A fusion protein or its variant and its application in preparing calcifediol

Abstract

The present invention provides a fusion protein or a variant thereof, which includes K1 and RhFR, wherein the amino acid sequence of the K1 is shown in SEQ ID NO:1, and the amino acid sequence of the RhFR is shown in the amino acids at positions 466-773 of SEQ ID NO:5. The present invention also provides a preparation method and application of the fusion protein or a variant thereof. When the fusion protein or a variant thereof of the present invention is applied to catalyze VD3 to synthesize 25-hydroxyvitamin D3 (calcifediol), no additional electron transfer-related protein needs to be added, the operation is simple, and the electron transfer efficiency and catalytic efficiency in the fusion protein are high, the yield of the obtained calcifediol is significantly improved, and the production cost is reduced, which is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein or its variant and its gene, a preparation method thereof, and use of the fusion protein or its variant in catalyzing VD3 to prepare calcifediol. Background Art

[0002] 25-hydroxyvitamin D3, also known as calcifediol, is an active metabolite of vitamin D3 (VD3) with strong physiological activity. The traditional method for synthesizing 25-hydroxyvitamin D3 is chemical synthesis, which requires multiple steps of group protection and deprotection, and then photoreaction, ring opening and isomerization to obtain 25-hydroxyvitamin D3. In recent years, research on microbial transformation of VD3 has developed rapidly, but the conversion rate is low. One of the keys to increasing the yield of active VD3 is to screen and obtain key metabolic enzymes with high conversion efficiency, cytochrome P450 enzymes.

[0003] Cytochrome P450 enzymes are widely present in prokaryotes and eukaryotes. The monooxygenation reactions mediated by them usually require the participation of ferredoxin and ferredoxin reductase in the electron transport chain, and an efficient electron transfer system can effectively improve the reaction efficiency of P450. There are also reports of self-consistent P450, which does not require the addition of electron transfer-related proteins during the reaction. At present, two self-consistent P450s have been reported, namely P450BM3 from Bacillus megaterium and P450 RhFRed from Rhodococcus. Their protein structures contain P450 domains and electron transfer domains, and no electron transfer-related proteins need to be added during the reaction. However, there are no reports of self-consistent P450 being used to catalyze VD3 to produce calcifediol.

[0004] Tamura et al. (BioChemical and Biophysical Research Communications 2009, 385, 170–175) modified the P450 enzyme from Pseudonocardia autotrophica to obtain a mutant Vdh-K1 with four mutation sites (T70R, V156L, E216M and E384R), and found that the activity of Vdh-K1 in the catalysis of VD3 was 21.6 times that of the wild-type Vdhwt. However, the catalysis of VD3 requires the participation of the electron transfer system, which is cumbersome and costly, and the catalytic efficiency is still not very high. Tamura et al. (ChemBioChem 2013, 14, 2284-2291) modified the P450 enzyme from Pseudonocardia autotrophica (through mutations such as T107A) and expressed it heterologously in Rhodococcus. By adding nisin to increase the permeability of the cell membrane and reduce the mass transfer resistance of the substrate VD3, the final yield of calcifediol obtained by catalyzing VD3 for two hours reached 573 μg / mL. The electron transfer systems AciFdx and AciFdxR were used in the method, but the yield needs to be improved.

[0005] Therefore, there is an urgent need in the prior art for a method which has high catalytic efficiency, high yield of the obtained product, simple operation and low cost when catalyzing VD3 to prepare calcifediol. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a fusion protein or its variant and its gene, a preparation method and the application of catalyzing VD3 to prepare calcifediol in view of the defects of low yield, high cost and complicated operation when cytochrome P450 enzyme is used to catalyze VD3 to prepare calcifediol in the prior art. When the fusion protein or its variant of the present invention is used to catalyze VD3 to synthesize 25-hydroxyvitamin D3 (calcifediol), no additional electron transfer-related protein needs to be added, the operation is simple, and the electron transfer efficiency and catalytic efficiency in the fusion protein are high, the yield of the obtained calcifediol is significantly improved, and the production cost is reduced, which is suitable for industrial production.

