 25% hydroxypropyl-. beta. -cyclodextrin. The crude enzyme solution of example 2 was used to perform an in vitro enzymatic reaction, and the reaction system is shown in Table 5.
TABLE 5
Figure BDA0002477627290000091
Adding crude enzyme solution of protein as K1-Gro7 to react
② adding crude enzyme solution of protein as reaction system of each fusion protein
After 14h reaction at 28 ℃ and subsequent 100. mu.L sampling, 500. mu.L ethanol and 400. mu.L acetonitrile were added, centrifuged at 12000rpm for 3min, and the supernatant was subjected to HPLC analysis after removal of impurities by filtration. The results are shown in table 6 below:
TABLE 6
Figure BDA0002477627290000092
Figure BDA0002477627290000101
Among them, the catalytic capacities of K1-RhFR and K1-RhFR-Gro7 are higher than those of K1-BM3R and K1-BM3R-Gro7, so that Linker optimization is carried out on the fusion protein K1-RhFR subsequently.
Example 6 fusion protein Linker optimization
In order to further improve the activity of the fusion protein K1-RhFR, primers were designed to extend or shorten the native Linker of K1-RhFR on the fusion protein. I3 and I6 show that 3 and 6 amino acids are respectively inserted into the N end of a natural Linker; d3 and D6 show that 3 and 6 amino acids are respectively deleted at the N terminal of the natural Linker; i14 shows the insertion of 14 amino acids at the N-terminus, the specific sequence is shown in Table 7.
TABLE 7
Figure BDA0002477627290000102
Figure BDA0002477627290000111
PCR was carried out using pET28a-K1-RhFR as a template and I3F and I3R as primers, and the PCR product was digested with Dpn1 at 37 ℃ for 2 hours. After completion of the reaction, the PCR product was transformed into BL21 competent cells, plated on LB medium containing 50. mu.g/mL of kanamycin, and cultured overnight at 37 ℃ to give BL21-pET28a-K1-RhFR-I3 transformant. BL21-pET28a-K1-RhFR-I3 transformant is selected and inoculated into 5mL LB liquid culture medium containing 50 mu g/mL kanamycin, shaking culture is carried out for 6h at 37 ℃, pET28a-K1-RhFR-I3 plasmid is extracted, pET28a-K1-RhFR-I3 plasmid and Gro7 chaperone plasmid are co-transformed into BL21, and BL21-pET28a-K1-RhFR-I3-Gro7 engineering bacteria are obtained. The primers and their sequences are shown in Table 3.
BL21-pET28a-K1-RhFR-I6-Gro7, BL21-pET28a-K1-RhFR-D3-Gro7, BL21-pET28a-K1-RhFR-D6-Gro7, BL21-pET28a-K1-RhFR-I14-Gro7 engineering bacteria, namely engineering bacteria with different Linker lengths, are obtained by the same method.
According to the same manner as in example 2, a crude enzyme solution of the fusion protease shown in Table 8 below was obtained.
The crude enzyme solution of the fusion protease obtained was used for in vitro catalytic VD3 in the same manner as in example 5, and the results are shown in Table 7 below. In the case of K1-RhFR-I6-Gro7, the detection spectrum of the product obtained after catalysis is shown in FIG. 1, the retention time 19.050min is VD3, and 10.884min is calcifediol. FIG. 2 is a graph of VD3 control (from Shanghai Demer medicine science and technology Co., Ltd.) with retention time of 19.020min, and FIG. 3 is a graph of calcifediol control (from national standards network) with retention time of 10.920 min. It can be seen that the peak time of VD3 and the product of calcifediol in this example is substantially consistent with that of the respective control, the peak time of calcifediol prepared in this example is also consistent with that of K1-RhFR-I6-Gro7 in the other crude enzyme solutions, and the peak time of VD3 and the peak time of calcifediol in the obtained product are substantially consistent with that of the respective control.
TABLE 8
Figure BDA0002477627290000121
SEQUENCE LISTING
<110> Korea chess, Korea biological medicine science and technology Limited
<120> fusion protein or variant thereof and application thereof in preparation of calcifediol
<130> P19014220C
<160> 38
<170> PatentIn version 3.5
<210> 1
<211> 403
<212> PRT
<213> Pseudonocardia autotrophica
<400> 1
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala
<210> 2
<211> 1209
<212> DNA
<213> Pseudonocardia autotrophica
<400> 2
atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60
aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120
ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180
ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240
gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300
ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360
attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420
tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480
cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540
aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600
gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660
ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720
accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780
ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840
tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900
accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960
tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020
ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080
attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140
gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200
cgttcagca 1209
<210> 3
<211> 1049
<212> PRT
<213> Bacillus megaterium
<400> 3
Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys
1 5 10 15
Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys
20 25 30
Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg
35 40 45
Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp
50 55 60
Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg
65 70 75 80
Asp Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn
85 90 95
Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala
100 105 110
Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val
115 120 125
Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu
130 135 140
Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn
145 150 155 160
Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr
165 170 175
Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala
180 185 190
Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu
195 200 205
Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg
210 215 220
Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn
225 230 235 240
Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg
245 250 255
Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly
260 265 270
Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu
275 280 285
Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro
290 295 300
Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn
305 310 315 320
Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala
325 330 335
Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp
340 345 350
Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp
355 360 365
Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser
370 375 380
Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala
385 390 395 400
Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly
405 410 415
Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu
420 425 430
Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys
435 440 445
Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr
450 455 460
Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn
465 470 475 480
Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly
485 490 495
Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro
500 505 510
Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly
515 520 525
Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn
530 535 540
Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val
545 550 555 560
Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala
565 570 575
Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala
580 585 590
Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp
595 600 605
Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp
610 615 620
Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys
625 630 635 640
Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu
645 650 655
Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu
660 665 670
Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu
675 680 685
Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile
690 695 700
Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly
705 710 715 720
Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu
725 730 735
Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln
740 745 750
Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met
755 760 765
Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu
770 775 780
Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr
785 790 795 800
Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser
805 810 815
Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile
820 825 830
Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser
835 840 845
Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile
850 855 860
Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys
865 870 875 880
Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu
885 890 895
Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg
900 905 910
Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu
915 920 925
Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr
930 935 940
Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr
945 950 955 960
Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val
965 970 975
Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp
980 985 990
Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro
995 1000 1005
Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln
1010 1015 1020
Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu
1025 1030 1035
Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly
1040 1045
<210> 4
<211> 3147
<212> DNA
<213> Bacillus megaterium
<400> 4
atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60
ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120
tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180
gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240
gattttgcag gagacgggtt atttacaagc tggacgcacg aaaaaaattg gaaaaaagcg 300
cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360
gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420
gaagtacccg aagatatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480
tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540
gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600
gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660
attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720
ggaaaagatc cagaaacagg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780
acattcttaa ttgcgggaca cgaaacaaca agcggtcttt tatcatttgc gctgtatttc 840
ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900
gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaaac 960
gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020
gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080
cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140
gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200
tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260
cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320
aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380
tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440
acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500
ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560
gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620
ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680
aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740
aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800
cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860
atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920
tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980
ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040
agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100
ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160
ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220
ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280
acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340
cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400
atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460
cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520
aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580
tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640
tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700
atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760
ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820
catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880
cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940
gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000
ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060
gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120
cgatacgcaa aagacgtgtg ggctggg 3147
<210> 5
<211> 773
<212> PRT
<213> Rhodococcus
<400> 5
Met Ser Ala Ser Val Pro Ala Ser Ala Pro Ala Cys Pro Val Asp His
1 5 10 15
Ala Ala Leu Ala Gly Gly Cys Pro Val Ser Ala Asn Ala Ala Ala Phe
20 25 30
Asp Pro Phe Gly Ser Ala Tyr Gln Thr Asp Pro Ala Glu Ser Leu Arg
35 40 45
Trp Ser Arg Asp Glu Glu Pro Val Phe Tyr Ser Pro Glu Leu Gly Tyr
50 55 60
Trp Val Val Thr Arg Tyr Glu Asp Val Lys Ala Val Phe Arg Asp Asn
65 70 75 80
Ile Leu Phe Ser Pro Ala Ile Ala Leu Glu Lys Ile Thr Pro Val Ser
85 90 95
Ala Glu Ala Thr Ala Thr Leu Ala Arg Tyr Asp Tyr Ala Met Ala Arg
100 105 110
Thr Leu Val Asn Glu Asp Glu Pro Ala His Met Pro Arg Arg Arg Ala
115 120 125
Leu Met Asp Pro Phe Thr Pro Lys Glu Leu Ala His His Glu Ala Met
130 135 140
Val Arg Arg Leu Thr Arg Glu Tyr Val Asp Arg Phe Val Glu Ser Gly
145 150 155 160
Lys Ala Asp Leu Val Asp Glu Met Leu Trp Glu Val Pro Leu Thr Val
165 170 175
Ala Leu His Phe Leu Gly Val Pro Glu Glu Asp Met Ala Thr Met Arg
180 185 190
Lys Tyr Ser Ile Ala His Thr Val Asn Thr Trp Gly Arg Pro Ala Pro
195 200 205
Glu Glu Gln Val Ala Val Ala Glu Ala Val Gly Arg Phe Trp Gln Tyr
210 215 220
Ala Gly Thr Val Leu Glu Lys Met Arg Gln Asp Pro Ser Gly His Gly
225 230 235 240
Trp Met Pro Tyr Gly Ile Arg Lys Gln Arg Glu Met Pro Asp Val Val
245 250 255
Thr Asp Ser Tyr Leu His Ser Met Met Met Ala Gly Ile Val Ala Ala
260 265 270
His Glu Thr Thr Ala Asn Ala Ser Ala Asn Ala Phe Lys Leu Leu Leu
275 280 285
Glu Asn Arg Ala Val Trp Glu Glu Ile Cys Ala Asp Pro Ser Leu Ile
290 295 300
Pro Asn Ala Val Glu Glu Cys Leu Arg His Ser Gly Ser Val Ala Ala
305 310 315 320
Trp Arg Arg Val Ala Thr Ala Asp Thr Arg Ile Gly Asp Val Asp Ile
325 330 335
Pro Ala Gly Ala Lys Leu Leu Val Val Asn Ala Ser Ala Asn His Asp
340 345 350
Glu Arg His Phe Glu Arg Pro Asp Glu Phe Asp Ile Arg Arg Pro Asn
355 360 365
Ser Ser Asp His Leu Thr Phe Gly Tyr Gly Ser His Gln Cys Met Gly
370 375 380
Lys Asn Leu Ala Arg Met Glu Met Gln Ile Phe Leu Glu Glu Leu Thr
385 390 395 400
Thr Arg Leu Pro His Met Glu Leu Val Pro Asp Gln Glu Phe Thr Tyr
405 410 415
Leu Pro Asn Thr Ser Phe Arg Gly Pro Asp His Val Trp Val Gln Trp
420 425 430
