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Fusion protein or variant thereof and application of fusion protein or variant thereof in preparation of calcifediol

A fusion protein and variant technology, applied in the fusion protein or its variants to catalyze VD3 to prepare calcifediol, the fusion protein or its variants and its gene, and its preparation field, which can solve the problems of cumbersome operation, high cost and high production cost. Low efficiency and other problems, to achieve the effect of high catalytic efficiency, easy operation, and cost reduction

Pending Publication Date: 2021-11-02
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a fusion protein or its variant and its Gene, its preparation method and its application to catalyze VD3 to prepare calcifediol

Method used

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  • Fusion protein or variant thereof and application of fusion protein or variant thereof in preparation of calcifediol
  • Fusion protein or variant thereof and application of fusion protein or variant thereof in preparation of calcifediol
  • Fusion protein or variant thereof and application of fusion protein or variant thereof in preparation of calcifediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of Strains Protease Fusion

[0046] 1.1 protein sequence analysis

[0047] The present embodiment is capable of catalyzing VD3 P450 enzymes Vdh-K1 P450 BM3, and with the reduction zone P450 RhFRed fusion construct self-consistent model P450 fusion proteins, the following steps:

[0048] The NCBI reported encoded cytochrome P450 enzyme SEQ ID NO: gene sequence of SEQ 1,3,5 IDNO: 2,4,6 total gene synthesis of each gene. Gene synthesis companies for gold intellectualism Biotechnology Co., Ltd. Suzhou (Suzhou Industrial Park, Star Lake Street, 218 C3 BioBay floor). Each gene information as shown in Table 1.

[0049] Table 1

[0050]

[0051] Analysis of the amino acid sequence of the protein BM3 software using Discovery studio, find the first 1-459 amino acids of the protein N-terminal domain of P450; P450 to amino acids 460-479 of domain Linker electron transfer domain; a first position 480-1048 electron transfer protein amino acid domain.

[0052] Ana...

Embodiment 2

[0063] Preparation Example 2 Protease Fusion

[0064] The constructed good BL21-pET28a-K1-Gro7 in Example 1, BL21-pET28a-FdR-Gro7, BL21-pET28a-Fdx and fusion proteins engineered bacteria engineered strain BL21-pET28a-K1-RhFR, BL21-pET28a-K1 -RhFR-Gro7, BL21-pET28a-K1-BM3R, BL21-pET28a-K1-BM3R-Gro7 single colony was inoculated into 5ml LB broth containing 50μg / ml kanamycin and 50μg / ml chloramphenicol, 37 [deg.] C shaking culture 12h. When divert Press 2v / v% inoculum into fresh LB liquid medium likewise containing 50μg / ml kanamycin and 50μg / ml chloramphenicol in a 50ml, 37 [deg.] C shaking until OD600 reached about 0.8, IPTG was added to a final concentration of 0.5mM, 22 ℃ induced culture 22h. After incubation, the culture was centrifuged 10000rpm 10min, the supernatant was discarded, cells were collected (i.e. bacterial sludge), placed in -20 ℃ stored in a refrigerator and set aside.

[0065] Each bacteria were fusion proteins with 100mM PBS7.4 mud to 1: homogeneous homo...

Embodiment 3

[0066] Example 3 Determination of P450 activity of the fusion protein

[0067] K1 P450 protein concentration determination using a fusion CO difference spectroscopy.

[0068] Determination Methods: A sample to be tested (i.e. the fusion protein of Example 2 a crude enzyme solution Embodiment) 2 in 1 mL 10mL centrifuge tubes, labeled control tube, sample tube. The sample to take the hood, means to take a suitable amount of water tubes, into the water conduit of CO, the three-way valve to the outlet velocity of about 1 second a CO bubble. Control and sample tube were added 1mg of sodium dithionite powder, repeatedly reversed as sodium dithionite dissolved completely and mixed well. Care will be respectively transferred to a sample tube and liquid cuvettes, scanning the absorbance in the 400-500nm UV spectrophotometer.

[0069] The enzyme concentration is calculated:

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

[0071] in:

[0072] C P450 , Concentration in the test sample P450 enz...

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Abstract

The invention provides a fusion protein or a variant thereof, the fusion protein comprises K1 and RhFR, the amino acid sequence of the K1 is as shown in SEQ ID NO: 1, and the amino acid sequence of the RhFR is as shown in amino acids from the 466th site to the 773rd site of SEQ ID NO:5. The invention also provides a preparation method and application of the fusion protein or the variant thereof. When the fusion protein or the variant of the fusion protein is applied to catalysis of VD3 to synthesize 25-hydroxyvitamin D3 (calcifediol), no extra electron transfer related protein needs to be added, the operation is simple and convenient, the electron transfer efficiency in the fusion protein is high, the catalytic efficiency is high, the yield of the obtained calcifediol is remarkably improved, the production cost is reduced, and the fusion protein or the variant of the fusion protein is suitable for industrial production.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a fusion protein or variants thereof, and genes, methods for their preparation, as well as fusion proteins or variants thereof in the preparation of the catalytic VD3 of calcitriol. Background technique [0002] 25-hydroxy vitamin D3, also known as calcitriol, vitamin D3 (VD3) active metabolite, has a strong physiological activity. The traditional method of synthesis of 25-hydroxyvitamin D3 chemical synthesis, the need for protection and deprotection of the group of multi-step, and then the reaction by light, ring opening and isomerization of the 25-hydroxy vitamin D3. In recent years the study of microbial transformation of VD3 has been rapid development, but one of the key conversion rate is low, and increase the activity of VD3 production is screened to obtain a high conversion efficiency of key metabolic enzyme cytochrome P450 enzymes. [0003] Cytochrome P450 enzyme widely ex...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P7/02C12R1/19
CPCC12N9/0081C12N15/70C12P7/02C07K2319/00
Inventor 吴燕田振华
Owner ABIOCHEM BIOTECH CO LTD
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