[0007] There are many methods to improve the yield of VD3 catalyzed by P450 enzymes, including seeking efficient P450 enzymes, optimizing reaction conditions, etc. After a lot of exploration, the inventors unexpectedly discovered that when a specific type of P450 is transformed into a self-consistent P450 enzyme in a specific way, the catalytic efficiency of catalyzing VD3 to prepare calcifediol can be significantly improved, and the yield of calcifediol can be significantly increased.

[0008] In order to solve the above technical problems, the first aspect of 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 in SEQ ID NO:1, and the amino acid sequence of RhFR is shown in the amino acids at positions 466-773 of SEQ ID NO:5.

[0009] Preferably, the fusion protein comprises K1 and RhFR from N-terminus to C-terminus.

[0010] Preferably, the K1 and RhFR are connected via a linker, and the amino acid sequence of the linker is preferably as shown in the amino acids at positions 445-465 of SEQ ID NO:5.

[0011] Preferably, the fusion protein or its variant is co-expressed with a molecular chaperone, and the molecular chaperone is preferably Gro7. In a preferred embodiment of the present invention, when expressing the fusion protein, a molecular chaperone such as Gro7 chaperone protein may be added so that the fusion protein is co-expressed with the molecular chaperone. In the present invention, the Gro7 chaperone protein may be commercially available, for example, Gro7 purchased from Biovector Science Lab, Inc.

[0012] Preferably, the variant is an insertion or deletion of amino acids 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; further more preferably, the variant is an insertion or deletion of 3-6 amino acids at the N-terminus of the RhFR.

[0013] Preferably, the amino acid sequence of the fusion protein or its variant is shown in 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.

[0014] More preferably, the nucleotide sequence encoding the fusion protein or its variant is shown in 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.

[0015] In order to solve the above technical problem, the second aspect of the present invention provides a fusion gene, which encodes the fusion protein or a variant thereof as described in the first aspect of the present invention.

[0016] In order to solve the above technical problem, the third aspect of the present invention provides a recombinant expression vector, which contains the fusion gene as described in the second aspect of the present invention.

[0017] Preferably, the backbone vector of the recombinant expression vector is pET28a.

[0018] In order to solve the above technical problems, the fourth aspect of the present invention provides a transformant, which includes the fusion gene as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention.

[0019] Preferably, the transformant is obtained by introducing the fusion gene as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention into the host.

[0020] More preferably, the host is Escherichia coli, preferably E. coli BL21 (DE3) cells.

[0021] In order to solve the above technical problems, the fifth aspect of the present invention provides a method for preparing a fusion protein or a variant thereof, which comprises the following steps:

[0022] (1) obtaining the transformant according to the fourth aspect of the present invention;

[0023] (2) Screening the transformants, expressing and purifying the fusion protein or its variant.

[0024] In order to solve the above technical problems, the sixth aspect of the present invention provides a method for preparing calcifediol, which comprises the following steps: in the presence of a reaction solvent and reduced coenzyme NADH / NADPH, catalyzing the hydroxylation reaction of vitamin D3 by the fusion protein or its variant as described in the first aspect of the present invention.

[0025] Preferably, the vitamin D3 is vitamin D3 pre-dissolved in a co-solvent; the co-solvent preferably includes one or more of DMSO, Tween 80, Triton X100, methanol, ethanol, isopropanol and DMF, such as ethanol.

[0026] Preferably, the method further comprises the step of adding cyclodextrin to the reaction solvent before carrying out the hydroxylation reaction, wherein the cyclodextrin may be, for example, hydroxypropyl-β-cyclodextrin; the mass volume percentage of the hydroxypropyl-β-cyclodextrin in the reaction system is preferably 0.05%-0.4%, for example 0.25%.