Asp Pro Gln Ala Asn Pro Glu Arg Thr Asp Pro Ala Val Leu His Arg
435 440 445
His Gln Pro Val Thr Ile Gly Glu Pro Ala Ala Arg Ala Val Ser Arg
450 455 460
Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala Asp Asp Val Leu Arg
465 470 475 480
Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu Pro Thr Trp Thr Pro
485 490 495
Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser
500 505 510
Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu Ile Ala Val His Leu
515 520 525
Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile His Glu Gln Leu Glu
530 535 540
Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg Asn His Phe Ala Leu
545 550 555 560
Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly Ile
565 570 575
Thr Pro Val Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp Ser
580 585 590
Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu
595 600 605
Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser Glu
610 615 620
Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu Ala Glu Pro Ala Pro
625 630 635 640
Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu
645 650 655
Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala Leu His Val Glu His
660 665 670
Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp Val Glu His Ala Phe
675 680 685
Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro Thr
690 695 700
Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro Ser
705 710 715 720
Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu Val Ala Val Leu Asp
725 730 735
Gly Glu Val Asp His Arg Asp Thr Val Leu Thr Lys Ala Glu Arg Ala
740 745 750
Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg
755 760 765
Leu Ala Leu Arg Leu
770
<210> 6
<211> 2319
<212> DNA
<213> Rhodococcus
<400> 6
atgagtgcat cagttccggc gtcggcgccg gcgtgtcccg tcgaccacgc ggccctggcg 60
ggcggctgcc cggtgtcggc gaacgccgcg gcgttcgatc cgttcggttc cgcgtaccag 120
accgatccgg ccgagtcgct gcgctggtcc cgcgacgagg agccggtgtt ctacagcccc 180
gaactcggct actgggtcgt cacccggtac gaggatgtga aggcggtgtt ccgcgacaac 240
atcctgttct cgccggcgat cgcgctggag aagatcactc ccgtctcggc ggaggccacc 300
gccaccctcg cccggtacga ctacgccatg gcccggaccc tcgtgaacga ggacgagccc 360
gcccacatgc cgcgccgccg cgcgctcatg gatccgttca ccccgaagga actggcgcac 420
cacgaggcga tggtgcgacg gctcacgcgc gaatacgtcg accgcttcgt cgaatccggc 480
aaggccgacc tggtggacga gatgctgtgg gaggttccgc tcaccgtcgc cctgcacttc 540
ctcggcgtgc cggaggagga catggcgacg atgcgcaagt actcgatcgc gcacaccgtg 600
aacacctggg gccgccccgc gcccgaggag caggtggccg tcgccgaggc ggtcggcagg 660
ttctggcagt acgcgggcac ggtgctcgag aagatgcggc aggacccgtc gggacacggc 720
tggatgccct acgggatccg caagcagcgg gagatgccgg acgtcgtcac cgactcctac 780
ctgcactcga tgatgatggc cggcatcgtc gccgcgcacg agaccacggc caacgcgtcc 840
gcgaacgcgt tcaagctgct gctcgagaac cgcgcggtgt gggaggagat ctgcgcggat 900
ccgtcgctga tccccaacgc cgtcgaggag tgcctgcgcc actccgggtc cgtggcggcg 960
tggcgacggg tggccaccgc cgacacccgc atcggcgacg tcgacatccc cgccggcgcc 1020
aagctgctcg tcgtcaacgc gtccgccaac cacgacgagc gccacttcga gcgccccgac 1080
gagttcgaca tccggcgccc gaactcgagc gaccatctca ccttcgggta cggcagccac 1140
cagtgcatgg gcaagaacct ggcccgcatg gagatgcaga tcttcctcga ggaactcacc 1200
acgcggcttc cccacatgga actcgtaccc gatcaggagt tcacctacct gccgaatacg 1260
tccttccgcg gacccgacca cgtgtgggtg cagtgggatc cgcaggcgaa tcccgagcgc 1320
accgatcctg ctgtgctgca ccggcatcaa ccggtcacca tcggagaacc cgccgcccgg 1380
gcggtgtccc gcaccgtcac cgtcgagcgc ctggaccgga tcgccgacga cgtgctgcgc 1440
ctcgtcctgc gcgacgccgg cggaaagaca ttacccacgt ggactcccgg cgcccatatc 1500
gacctcgacc tcggcgcgct gtcgcgccag tactccctgt gcggcgcgcc cgatgcgccg 1560
agctacgaga ttgccgtgca cctggatccc gagagccgcg gcggttcgcg ctacatccac 1620
gaacagctcg aggtgggaag cccgctccgg atgcgcggcc ctcggaacca tttcgcgctc 1680
gaccccggcg ccgagcacta cgtgttcgtc gccggcggca tcggcatcac cccagtcctg 1740
gccatggccg accacgcccg cgcccggggg tggagctacg aactgcacta ctgcggccga 1800
aaccgttccg gcatggccta tctcgagcgt gtcgccgggc acggtgaccg ggccgccctg 1860
cacgtgtccg aggaaggcac ccggatcgac ctcgccgccc tcctcgccga gcccgccccc 1920
ggcgtccaga tctacgcgtg cgggcccggg cggctgctcg ccggactcga ggacgcgagc 1980
cggaactggc ccgacggggc gctgcacgtc gagcacttca cctcgtccct cgcggcgctc 2040
gatccggacg tcgagcacgc cttcgacctc gaactgcgtg actcggggct gaccgtgcgg 2100
gtcgaaccca cccagaccgt cctcgacgcg ttgcgcgcca acaacatcga cgtgcccagc 2160
gactgcgagg aaggcctctg cggctcgtgc gaggtcgccg tcctcgacgg cgaggtcgac 2220
catcgcgaca cggtgctgac caaggccgag cgggcggcga accggcagat gatgacctgc 2280
tgctcgcgtg cctgtggcga ccggctggcc ctgcgactc 2319
<210> 7
<211> 993
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-BM3R amino acid sequence
<400> 7
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Pro Ser Pro Ser Thr Glu Gln Ser Ala Lys Lys Val Arg
405 410 415
Lys Lys Ala Glu Asn Ala His Asn Thr Pro Leu Leu Val Leu Tyr Gly
420 425 430
Ser Asn Met Gly Thr Ala Glu Gly Thr Ala Arg Asp Leu Ala Asp Ile
435 440 445
Ala Met Ser Lys Gly Phe Ala Pro Gln Val Ala Thr Leu Asp Ser His
450 455 460
Ala Gly Asn Leu Pro Arg Glu Gly Ala Val Leu Ile Val Thr Ala Ser
465 470 475 480
Tyr Asn Gly His Pro Pro Asp Asn Ala Lys Gln Phe Val Asp Trp Leu
485 490 495
Asp Gln Ala Ser Ala Asp Glu Val Lys Gly Val Arg Tyr Ser Val Phe
500 505 510
Gly Cys Gly Asp Lys Asn Trp Ala Thr Thr Tyr Gln Lys Val Pro Ala
515 520 525
Phe Ile Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn Ile Ala Asp
530 535 540
Arg Gly Glu Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu Glu
545 550 555 560
Trp Arg Glu His Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu Asp
565 570 575
Ile Glu Asn Ser Glu Asp Asn Lys Ser Thr Leu Ser Leu Gln Phe Val
580 585 590
Asp Ser Ala Ala Asp Met Pro Leu Ala Lys Met His Gly Ala Phe Ser
595 600 605
Thr Asn Val Val Ala Ser Lys Glu Leu Gln Gln Pro Gly Ser Ala Arg
610 615 620
Ser Thr Arg His Leu Glu Ile Glu Leu Pro Lys Glu Ala Ser Tyr Gln
625 630 635 640
Glu Gly Asp His Leu Gly Val Ile Pro Arg Asn Tyr Glu Gly Ile Val
645 650 655
Asn Arg Val Thr Ala Arg Phe Gly Leu Asp Ala Ser Gln Gln Ile Arg
660 665 670
Leu Glu Ala Glu Glu Glu Lys Leu Ala His Leu Pro Leu Ala Lys Thr
675 680 685
Val Ser Val Glu Glu Leu Leu Gln Tyr Val Glu Leu Gln Asp Pro Val
690 695 700
Thr Arg Thr Gln Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro Pro
705 710 715 720
His Lys Val Glu Leu Glu Ala Leu Leu Glu Lys Gln Ala Tyr Lys Glu
725 730 735
Gln Val Leu Ala Lys Arg Leu Thr Met Leu Glu Leu Leu Glu Lys Tyr
740 745 750
Pro Ala Cys Glu Met Lys Phe Ser Glu Phe Ile Ala Leu Leu Pro Ser
755 760 765
Ile Arg Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Val Asp Glu
770 775 780
Lys Gln Ala Ser Ile Thr Val Ser Val Val Ser Gly Glu Ala Trp Ser
785 790 795 800
Gly Tyr Gly Glu Tyr Lys Gly Ile Ala Ser Asn Tyr Leu Ala Glu Leu
805 810 815
Gln Glu Gly Asp Thr Ile Thr Cys Phe Ile Ser Thr Pro Gln Ser Glu
820 825 830
Phe Thr Leu Pro Lys Asp Pro Glu Thr Pro Leu Ile Met Val Gly Pro
835 840 845
Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Val Gln Ala Arg Lys Gln
850 855 860
Leu Lys Glu Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe Gly
865 870 875 880
Cys Arg Ser Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu Asn
885 890 895
Ala Gln Ser Glu Gly Ile Ile Thr Leu His Thr Ala Phe Ser Arg Met
900 905 910
Pro Asn Gln Pro Lys Thr Tyr Val Gln His Val Met Glu Gln Asp Gly
915 920 925
Lys Lys Leu Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile Cys
930 935 940
Gly Asp Gly Ser Gln Met Ala Pro Ala Val Glu Ala Thr Leu Met Lys
945 950 955 960
Ser Tyr Ala Asp Val His Gln Val Ser Glu Ala Asp Ala Arg Leu Trp
965 970 975
Leu Gln Gln Leu Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala
980 985 990
Gly
<210> 8
<211> 2979
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-BM3R nucleotide sequence