[0027] Preferably, the reaction temperature is 20-33° C., for example, 22° C., 25° C., 28° C. or 30° C. The inventors found in the experiment that when the temperature is not within this range, the yield of the obtained product will be reduced.

[0028] Preferably, the pH of the reaction is 6.0-8.0, for example 7.4. The inventors found in the experiment that when the pH is not within the range specified by the present invention, the yield of the obtained product will be reduced.

[0029] Preferably, the concentration of vitamin D3 is 1g / L-10g / L, for example, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L or 10g / L. The inventors found during the experiment that if the concentration of VD3 is too high, it cannot be dissolved or the substrate may inhibit the enzyme activity, resulting in an incomplete reaction; if the concentration is too low, the yield is also very low.

[0030] Preferably, the molar ratio of the NADH / NADPH to the vitamin D3 is 0.001:1 to 2:1, for example 0.2:1.

[0031] Preferably, the preparation method further comprises the following steps: in the presence of a dehydrogenase and a hydrogen donor, oxidized coenzyme NAD + / NADP + Perform a reduction reaction to obtain the reduced coenzyme NADH / NADPH;

[0032] More preferably, the dehydrogenase is glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase; and / or, the hydrogen donor is glucose, isopropanol or formate;

[0033] Further 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.

[0034] In order to solve the above technical problems, the seventh aspect of the present invention provides a fusion protein or its variant as described in the first aspect of the present invention, the fusion gene as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, or the transformant as described in the fourth aspect of the present invention for use in the preparation of calcifediol.

[0035] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0036] The reagents and raw materials used in the present invention are commercially available.

[0037] The positive and progressive effects of the present invention are: when the fusion protein of the present invention or its variant is used to catalyze VD3 to synthesize 25-hydroxyvitamin D3 (calcifediol), no additional electron transfer-related proteins need to be added, the operation is simple, and the electron transfer efficiency and catalytic efficiency in the fusion protein are high, the yield of the obtained calcifediol is significantly improved, and the production cost is reduced, which is suitable for industrial production. In a preferred embodiment of the present invention, when the fusion protein of the present invention or its variant catalyzes VD3, the yield of the obtained calcifediol is as high as 4.427 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the detection spectrum of K1-RhFR-I6-Gro7 in Example 6.

[0039] Figure 2 This is the spectrum result of VD3 substrate reference substance.

[0040] Figure 3 This is the chromatographic result of the calcifediol reference substance. DETAILED DESCRIPTION

[0041] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0042] HPLC analysis of products

[0043] Chromatographic conditions: chromatographic column: Poroshell EC-C18 (4.0μm, 4.6×150mm); detection wavelength: 265nm; flow rate: 1mL / min; column temperature: 35℃; injection volume: 10μL. Gradient elution program is as follows: 0-8min, H2O: acetonitrile = 85%: 15%; 8-20min, H2O: acetonitrile = 0: 100%; 20-21min, H2O: acetonitrile = 85%: 15%; 21-27min, H2O: acetonitrile = 85%: 15%.

[0044] pET28a was purchased from Novagen; DpnI enzyme, NdeI enzyme, and HindIII enzyme were purchased from Thermo Fisher; ExnaseⅡ enzyme was purchased from Nanjing Novagen Biotechnology Co., Ltd.; E.coli BL21 (DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; NAD+ was purchased from Shenzhen Bangtai Biotechnology Co., Ltd.; vitamin D3 and plasmid extraction kits were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0045] Example 1 Construction of fusion protease strain

[0046] 1.1 Protein sequence analysis

[0047] In this example, the P450 enzyme Vdh-K1 that can catalyze VD3 is fused with the reducing regions of P450 BM3 and P450 RhFRed to construct a self-consistent P450 fusion protein. The specific steps are as follows:

[0048] Each gene was synthesized according to the gene sequence SEQ ID NO: 2, 4, 6 encoding the cytochrome P450 enzyme SEQ ID NO: 1, 3, 5 reported on NCBI. The company that synthesized the gene was Suzhou Jinweizhi Biotechnology Co., Ltd. (Building C3, Bio-Nano Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park). The information of each gene is shown in Table 1.