<400> 8
atggctctga ccaccaccgg taccgaacag cacgacctgt tctctggtac cttctggcag 60
aacccgcacc cggcttacgc tgctctgcgt gctgaagacc cggttcgtaa actggctctg 120
ccggacggtc cggtttggct gctgacccgt tacgctgacg ttcgtgaagc tttcgttgac 180
ccgcgtctgt ctaaagactg gcgtcaccgt ctgccggaag accagcgtgc tgacatgccg 240
gctaccccga ccccgatgat gatcctgatg gacccgccgg accacacccg tctgcgtaaa 300
ctggttggtc gttctttcac cgttcgtcgt atgaacgaac tggaaccgcg tatcaccgaa 360
atcgctgacg gtctgctggc tggtctgccg accgacggtc cggttgacct gatgcgtgaa 420
tacgctttcc agatcccggt tcaggttatc tgcgaactgc tgggtctgcc ggctgaagac 480
cgtgacgact tctctgcttg gtcttctgtt ctggttgacg actctccggc tgacgacaaa 540
aacgctgcta tgggtaaact gcacggttac ctgtctgacc tgctggaacg taaacgtacc 600
gaaccggacg acgctctgct gtcttctctg ctggctgttt ctgacatgga cggtgaccgt 660
ctgtctcagg aagaactggt tgctatggct atgctgctgc tgatcgctgg tcacgaaacc 720
accgttaacc tgatcggtaa cggtgttctg gctctgctga cccacccgga ccagcgtaaa 780
ctgctggctg aagacccgtc tctgatctct tctgctgttg aagaattcct gcgtttcgac 840
tctccggttt ctcaggctcc gatccgtttc accgctgaag acgttaccta ctctggtgtt 900
accatcccgg ctggtgaaat ggttatgctg ggtctggctg ctgctaaccg tgacgctgac 960
tggatgccgg aaccggaccg tctggacatc acccgtgacg cttctggtgg tgttttcttc 1020
ggtcacggta tccacttctg cctgggtgct cagctggctc gtctggaagg tcgtgttgct 1080
atcggtcgtc tgttcgctga ccgtccggaa ctggctctgg ctgttggtct ggacgaactg 1140
gtttaccgtc gttctaccct ggttcgtggt ctgtctcgta tgccggttac catgggtccg 1200
cgttctgctc cgtctccgtc taccgaacag tctgctaaaa aagttcgtaa aaaagctgaa 1260
aacgctcaca acaccccgct gctggttctg tacggttcta acatgggtac cgctgaaggt 1320
accgctcgtg acctggctga catcgctatg tctaaaggtt tcgctccgca ggttgctacc 1380
ctggactctc acgctggtaa cctgccgcgt gaaggtgctg ttctgatcgt taccgcttct 1440
tacaacggtc acccgccgga caacgctaaa cagttcgttg actggctgga ccaggcttct 1500
gctgacgaag ttaaaggtgt tcgttactct gttttcggtt gcggtgacaa aaactgggct 1560
accacctacc agaaagttcc ggctttcatc gacgaaaccc tggctgctaa aggtgctgaa 1620
aacatcgctg accgtggtga agctgacgct tctgacgact tcgaaggtac ctacgaagaa 1680
tggcgtgaac acatgtggtc tgacgttgct gcttacttca acctggacat cgaaaactct 1740
gaagacaaca aatctaccct gtctctgcag ttcgttgact ctgctgctga catgccgctg 1800
gctaaaatgc acggtgcttt ctctaccaac gttgttgctt ctaaagaact gcagcagccg 1860
ggttctgctc gttctacccg tcacctggaa atcgaactgc cgaaagaagc ttcttaccag 1920
gaaggtgacc acctgggtgt tatcccgcgt aactacgaag gtatcgttaa ccgtgttacc 1980
gctcgtttcg gtctggacgc ttctcagcag atccgtctgg aagctgaaga agaaaaactg 2040
gctcacctgc cgctggctaa aaccgtttct gttgaagaac tgctgcagta cgttgaactg 2100
caggacccgg ttacccgtac ccagctgcgt gctatggctg ctaaaaccgt ttgcccgccg 2160
cacaaagttg aactggaagc tctgctggaa aaacaggctt acaaagaaca ggttctggct 2220
aaacgtctga ccatgctgga actgctggaa aaatacccgg cttgcgaaat gaaattctct 2280
gaattcatcg ctctgctgcc gtctatccgt ccgcgttact actctatctc ttcttctccg 2340
cgtgttgacg aaaaacaggc ttctatcacc gtttctgttg tttctggtga agcttggtct 2400
ggttacggtg aatacaaagg tatcgcttct aactacctgg ctgaactgca ggaaggtgac 2460
accatcacct gcttcatctc taccccgcag tctgaattca ccctgccgaa agacccggaa 2520
accccgctga tcatggttgg tccgggtacc ggtgttgctc cgttccgtgg tttcgttcag 2580
gctcgtaaac agctgaaaga acagggtcag tctctgggtg aagctcacct gtacttcggt 2640
tgccgttctc cgcacgaaga ctacctgtac caggaagaac tggaaaacgc tcagtctgaa 2700
ggtatcatca ccctgcacac cgctttctct cgtatgccga accagccgaa aacctacgtt 2760
cagcacgtta tggaacagga cggtaaaaaa ctgatcgaac tgctggacca gggtgctcac 2820
ttctacatct gcggtgacgg ttctcagatg gctccggctg ttgaagctac cctgatgaaa 2880
tcttacgctg acgttcacca ggtttctgaa gctgacgctc gtctgtggct gcagcagctg 2940
gaagaaaaag gtcgttacgc taaagacgtt tgggctggt 2979
<210> 9
<211> 732
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-RhFR amino acid sequence
<400> 9
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Val Leu His Arg His Gln Pro Val Thr Ile Gly Glu Pro
405 410 415
Ala Ala Arg Ala Val Ser Arg Thr Val Thr Val Glu Arg Leu Asp Arg
420 425 430
Ile Ala Asp Asp Val Leu Arg Leu Val Leu Arg Asp Ala Gly Gly Lys
435 440 445
Thr Leu Pro Thr Trp Thr Pro Gly Ala His Ile Asp Leu Asp Leu Gly
450 455 460
Ala Leu Ser Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser
465 470 475 480
Tyr Glu Ile Ala Val His Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg
485 490 495
Tyr Ile His Glu Gln Leu Glu Val Gly Ser Pro Leu Arg Met Arg Gly
500 505 510
Pro Arg Asn His Phe Ala Leu Asp Pro Gly Ala Glu His Tyr Val Phe
515 520 525
Val Ala Gly Gly Ile Gly Ile Thr Pro Val Leu Ala Met Ala Asp His
530 535 540
Ala Arg Ala Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn
545 550 555 560
Arg Ser Gly Met Ala Tyr Leu Glu Arg Val Ala Gly His Gly Asp Arg
565 570 575
Ala Ala Leu His Val Ser Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala
580 585 590
Leu Leu Ala Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro
595 600 605
Gly Arg Leu Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp
610 615 620
Gly Ala Leu His Val Glu His Phe Thr Ser Ser Leu Ala Ala Leu Asp
625 630 635 640
Pro Asp Val Glu His Ala Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu
645 650 655
Thr Val Arg Val Glu Pro Thr Gln Thr Val Leu Asp Ala Leu Arg Ala
660 665 670
Asn Asn Ile Asp Val Pro Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser
675 680 685
Cys Glu Val Ala Val Leu Asp Gly Glu Val Asp His Arg Asp Thr Val
690 695 700
Leu Thr Lys Ala Glu Arg Ala Ala Asn Arg Gln Met Met Thr Cys Cys
705 710 715 720
Ser Arg Ala Cys Gly Asp Arg Leu Ala Leu Arg Leu
725 730
<210> 10
<211> 2196
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-RhFR nucleotide sequence
<400> 10
atggctctga ccaccaccgg taccgaacag cacgacctgt tctctggtac cttctggcag 60
aacccgcacc cggcttacgc tgctctgcgt gctgaagacc cggttcgtaa actggctctg 120
ccggacggtc cggtttggct gctgacccgt tacgctgacg ttcgtgaagc tttcgttgac 180
ccgcgtctgt ctaaagactg gcgtcaccgt ctgccggaag accagcgtgc tgacatgccg 240
gctaccccga ccccgatgat gatcctgatg gacccgccgg accacacccg tctgcgtaaa 300
ctggttggtc gttctttcac cgttcgtcgt atgaacgaac tggaaccgcg tatcaccgaa 360
atcgctgacg gtctgctggc tggtctgccg accgacggtc cggttgacct gatgcgtgaa 420
tacgctttcc agatcccggt tcaggttatc tgcgaactgc tgggtctgcc ggctgaagac 480
cgtgacgact tctctgcttg gtcttctgtt ctggttgacg actctccggc tgacgacaaa 540
aacgctgcta tgggtaaact gcacggttac ctgtctgacc tgctggaacg taaacgtacc 600
gaaccggacg acgctctgct gtcttctctg ctggctgttt ctgacatgga cggtgaccgt 660
ctgtctcagg aagaactggt tgctatggct atgctgctgc tgatcgctgg tcacgaaacc 720
accgttaacc tgatcggtaa cggtgttctg gctctgctga cccacccgga ccagcgtaaa 780
ctgctggctg aagacccgtc tctgatctct tctgctgttg aagaattcct gcgtttcgac 840
tctccggttt ctcaggctcc gatccgtttc accgctgaag acgttaccta ctctggtgtt 900
accatcccgg ctggtgaaat ggttatgctg ggtctggctg ctgctaaccg tgacgctgac 960
tggatgccgg aaccggaccg tctggacatc acccgtgacg cttctggtgg tgttttcttc 1020
ggtcacggta tccacttctg cctgggtgct cagctggctc gtctggaagg tcgtgttgct 1080
atcggtcgtc tgttcgctga ccgtccggaa ctggctctgg ctgttggtct ggacgaactg 1140
gtttaccgtc gttctaccct ggttcgtggt ctgtctcgta tgccggttac catgggtccg 1200
cgttctgctg ttctgcaccg tcaccagccg gttaccatcg gtgaaccggc tgctcgtgct 1260
gtttctcgta ccgttaccgt tgaacgtctg gaccgtatcg ctgacgacgt tctgcgtctg 1320
gttctgcgtg acgctggtgg taaaaccctg ccgacctgga ccccgggtgc tcacatcgac 1380
ctggacctgg gtgctctgtc tcgtcagtac tctctgtgcg gtgctccgga cgctccgtct 1440
tacgaaatcg ctgttcacct ggacccggaa tctcgtggtg gttctcgtta catccacgaa 1500
cagctggaag ttggttctcc gctgcgtatg cgtggtccgc gtaaccactt cgctctggac 1560
ccgggtgctg aacactacgt tttcgttgct ggtggtatcg gtatcacccc ggttctggct 1620
atggctgacc acgctcgtgc tcgtggttgg tcttacgaac tgcactactg cggtcgtaac 1680
cgttctggta tggcttacct ggaacgtgtt gctggtcacg gtgaccgtgc tgctctgcac 1740
gtttctgaag aaggtacccg tatcgacctg gctgctctgc tggctgaacc ggctccgggt 1800
gttcagatct acgcttgcgg tccgggtcgt ctgctggctg gtctggaaga cgcttctcgt 1860