[0049] Table 1

[0050]

[0051] The amino acid sequence of BM3 protein was analyzed using Discovery studio software, and it was found that the 1st to 459th amino acids at the N-terminus were the P450 domain of the protein; the 460th to 479th amino acids were the linker between the P450 domain and the electron transfer domain; and the 480th to 1048th amino acids were the electron transfer domain of the protein.

[0052] The amino acid sequence of RhFRed protein was analyzed using Discovery studio software, and it was found that the 24-444 amino acids at the N-terminus were the P450 domain of the protein; the 445-465 amino acids were the linker between the P450 domain and the electron transfer domain; and the 466-773 amino acids were the electron transfer domain of the protein.

[0053] 1.2 Construction of K1, FdR, Fdx protein engineering strains and fusion protein engineering strains

[0054] The K1, BM3, RhFRed synthesized in Example 1.1 and the synthesized electron transfer system AciFdR, AciFdx (as shown in Table 2 below) genes were connected to pET28a, restriction sites NdeI & HindIII, and the enzyme-linked vectors were transformed into host E. coli BL21 (DE3) competent cells to obtain engineering strains containing K1, BM3, RhFRed, AciFdR, and AciFdx, respectively. The plasmids were extracted using the plasmid extraction kit of Shanghai Biotech, and pET28a-K1, pET28a-BM3, pET28a-RhFRed, pET28a-FdR, and pET28a-Fdx plasmids were obtained, respectively.

[0055] The obtained pET28a-K1 and pET28a-FdR plasmids were co-transformed with the Gro7 companion plasmid into E. coli BL21 to obtain BL21-pET28a-K1-Gro7 and BL21-pET28a-FdR-Gro7 engineered bacteria. The pET28a-Fdx plasmid was transformed into BL21 to obtain BL21-pET28a-Fdx engineered bacteria.

[0056] Table 2

[0057] Enzyme number Gene source NCBI accession number AciFd Acinetobacter sp.OC4 BAE78451.1 FWf Acinetobacter sp.OC4 BAE78453.1

[0058] The target fragment ΔK1-1 was amplified using the original plasmid pET28a-K1 as a template and K1-Rh-F1 and K1-Rh-R1 as primers. The vector fragment RhFR with a 15bP homology arm at the 5' end and the 15bP homology arm at the 3' end of ΔK1-1 and a 15bP homology arm at the 3' end and the 5' end of ΔK1-1 was amplified using the pET28a-RhFRed plasmid as a template and RhFR-F1 and RhFR-R1 as primers. The PCR product was digested with Dpn1 at 37°C for 2 hours. After the reaction was completed, ΔK1-1 and RhFR were recombined with recombinase ExnaseⅡ at 37°C for 0.5 hours, and the recombinant product was transformed into BL21 competent cells, coated in LB medium containing 50μg / mL kanamycin, and cultured at 37°C overnight to obtain BL21-pET28a-K1-RhFR transformants, i.e. BL21-pET28a-K1-RhFR engineering bacteria. Pick BL21-pET28a-K1-RhFR transformants and inoculate them into 5ml LB liquid culture medium containing 50μg / mL kanamycin, shake culture at 37°C for 6h, extract pET28a-K1-RhFR plasmid, co-transform pET28a-K1-RhFR plasmid with Gro7 companion plasmid into BL21, and obtain BL21-pET28a-K1-RhFR-Gro7 engineering bacteria. Primers and their sequences are shown in Table 2.