aactggccgg acggtgctct gcacgttgaa cacttcacct cttctctggc tgctctggac 1920
ccggacgttg aacacgcttt cgacctggaa ctgcgtgact ctggtctgac cgttcgtgtt 1980
gaaccgaccc agaccgttct ggacgctctg cgtgctaaca acatcgacgt tccgtctgac 2040
tgcgaagaag gtctgtgcgg ttcttgcgaa gttgctgttc tggacggtga agttgaccac 2100
cgtgacaccg ttctgaccaa agctgaacgt gctgctaacc gtcagatgat gacctgctgc 2160
tctcgtgctt gcggtgaccg tctggctctg cgtctg 2196
<210> 11
<211> 735
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-RhFR-I3 amino acid sequence
<400> 11
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Gly Gly Ser Val Leu His Arg His Gln Pro Val Thr Ile
405 410 415
Gly Glu Pro Ala Ala Arg Ala Val Ser Arg Thr Val Thr Val Glu Arg
420 425 430
Leu Asp Arg Ile Ala Asp Asp Val Leu Arg Leu Val Leu Arg Asp Ala
435 440 445
Gly Gly Lys Thr Leu Pro Thr Trp Thr Pro Gly Ala His Ile Asp Leu
450 455 460
Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp
465 470 475 480
Ala Pro Ser Tyr Glu Ile Ala Val His Leu Asp Pro Glu Ser Arg Gly
485 490 495
Gly Ser Arg Tyr Ile His Glu Gln Leu Glu Val Gly Ser Pro Leu Arg
500 505 510
Met Arg Gly Pro Arg Asn His Phe Ala Leu Asp Pro Gly Ala Glu His
515 520 525
Tyr Val Phe Val Ala Gly Gly Ile Gly Ile Thr Pro Val Leu Ala Met
530 535 540
Ala Asp His Ala Arg Ala Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys
545 550 555 560
Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu Glu Arg Val Ala Gly His
565 570 575
Gly Asp Arg Ala Ala Leu His Val Ser Glu Glu Gly Thr Arg Ile Asp
580 585 590
Leu Ala Ala Leu Leu Ala Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala
595 600 605
Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn
610 615 620
Trp Pro Asp Gly Ala Leu His Val Glu His Phe Thr Ser Ser Leu Ala
625 630 635 640
Ala Leu Asp Pro Asp Val Glu His Ala Phe Asp Leu Glu Leu Arg Asp
645 650 655
Ser Gly Leu Thr Val Arg Val Glu Pro Thr Gln Thr Val Leu Asp Ala
660 665 670
Leu Arg Ala Asn Asn Ile Asp Val Pro Ser Asp Cys Glu Glu Gly Leu
675 680 685
Cys Gly Ser Cys Glu Val Ala Val Leu Asp Gly Glu Val Asp His Arg
690 695 700
Asp Thr Val Leu Thr Lys Ala Glu Arg Ala Ala Asn Arg Gln Met Met
705 710 715 720
Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg Leu Ala Leu Arg Leu
725 730 735
<210> 12
<211> 2205
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-RhFR-I3 nucleotide sequence
<400> 12
atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60
aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120
ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180
ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240
gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300
ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360
attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420
tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480
cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540
aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600
gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660
ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720
accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780
ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840
tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900
accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960
tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020
ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080
attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140
gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200
cgttcagcag gcggaagtgt gctgcaccgg catcaaccgg tcaccatcgg agaacccgcc 1260
gcccgggcgg tgtcccgcac cgtcaccgtc gagcgcctgg accggatcgc cgacgacgtg 1320
ctgcgcctcg tcctgcgcga cgccggcgga aagacattac ccacgtggac tcccggcgcc 1380
catatcgacc tcgacctcgg cgcgctgtcg cgccagtact ccctgtgcgg cgcgcccgat 1440
gcgccgagct acgagattgc cgtgcacctg gatcccgaga gccgcggcgg ttcgcgctac 1500
atccacgaac agctcgaggt gggaagcccg ctccggatgc gcggccctcg gaaccatttc 1560
gcgctcgacc ccggcgccga gcactacgtg ttcgtcgccg gcggcatcgg catcacccca 1620
gtcctggcca tggccgacca cgcccgcgcc cgggggtgga gctacgaact gcactactgc 1680
ggccgaaacc gttccggcat ggcctatctc gagcgtgtcg ccgggcacgg tgaccgggcc 1740
gccctgcacg tgtccgagga aggcacccgg atcgacctcg ccgccctcct cgccgagccc 1800
gcccccggcg tccagatcta cgcgtgcggg cccgggcggc tgctcgccgg actcgaggac 1860
gcgagccgga actggcccga cggggcgctg cacgtcgagc acttcacctc gtccctcgcg 1920
gcgctcgatc cggacgtcga gcacgccttc gacctcgaac tgcgtgactc ggggctgacc 1980
gtgcgggtcg aacccaccca gaccgtcctc gacgcgttgc gcgccaacaa catcgacgtg 2040
cccagcgact gcgaggaagg cctctgcggc tcgtgcgagg tcgccgtcct cgacggcgag 2100
gtcgaccatc gcgacacggt gctgaccaag gccgagcggg cggcgaaccg gcagatgatg 2160
acctgctgct cgcgtgcctg tggcgaccgg ctggccctgc gactc 2205
<210> 13
<211> 738
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-RhFR-I6 amino acid sequence
<400> 13
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Gly Gly Ser Gly Gly Ser Val Leu His Arg His Gln Pro
405 410 415
Val Thr Ile Gly Glu Pro Ala Ala Arg Ala Val Ser Arg Thr Val Thr
420 425 430
Val Glu Arg Leu Asp Arg Ile Ala Asp Asp Val Leu Arg Leu Val Leu
435 440 445
Arg Asp Ala Gly Gly Lys Thr Leu Pro Thr Trp Thr Pro Gly Ala His
450 455 460
Ile Asp Leu Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser Leu Cys Gly
465 470 475 480
Ala Pro Asp Ala Pro Ser Tyr Glu Ile Ala Val His Leu Asp Pro Glu
485 490 495
Ser Arg Gly Gly Ser Arg Tyr Ile His Glu Gln Leu Glu Val Gly Ser
500 505 510
Pro Leu Arg Met Arg Gly Pro Arg Asn His Phe Ala Leu Asp Pro Gly
515 520 525
Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly Ile Thr Pro Val
530 535 540
Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp Ser Tyr Glu Leu
545 550 555 560
His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu Glu Arg Val
565 570 575
Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser Glu Glu Gly Thr
580 585 590
Arg Ile Asp Leu Ala Ala Leu Leu Ala Glu Pro Ala Pro Gly Val Gln
595 600 605
Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu Glu Asp Ala
610 615 620
Ser Arg Asn Trp Pro Asp Gly Ala Leu His Val Glu His Phe Thr Ser
625 630 635 640
Ser Leu Ala Ala Leu Asp Pro Asp Val Glu His Ala Phe Asp Leu Glu
645 650 655
Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro Thr Gln Thr Val
660 665 670
Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro Ser Asp Cys Glu
675 680 685
Glu Gly Leu Cys Gly Ser Cys Glu Val Ala Val Leu Asp Gly Glu Val
690 695 700
Asp His Arg Asp Thr Val Leu Thr Lys Ala Glu Arg Ala Ala Asn Arg
705 710 715 720
Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg Leu Ala Leu
725 730 735
Arg Leu
<210> 14
<211> 2214
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-RhFR-I6
<400> 14
atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60
aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120
ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180
ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240
gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300
ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360
attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420
tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480
cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540
aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600
gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660
ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720
accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780
ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840
tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900
accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960
tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020
ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080
attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140
gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200
cgttcagcag gcggaagtgg cggaagtgtg ctgcaccggc atcaaccggt caccatcgga 1260
gaacccgccg cccgggcggt gtcccgcacc gtcaccgtcg agcgcctgga ccggatcgcc 1320
gacgacgtgc tgcgcctcgt cctgcgcgac gccggcggaa agacattacc cacgtggact 1380
cccggcgccc atatcgacct cgacctcggc gcgctgtcgc gccagtactc cctgtgcggc 1440
gcgcccgatg cgccgagcta cgagattgcc gtgcacctgg atcccgagag ccgcggcggt 1500
tcgcgctaca tccacgaaca gctcgaggtg ggaagcccgc tccggatgcg cggccctcgg 1560
aaccatttcg cgctcgaccc cggcgccgag cactacgtgt tcgtcgccgg cggcatcggc 1620
atcaccccag tcctggccat ggccgaccac gcccgcgccc gggggtggag ctacgaactg 1680
cactactgcg gccgaaaccg ttccggcatg gcctatctcg agcgtgtcgc cgggcacggt 1740
gaccgggccg ccctgcacgt gtccgaggaa ggcacccgga tcgacctcgc cgccctcctc 1800
gccgagcccg cccccggcgt ccagatctac gcgtgcgggc ccgggcggct gctcgccgga 1860
ctcgaggacg cgagccggaa ctggcccgac ggggcgctgc acgtcgagca cttcacctcg 1920
tccctcgcgg cgctcgatcc ggacgtcgag cacgccttcg acctcgaact gcgtgactcg 1980
gggctgaccg tgcgggtcga acccacccag accgtcctcg acgcgttgcg cgccaacaac 2040
atcgacgtgc ccagcgactg cgaggaaggc ctctgcggct cgtgcgaggt cgccgtcctc 2100
gacggcgagg tcgaccatcg cgacacggtg ctgaccaagg ccgagcgggc ggcgaaccgg 2160
cagatgatga cctgctgctc gcgtgcctgt ggcgaccggc tggccctgcg actc 2214
<210> 15
<211> 729
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-RhFR-D3 amino acid sequence
<400> 15
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Arg His Gln Pro Val Thr Ile Gly Glu Pro Ala Ala Arg
405 410 415
Ala Val Ser Arg Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala Asp
420 425 430
Asp Val Leu Arg Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu Pro
435 440 445
Thr Trp Thr Pro Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu Ser
450 455 460
Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu Ile
465 470 475 480
Ala Val His Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile His
485 490 495
Glu Gln Leu Glu Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg Asn
500 505 510
His Phe Ala Leu Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala Gly
515 520 525
Gly Ile Gly Ile Thr Pro Val Leu Ala Met Ala Asp His Ala Arg Ala
530 535 540
Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser Gly
545 550 555 560
Met Ala Tyr Leu Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala Leu
565 570 575
His Val Ser Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu Ala
580 585 590
Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu
595 600 605
Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala Leu
610 615 620
His Val Glu His Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp Val
625 630 635 640
Glu His Ala Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val Arg
645 650 655
Val Glu Pro Thr Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn Ile
660 665 670
Asp Val Pro Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu Val
675 680 685
Ala Val Leu Asp Gly Glu Val Asp His Arg Asp Thr Val Leu Thr Lys
690 695 700
Ala Glu Arg Ala Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg Ala
705 710 715 720
Cys Gly Asp Arg Leu Ala Leu Arg Leu
725
<210> 16
<211> 2187
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-RhFR-D3 nucleotide sequence
<400> 16
atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60
aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120
ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180
ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240
gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300
ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360
attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420
tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480
cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540
aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600
gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660
ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720
accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780
ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840
tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900
accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960
tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020
ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080
attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140
gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200
cgttcagcac ggcatcaacc ggtcaccatc ggagaacccg ccgcccgggc ggtgtcccgc 1260
accgtcaccg tcgagcgcct ggaccggatc gccgacgacg tgctgcgcct cgtcctgcgc 1320
gacgccggcg gaaagacatt acccacgtgg actcccggcg cccatatcga cctcgacctc 1380
ggcgcgctgt cgcgccagta ctccctgtgc ggcgcgcccg atgcgccgag ctacgagatt 1440
gccgtgcacc tggatcccga gagccgcggc ggttcgcgct acatccacga acagctcgag 1500
gtgggaagcc cgctccggat gcgcggccct cggaaccatt tcgcgctcga ccccggcgcc 1560
gagcactacg tgttcgtcgc cggcggcatc ggcatcaccc cagtcctggc catggccgac 1620
cacgcccgcg cccgggggtg gagctacgaa ctgcactact gcggccgaaa ccgttccggc 1680
atggcctatc tcgagcgtgt cgccgggcac ggtgaccggg ccgccctgca cgtgtccgag 1740
gaaggcaccc ggatcgacct cgccgccctc ctcgccgagc ccgcccccgg cgtccagatc 1800
tacgcgtgcg ggcccgggcg gctgctcgcc ggactcgagg acgcgagccg gaactggccc 1860
gacggggcgc tgcacgtcga gcacttcacc tcgtccctcg cggcgctcga tccggacgtc 1920
gagcacgcct tcgacctcga actgcgtgac tcggggctga ccgtgcgggt cgaacccacc 1980
cagaccgtcc tcgacgcgtt gcgcgccaac aacatcgacg tgcccagcga ctgcgaggaa 2040
ggcctctgcg gctcgtgcga ggtcgccgtc ctcgacggcg aggtcgacca tcgcgacacg 2100
gtgctgacca aggccgagcg ggcggcgaac cggcagatga tgacctgctg ctcgcgtgcc 2160
tgtggcgacc ggctggccct gcgactc 2187
<210> 17
<211> 726
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-RhFR-D6 amino acid sequence
<400> 17
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Pro Val Thr Ile Gly Glu Pro Ala Ala Arg Ala Val Ser
405 410 415
Arg Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala Asp Asp Val Leu
420 425 430
Arg Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu Pro Thr Trp Thr
435 440 445
Pro Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu Ser Arg Gln Tyr
450 455 460
Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu Ile Ala Val His
465 470 475 480
Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile His Glu Gln Leu
485 490 495
Glu Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg Asn His Phe Ala
500 505 510
Leu Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly
515 520 525
Ile Thr Pro Val Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp
530 535 540
Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr
545 550 555 560
Leu Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser
565 570 575
Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu Ala Glu Pro Ala
580 585 590
Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly
595 600 605
Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala Leu His Val Glu
610 615 620
His Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp Val Glu His Ala
625 630 635 640
Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro
645 650 655
Thr Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro
660 665 670
Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu Val Ala Val Leu
675 680 685
Asp Gly Glu Val Asp His Arg Asp Thr Val Leu Thr Lys Ala Glu Arg
690 695 700
Ala Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp
705 710 715 720
Arg Leu Ala Leu Arg Leu
725
<210> 18
<211> 2178
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-RhFR-D6 nucleotide sequence
<400> 18
atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60
aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120
ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180
ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240
gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300
ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360
attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420
tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480
cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540
aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600
gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660
ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720
accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780
ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840
tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900
accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960
tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020
ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080
attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140
gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200
cgttcagcac cggtcaccat cggagaaccc gccgcccggg cggtgtcccg caccgtcacc 1260
gtcgagcgcc tggaccggat cgccgacgac gtgctgcgcc tcgtcctgcg cgacgccggc 1320
ggaaagacat tacccacgtg gactcccggc gcccatatcg acctcgacct cggcgcgctg 1380
tcgcgccagt actccctgtg cggcgcgccc gatgcgccga gctacgagat tgccgtgcac 1440
ctggatcccg agagccgcgg cggttcgcgc tacatccacg aacagctcga ggtgggaagc 1500
ccgctccgga tgcgcggccc tcggaaccat ttcgcgctcg accccggcgc cgagcactac 1560
gtgttcgtcg ccggcggcat cggcatcacc ccagtcctgg ccatggccga ccacgcccgc 1620
gcccgggggt ggagctacga actgcactac tgcggccgaa accgttccgg catggcctat 1680
ctcgagcgtg tcgccgggca cggtgaccgg gccgccctgc acgtgtccga ggaaggcacc 1740
cggatcgacc tcgccgccct cctcgccgag cccgcccccg gcgtccagat ctacgcgtgc 1800
gggcccgggc ggctgctcgc cggactcgag gacgcgagcc ggaactggcc cgacggggcg 1860
ctgcacgtcg agcacttcac ctcgtccctc gcggcgctcg atccggacgt cgagcacgcc 1920
ttcgacctcg aactgcgtga ctcggggctg accgtgcggg tcgaacccac ccagaccgtc 1980
ctcgacgcgt tgcgcgccaa caacatcgac gtgcccagcg actgcgagga aggcctctgc 2040
ggctcgtgcg aggtcgccgt cctcgacggc gaggtcgacc atcgcgacac ggtgctgacc 2100
aaggccgagc gggcggcgaa ccggcagatg atgacctgct gctcgcgtgc ctgtggcgac 2160
cggctggccc tgcgactc 2178
<210> 19
<211> 746
<212> PRT
<213> Artificial Sequence
<220>
<223> K1-RhFR-I14 amino acid sequence
<400> 19
Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly
1 5 10 15
Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu
20 25 30
Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu
35 40 45
Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser
50 55 60
Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro
65 70 75 80
Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr
85 90 95
Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn
100 105 110
Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly
115 120 125
Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln
130 135 140
Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp
145 150 155 160
Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro
165 170 175
Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser
180 185 190
Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser
195 200 205
Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu
210 215 220
Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr
225 230 235 240
Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro
245 250 255
Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala
260 265 270
Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile
275 280 285
Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala
290 295 300
Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp
305 310 315 320
Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly
325 330 335
Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu
340 345 350
Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg
355 360 365
Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg
370 375 380
Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro
385 390 395 400
Arg Ser Ala Glu Leu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu
405 410 415
Asn Val Leu His Arg His Gln Pro Val Thr Ile Gly Glu Pro Ala Ala
420 425 430
Arg Ala Val Ser Arg Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala
435 440 445
Asp Asp Val Leu Arg Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu
450 455 460
Pro Thr Trp Thr Pro Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu
465 470 475 480
Ser Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu
485 490 495
Ile Ala Val His Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile
500 505 510
His Glu Gln Leu Glu Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg
515 520 525
Asn His Phe Ala Leu Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala
530 535 540
Gly Gly Ile Gly Ile Thr Pro Val Leu Ala Met Ala Asp His Ala Arg
545 550 555 560
Ala Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser
565 570 575
Gly Met Ala Tyr Leu Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala
580 585 590
Leu His Val Ser Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu
595 600 605
Ala Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg
610 615 620
Leu Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala
625 630 635 640
Leu His Val Glu His Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp
645 650 655
Val Glu His Ala Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val
660 665 670
Arg Val Glu Pro Thr Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn
675 680 685
Ile Asp Val Pro Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu
690 695 700
Val Ala Val Leu Asp Gly Glu Val Asp His Arg Asp Thr Val Leu Thr
705 710 715 720
Lys Ala Glu Arg Ala Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg
725 730 735
Ala Cys Gly Asp Arg Leu Ala Leu Arg Leu
740 745
<210> 20
<211> 2238
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-RhFR-I14 nucleotide sequence
<400> 20
atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60
aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120
ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180
ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240
gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300
ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360
attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420
tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480
cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540
aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600
gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660
ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720
accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780
ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840
tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900
accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960
tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020
ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080
attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140
gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200
cgttcagcag aactgcagag tgcaaaaaaa gttcgtaaaa aagcagaaaa tgtgctgcac 1260
cggcatcaac cggtcaccat cggagaaccc gccgcccggg cggtgtcccg caccgtcacc 1320
gtcgagcgcc tggaccggat cgccgacgac gtgctgcgcc tcgtcctgcg cgacgccggc 1380
ggaaagacat tacccacgtg gactcccggc gcccatatcg acctcgacct cggcgcgctg 1440
tcgcgccagt actccctgtg cggcgcgccc gatgcgccga gctacgagat tgccgtgcac 1500
ctggatcccg agagccgcgg cggttcgcgc tacatccacg aacagctcga ggtgggaagc 1560
ccgctccgga tgcgcggccc tcggaaccat ttcgcgctcg accccggcgc cgagcactac 1620
gtgttcgtcg ccggcggcat cggcatcacc ccagtcctgg ccatggccga ccacgcccgc 1680