[0059] Using the original plasmid pET28a-K1 as a template, K1-BM3-F1 and K1-BM3-R1 as primers, the target fragment ΔK1-2 was amplified. Using the pET28a-BM3 plasmid as a template, BM3R-F1 and BM3R-R1 as primers, the vector fragment BM3R with a 15bP homology arm at the 5' end and the 3' end and the 15bP homology arm at the 5' end of K1-2 was amplified. The PCR product was digested with Dpn1 at 37°C for 2 hours. After the reaction was completed, ΔK1-2 and BM3R were recombined with recombinase ExnaseⅡ at 37°C for 0.5 hours, and the recombinant product was transformed into BL21 competent cells, spread on LB medium containing 50μg / mL kanamycin, and cultured at 37°C overnight to obtain BL21-pET28a-K1-BM3R transformants, i.e. BL21-pET28a-K1-BM3R engineered bacteria. BL21-pET28a-K1-BM3R transformants were picked and inoculated into 5ml LB liquid medium containing 50μg / mL kanamycin, shaken and cultured at 37°C for 6h, pET28a-K1-BM3R plasmids were extracted, and pET28a-K1-BM3R plasmids were co-transformed into BL21 with Gro7 companion plasmids (purchased from Biovector Science Lab, Inc) to obtain BL21-pET28a-K1-BM3R-Gro7 engineered bacteria. The primers and their sequences are shown in Table 3.

[0060] Table 3

[0061]

[0062]

[0063] Example 2 Preparation of fusion protease

[0064] The single colonies of the BL21-pET28a-K1-Gro7, BL21-pET28a-FdR-Gro7, BL21-pET28a-Fdx engineered bacteria and the fusion protein engineered bacteria BL21-pET28a-K1-RhFR, BL21-pET28a-K1-RhFR-Gro7, BL21-pET28a-K1-BM3R, and BL21-pET28a-K1-BM3R-Gro7 constructed in Example 1 were inoculated into 5 ml LB liquid culture medium containing 50 μg / ml kanamycin and 50 μg / ml chloramphenicol, and cultured at 37°C with shaking for 12 h. The inoculum was transferred to 50 ml of fresh LB liquid medium containing 50 μg / ml kanamycin and 50 μg / ml chloramphenicol at a 2 v / v% inoculum, and shaken at 37°C until OD600 reached about 0.8, and IPTG was added to a final concentration of 0.5 mM, and induced at 22°C for 22 h. After the culture was completed, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial cells (i.e., bacterial mud) were collected and stored in a -20°C refrigerator for later use.

[0065] 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. The supernatant was flocculated with 2‰ PEI and centrifuged at 4000rpm for 20min. The supernatant was the crude enzyme solution of K1-Gro7, AciFdR-Gro7, AciFdx protein and fusion protein K1-BM3R, K1-BM3R-Gro7, K1-RhFR, K1-RhFR-Gro7.

[0066] Example 3 Determination of fusion protein P450 activity

[0067] The concentration of K1 fusion protein P450 was determined by CO difference spectroscopy.

[0068] Determination method: Take 1 mL of the sample to be tested (i.e., the crude enzyme solution of the fusion protein in Example 2) in two 10 mL centrifuge tubes, respectively, and mark them as control tube and sample tube. Take the sample to a fume hood, first take a centrifuge tube to fill it with an appropriate amount of water, insert the CO pipeline into the water, and adjust the three-way valve to a CO gas outlet speed of about one bubble per second. Add 1 mg of sodium dithionite powder to the control tube and the sample tube respectively, and repeatedly invert to dissolve the sodium dithionite completely and mix evenly. Transfer the liquid of the control tube and the sample tube to a cuvette respectively, and scan the absorbance value of 400-500nm on a UV spectrophotometer.

[0069] Enzyme concentration calculation:

[0070] C P450 =(ΔA450-ΔA490) / (ε 450 ·L)

[0071] in:

[0072] C P450 , the concentration of P450 enzyme in the measured sample, unit: nmol / mL;

[0073] ΔA450,A450 样品 -A450 对照 The difference between

[0074] ΔA490,A490 样品 -A490 对照 The difference between

[0075] ε 450 , P450 molar absorption coefficient, is 0.091mL / nmol -1 cm -1 .

[0076] L, optical path, 1 cm.

[0077] The measurement results are shown in Table 4 below:

[0078] Table 4

[0079]

[0080] The above-obtained fusion proteins all have P450 activity, indicating that the fusion proteins were successfully constructed.

[0081] Example 4 Acquisition and expression of glucose dehydrogenase (GDH) gene

[0082] The glucose dehydrogenase gene was fully synthesized based on the glucose dehydrogenase gene sequence from Bacillus subtilis 168 (NCBI accession number NP_388275.1).

[0083] The glucose dehydrogenase gene was linked to pET21a, with restriction sites NdeI & HindIII. The enzyme-linked vector was transformed into the host E. coli BL21 (DE3) competent cells to obtain an engineered strain containing the glucose dehydrogenase gene. After the engineered bacteria containing the glucose dehydrogenase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 ml LB liquid culture medium containing 100 μg / ml ampicillin, and cultured at 37°C for 12 hours. The inoculation volume was transferred to 50 ml of fresh LB liquid culture medium containing 100 μg / ml ampicillin at 2% (v / v), and the culture was shaken at 37°C until the OD 600When the pH value reached about 0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 18°C ​​for 16 h. After the culture was completed, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, the bacteria (i.e., glucose dehydrogenase bacterial sludge) was collected, and stored in a -20°C refrigerator for later use.

[0084] Example 5 Fusion protease catalyzes VD3 in vitro

[0085] The substrate VD3 (purchased from Shanghai Demo Pharmaceutical Technology Co., Ltd.) was prepared into a mother solution with a concentration of 50 g / L using ethanol, and 25% hydroxypropyl-β-cyclodextrin was added to aid dissolution. The crude enzyme solution in Example 2 was used to carry out an in vitro enzyme catalytic reaction, and the reaction system is shown in Table 5.

[0086] Table 5

[0087]

[0088] ① Add protein crude enzyme solution to the reaction system of K1-Gro7 crude enzyme solution

[0089] ② Add protein crude enzyme solution as the reaction system for each fusion protein

[0090] The reaction was carried out at 28°C for 14 hours, and then 100 μL of the sample was taken, 500 μL of ethanol and 400 μL of acetonitrile were added, and the mixture was centrifuged at 12000 rpm for 3 minutes. The supernatant was filtered through a membrane to remove impurities and then tested by HPLC. The test results are shown in Table 6 below:

[0091] Table 6

[0092]

[0093]

[0094] Among them, the catalytic abilities of K1-RhFR and K1-RhFR-Gro7 were higher than those of K1-BM3R and K1-BM3R-Gro7, so the Linker was subsequently optimized for the fusion protein K1-RhFR.

[0095] Example 6 Fusion Protein Linker Optimization

[0096] In order to further improve the activity of the fusion protein K1-RhFR, primers were designed to extend or shorten the natural linker of K1-RhFR on the fusion protein. I3 and I6 indicate the insertion of 3 and 6 amino acids at the N-terminus of the natural linker, respectively; D3 and D6 indicate the deletion of 3 and 6 amino acids at the N-terminus of the natural linker, respectively; I14 indicates the insertion of 14 amino acids at the N-terminus. The specific sequences are shown in Table 7.

[0097] Table 7

[0098]

[0099]

[0100] Using pET28a-K1-RhFR as a template and I3F and I3R as primers, PCR was performed, and the PCR product was digested with Dpn1 at 37°C for 2 hours. After the reaction was completed, the PCR product was transformed into BL21 competent cells, spread on LB medium containing 50 μg / mL kanamycin, and cultured at 37°C overnight to obtain BL21-pET28a-K1-RhFR-I3 transformants. Pick the BL21-pET28a-K1-RhFR-I3 transformant and inoculate it into 5 ml LB liquid medium containing 50 μg / mL kanamycin, shake and culture at 37°C for 6 hours, extract the pET28a-K1-RhFR-I3 plasmid, and co-transform the pET28a-K1-RhFR-I3 plasmid with the Gro7 companion plasmid into BL21 to obtain the BL21-pET28a-K1-RhFR-I3-Gro7 engineered bacteria. The primers and their sequences are shown in Table 3.