gcccgggggt ggagctacga actgcactac tgcggccgaa accgttccgg catggcctat 1740
ctcgagcgtg tcgccgggca cggtgaccgg gccgccctgc acgtgtccga ggaaggcacc 1800
cggatcgacc tcgccgccct cctcgccgag cccgcccccg gcgtccagat ctacgcgtgc 1860
gggcccgggc ggctgctcgc cggactcgag gacgcgagcc ggaactggcc cgacggggcg 1920
ctgcacgtcg agcacttcac ctcgtccctc gcggcgctcg atccggacgt cgagcacgcc 1980
ttcgacctcg aactgcgtga ctcggggctg accgtgcggg tcgaacccac ccagaccgtc 2040
ctcgacgcgt tgcgcgccaa caacatcgac gtgcccagcg actgcgagga aggcctctgc 2100
ggctcgtgcg aggtcgccgt cctcgacggc gaggtcgacc atcgcgacac ggtgctgacc 2160
aaggccgagc gggcggcgaa ccggcagatg atgacctgct gctcgcgtgc ctgtggcgac 2220
cggctggccc tgcgactc 2238
<210> 21
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-Rh-F1
<400> 21
accggctggc cctgcgactc taaaagcttg cggccgcact 40
<210> 22
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-Rh-R1
<400> 22
ggttgatgcc ggtgcagcac tgctgaacgc ggacccattg 40
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> RhFR-F1
<400> 23
caatgggtcc gcgttcagca gtgctgcacc ggcatcaacc 40
<210> 24
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> RhFR-R1
<400> 24
agtgcggccg caagctttta gagtcgcagg gccagccggt 40
<210> 25
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-BM3-F1
<400> 25
caaaagacgt gtgggctggg taaaagcttg cggccgcact 40
<210> 26
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> K1-BM3-R1
<400> 26
tgttcagtgc taggtgaagg tgctgaacgc ggacccattg 40
<210> 27
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> BM3R-F1
<400> 27
caatgggtcc gcgttcagca ccttcaccta gcactgaaca 40
<210> 28
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> BM3R-R1
<400> 28
agtgcggccg caagctttta cccagcccac acgtcttttg 40
<210> 29
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> I3-F
<400> 29
ccgcgttcag caggcggaag tgtgctgcac cgg 33
<210> 30
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> I3-R
<400> 30
ccggtgcagc acacttccgc ctgctgaacg cgg 33
<210> 31
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> I6-F
<400> 31
gcgttcagca ggcggaagtg gcggaagtgt gctgcacc 38
<210> 32
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> I6-R
<400> 32
ggtgcagcac acttccgcca cttccgcctg ctgaacgc 38
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> D3-F
<400> 33
ggtccgcgtt cagcacggca tcaaccggtc 30
<210> 34
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> D3-R
<400> 34
gaccggttga tgccgtgctg aacgcggacc 30
<210> 35
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> D6-F
<400> 35
ggtccgcgtt cagcaccggt caccatcgga 30
<210> 36
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> D6-R
<400> 36
tccgatggtg accggtgctg aacgcggacc 30
<210> 37
<211> 72
<212> DNA
<213> Artificial Sequence
<220>
<223> I14-F
<400> 37
atgccggtgc agcacatttt ctgctttttt acgaactttt tttgcactct gcagttctgc 60
tgaacgcgga cc 72
<210> 38
<211> 72
<212> DNA
<213> Artificial Sequence
<220>
<223> I14-R
<400> 38
ggtccgcgtt cagcagaact gcagagtgca aaaaaagttc gtaaaaaagc agaaaatgtg 60
ctgcaccggc at 72

Claims (10)

1. A fusion protein or a variant thereof, which comprises K1 and RhFR, wherein the amino acid sequence of K1 is shown as SEQ ID NO. 1, and the amino acid sequence of RhFR is shown as amino acid 466-773 of SEQ ID NO. 5.
2. The fusion protein or variant thereof of claim 1, wherein the fusion protein is K1 and RhFR;
and/or, the K1 and the RhFR are connected through a linker, the amino acid sequence of the linker is preferably shown as the amino acid at the 445-465 position of SEQ ID NO. 5;
and/or, the fusion protein or variant thereof is co-expressed with a chaperone, preferably Gro 7.
3. The fusion protein or variant thereof according to claim 1 or 2, wherein the variant is an insertion or deletion of amino acids at the N-terminus of the RhFR, preferably an insertion or deletion of 1-14, more preferably 3-6 amino acids at the N-terminus of the RhFR;
preferably, the amino acid sequence of the fusion protein or the variant thereof is shown as SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17 or SEQ ID NO 19;
more preferably, the nucleotide sequence encoding the fusion protein or the variant thereof is shown as SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18 or SEQ ID NO 20.
4. A fusion gene encoding the fusion protein or the variant thereof according to any one of claims 1 to 3.
5. A recombinant expression vector comprising the fusion gene of claim 4;
preferably, the backbone vector of the recombinant expression vector is pET28 a.
6. A transformant comprising the fusion gene according to claim 4 or the recombinant expression vector according to claim 5;
preferably, the transformant is obtained by introducing the fusion gene or the recombinant expression vector into a host, preferably an E.coli, more preferably E.coli BL21(DE3) cell.
7. A method of making a fusion protein or variant thereof, comprising the steps of:
(1) obtaining the transformant of claim 6;
(2) screening said transformants, expressing and purifying said fusion protein or variant thereof.
8. A method for preparing a calcifediol, comprising the steps of: the fusion protein or the variant thereof according to any one of claims 1 to 3 is used for catalyzing the hydroxylation reaction of vitamin D3 in the presence of a reaction solvent and reduced coenzyme NADH/NADPH;
preferably:
the vitamin D3 is cosolvent pre-dissolved vitamin D3; the cosolvent preferably comprises one or more of DMSO, Tween 80, Triton X100, methanol, ethanol, isopropanol and DMF, such as ethanol;
and/or, the method further comprises the step of adding a cyclodextrin, such as hydroxypropyl- β -cyclodextrin, to the reaction solvent prior to performing the hydroxylation reaction; the hydroxypropyl-beta-cyclodextrin accounts for 0.05-0.4% of the reaction system by mass volume, such as 0.25%;
and/or the reaction temperature is 20-33 ℃, for example, 22 ℃, 25 ℃, 28 ℃ or 30 ℃;
and/or the pH of the reaction is 6.0-8.0, for example 7.4;
and/or the concentration of vitamin D3 is 1g/L to 10g/L, such as 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, or 9 g/L;
and/or the molar ratio of the NADH/NADPH to the vitamin D3 is 0.001:1 to 2:1, for example 0.2: 1.
9. The method of claim 8, wherein the method of making further comprises the steps of: in the presence of dehydrogenase and a hydrogen donor, NAD (oxidized coenzyme)+/NADP+Carrying out reduction reaction to obtain the NADH/NADPH;
preferably, the dehydrogenase is glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase; and/or the hydrogen donor is glucose, isopropanol or formate;
more preferably, when the dehydrogenase is alcohol dehydrogenase, the hydrogen donor is isopropanol; when the dehydrogenase is glucose dehydrogenase, the hydrogen donor is glucose; when the dehydrogenase is formate dehydrogenase, the hydrogen donor is formate.
10. Use of the fusion protein or variant thereof according to any one of claims 1 to 3, the fusion gene according to claim 4, the recombinant expression vector according to claim 5, or the transformant according to claim 6 for the preparation of calcifediol.
CN202010369514.XA 2020-04-30 2020-04-30 Fusion protein or variant thereof and application thereof in preparation of calcifediol Pending CN113583983A (en)

Priority Applications (1)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010369514.XA CN113583983A (en) 2020-04-30 2020-04-30 Fusion protein or variant thereof and application thereof in preparation of calcifediol

Publications (1)

Publication Number Publication Date
CN113583983A true CN113583983A (en) 2021-11-02

Family

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Country Status (1)

Country Link
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