[0101] Using the same method as above, BL21-pET28a-K1-RhFR-I6-Gro7, BL21-pET28a-K1-RhFR-D3-Gro7, BL21-pET28a-K1-RhFR-D6-Gro7, and BL21-pET28a-K1-RhFR-I14-Gro7 engineered bacteria, i.e., engineered bacteria with different linker lengths, were obtained respectively.

[0102] The same method as in Example 2 was used to obtain the crude fusion protease solution shown in Table 8 below.

[0103] According to the same method as in Example 5, the obtained crude enzyme solution of fusion protease was used to catalyze VD3 in vitro. The results are shown in Table 7. Taking K1-RhFR-I6-Gro7 as an example, the detection spectrum of the product obtained after catalysis is shown in Table 7. Figure 1 As shown, the retention time is 19.050 min for VD3 and 10.884 min for calcifediol. Figure 2 It is the spectrum of VD3 reference substance (purchased from Shanghai Demo Pharmaceutical Technology Co., Ltd.), with a retention time of 19.020min. Figure 3 It is the spectrum of calcifediol reference substance (purchased from the National Standard Material Network), and the retention time is 10.920min. It can be seen that the elution time of VD3 and the product calcifediol in this embodiment is basically consistent with the elution time of the respective reference substances, and calcifediol is prepared in this embodiment. The results of other crude enzyme solutions are also consistent with K1-RhFR-I6-Gro7, and the elution time of VD3 and calcifediol in the obtained product is basically consistent with the elution time of the respective reference substances.

[0104] Table 8

[0105] SEQUENCE LISTING <110> Shanghai Yikelai Biopharmaceutical Technology Co., Ltd. <120> A fusion protein or its variant and its application in preparing 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> Ms <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 Pro Lys Thr Tyr Val 965,970,975 Gln His Will Meet 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 Only Glu Only Thr Leu Met Lys Ser Tyr Only On Asp Only His Gln 1010 1015 1020 Val Ser Glu Ala Asp Ala Arg More Than Gln Gln More 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 ataaccggt tcaagcttg atgaaattg cggatgaatt aggaaatc 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 ataaaaacaat 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 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980. ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga agcacgcgac atcttgaat tgaacttcca aaagaagctt cttatcaaga aggagatcat ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagg 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 gccgctttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aggacagtc 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 cggctggcc ctgcgactc 2319 <210> 7 <211> 993 <212> PRT <213> Artificial Sequence <220> <223> K1-BM3R <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 tgctgggaacg 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 tgctgggaacg 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 <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 cgtcgtcgt 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 tgctgggaacg 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 gccccggcg 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 <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 gcaacccga 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 tgctgggaacg 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 gcaacccga 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 tgctgggaacg 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 Wing Leu Arg Leu 725 <210> 18 <211> 2178 <212> DNA <213> Artificial Sequence <220> <223> K1-RhFR-D6 <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 gcaacccga 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 tgctgggaacg 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> Ms <213> Artificial Sequence <220> <223> K1-RhFR-I14 <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 gccgccccggg 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 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> D3-R <400> 34 30. gaccggttga tgccgtgctg aacgcggacc <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> D6-F <400> 35 ggtccgcgtt cagcaccggt caccatcgga <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> D6-R <400> 36 tccgatggtg accggtgctg aacgcggacc <210> 37 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> I14-F <400> 37 atgccggtgc agcacatttt ctgcttttttt acgaactttt tttgcactct gcagttctgc tgaacgcgga 72 cc <210> 38 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> I14-R <400> 38 ggtccgcgtt cagcagaact gcagagtgca aaaaaagttc gtaaaaaagc agaaaatgtg 60 ctgcaccggc and 72

Claims

1. A fusion protein, characterized in that The fusion protein is K1 and RhFR from N-terminus to C-terminus; the amino acid sequence of K1 is shown in SEQ ID NO: 1, and the amino acid sequence of RhFR is shown in amino acids 466-773 of SEQ ID NO: 5; The K1 and RhFR are connected via a linker, and the amino acid sequence of the linker is shown in the amino acids 445-465 of SEQ ID NO: 5; the fusion protein is an insertion of 3 or 6 amino acids at the N-terminus of the linker, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 11 or SEQ ID NO:

13.

2. The fusion protein according to claim 1, characterized in that The fusion protein is co-expressed with a molecular chaperone.

3. The fusion protein according to claim 2, characterized in that The molecular chaperone is Gro7.

4. The fusion protein according to claim 1, characterized in that The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 12 or SEQ ID NO:

14.

5. A fusion gene, characterized in that: It encodes the fusion protein according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the fusion gene as claimed in claim 5.

7. The recombinant expression vector according to claim 6, characterized in that The backbone vector of the recombinant expression vector is pET28a.

8. A transformant, characterized in that It comprises the fusion gene as claimed in claim 5 or the recombinant expression vector as claimed in claim 6 or 7; The transformant is obtained by introducing the fusion gene or the recombinant expression vector into a host, and the host is Escherichia coli.

9. The transformant according to claim 8, characterized in that The host is Escherichia coli E. coli BL21(DE3) cells.

10. A method for preparing a fusion protein, characterized in that: It includes the following steps: (1) obtaining the transformant according to claim 8 or 9; (2) Screening the transformants, expressing and purifying the fusion protein.

11. A method for preparing calcifediol, characterized in that: The preparation method comprises the following steps: in the presence of a reaction solvent and reduced coenzyme NADH / NADPH, catalyzing a hydroxylation reaction of vitamin D3 with the fusion protein according to any one of claims 1 to 4.

12. The preparation method according to claim 11, characterized in that: The vitamin D3 is vitamin D3 pre-dissolved in a solvent; And / or, the preparation method further comprises the step of adding cyclodextrin to the reaction solvent before carrying out the hydroxylation reaction; And / or, the reaction temperature is 20-33°C; And / or, the pH of the reaction is 6.0-8.0; And / or, the concentration of vitamin D3 is 1g / L-10g / L; And / or, the molar ratio of the NADH / NADPH to the vitamin D3 is 0.001:1~2:

1.

13. The preparation method according to claim 12, characterized in that The cosolvent includes one or more of DMSO, Tween 80, Triton X100, methanol, ethanol, isopropanol and DMF; And / or, the cyclodextrin is hydroxypropyl-β-cyclodextrin; and / or, the reaction temperature is 22°C, 25°C, 28°C or 30°C; and / or, the pH of the reaction is 7.4; And / or, the concentration of vitamin D3 is 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L or 9g / L; And / or, the molar ratio of the NADH / NADPH to the vitamin D3 is 0.2:

1.

14. The preparation method according to claim 13, characterized in that: The cosolvent is ethanol; And / or, the mass volume percentage of the hydroxypropyl-β-cyclodextrin in the reaction system is 0.05%-0.4%.

15. The preparation method according to claim 14, characterized in that: The mass volume percentage of the hydroxypropyl-β-cyclodextrin in the reaction system is 0.25%.

16. The preparation method according to claim 11, characterized in that: The preparation method further comprises the following steps: in the presence of a dehydrogenase and a hydrogen donor, oxidized coenzyme NAD + / NADP + Carry out reduction reaction to obtain the NADH / NADPH.

17. The preparation method according to claim 16, characterized in that: The dehydrogenase is glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase; and / or, the hydrogen donor is glucose, isopropanol or formate.

18. The preparation method according to claim 17, characterized in that: 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.

19. Use of the fusion protein according to any one of claims 1 to 4, the fusion gene according to claim 5, the recombinant expression vector according to claim 6 or 7, or the transformant according to claim 8 or 9 in the preparation of calcifediol.

Images