Patent Application
20190048044
The present invention relates to a method for preparation of four kinds of 2′, 3′-cNMPs (2′, 3′-cAMP, 2′, 3′-cGMP, 2′, 3′-cCMP and 2′, 3′-cUMP) simultaneously by a recombinant protein expressed in Escherichia coli. It belongs to biotechnological field.
Cyclic nucleotides are important second messengers in living cells, involving in a range of intracellular physiological pathways. Earl. W. Sutheland first proposed that 3′, 5′-cAMP is a second messenger in human body in 1965. Thereafter, 3′, 5′-cGMP, 3′, 5′-cCMP, 3′, 5′-cUMP, c-di-GMP, c-di-AMP and cGAMP were successively found in various biological cells to involve in the regulation of many intracellular signaling pathways as second messengers. Except 3′, 5′-cNMPs, 2′, 3′-cNMPs are also found in mammals, plants and bacteria. Compared to 3′, 5′-cNMPs, studies on the physiological roles of 2′, 3′-cNMPs are still limited. It was found that 2′, 3′-cNMPs may be involved in tissue and cells damage. In damaged tissue or cells, 2′, 3′-cNMPs increase significantly. It was also found that 2′, 3′-cAMP can induce apoptosis by activate the mitochondrial permeability transition pores. In addition, the phosphorylation level of 2′, 3′-cyclic nucleotide-3′ phosphodiesterase increased in brain cells during HIV infection. These findings on 2′, 3′-cNMPs are arousing more and more interests of researchers.
As 2′, 3′-cNMPs attract more and more attention, there will be an increasing demand of 2′, 3′-cNMPs in scientific research and pharmaceutical application. Now, all available 2′, 3′-cNMPs are chemically synthesized. However, chemical synthesis have disadvantages such as low yield and high price, which limit the application of 2′, 3′-cNMPs to some extent.
To overcome the limitation of current technology, here a method is provided to prepare four kinds of 2′, 3′-cNMPs simultaneously by biotechnology. In this method, four kinds of 2′, 3′-cNMPs can be denovo synthesized by the growth of recombinant Escherichia coli, without the need of a specific precursor, and can be prepared rapidly by purifying the recombinant protein IF3 from recombinant E. coli.
In this invention, preparation of 2′, 3′-cNMPs includes the following steps:
The preferred PCR amplification system (50 μL) in step (1) of the present invention is as follows:
PCR reaction conditions in step 1 are as follows:
Pre-denature at 95° C. for 5 min; denature at 95° C. for 30 sec; anneal at 55° C. for 30 sec; extend at 72° C. for 30 sec; 30 cycles; finally extend at 72° C. for 5 min.
The specific restriction enzymes used for DNA digestion in step (2) are Nde I and Xho I.
The preferred digestion reaction system in step (2) is as follows:
Reaction condition: Incubation at 37° C. for 30 min.
ligation reaction system in step (2) is as follows:
Reaction condition: Incubation at 16° C. overnight
The preferred procedure for strain cultivation in step (2) is as below:
Cultivate the recombinant strain at 35-38° C. and 150-200 rpm until the OD600 reaches 0.8; then incubate the culture at 18-22° C. and 100-140 rpm for 30 min; add 0.1 mM isopropul-β-D-thiogalactopyranoside to the culture as an inducer and continue the cultivation at 18-22° C. and 100-140 rpm for 22-25 hours.
The preferred culture medium in step (2) is Luria-Bertani (LB) medium (one liter medium contains 10 g NaCl, 10 g peptone and 5 g yeast extract dissolved in distilled water, pH 8.0).
The preferred cell disruption procedure in step (3) is as follows:
Cells are collected from the fermentation broth and suspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). Then the cells are broken under pressure of 950˜1050 bar. After centrifugation at 12,000 rpm for 50 min, the supernatant containing the recombinant protein is collected.
The preferred purification procedure for recombinant protein IF3 by Ni2+-nitrilotriacetic acid resin in step (3) is as follows:
Load the supernatant from step (3) on a nickel column. After the supernatant flowed through the nickel column, equilibrate the column with 20 ml lysis buffer. Wash the column with 20 ml washing buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8.0). Elute the column with 10 ml elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0). Collect the eluent containing recombinant protein IF3.
The preferred molecular weight cutoff for ultrafiltration in step (4) is 3000 Da.
The preferred protocol for sample separation in step (4) is as follows (the values are times in minutes and percentage of buffer B used): 0.0, 0; 2.5, 0; 5.0, 30; 10.0, 60; 14.0, 100; 21.0, 100; 22.0, 50 and 23.0, 0 at a flow rate of 10 ml/min. The detection wavelength is 254 nm.
The preferred mobile phases in step (4) used in the gradient program are as follows: buffer A (10 mM ammonium acetate, pH 5.0) and buffer B (75% (v/v) buffer A, 25% (v/v) methanol).
Beneficial Effects
1. The present invention offers a method for the preparation of four kinds of 2′, 3′-cNMPs simultaneously from recombinant E. coli. Four kinds of 2′, 3′-cNMPs can be denovo synthesized by bacterial fermentation without the need of a specific precursor. This method is simple, convenient and has less contamination compared to chemical synthesis. Five milligram 2′, 3′-cNMPs can be obtained from 1 L fermentation broth.
2. In the present invention, E. coli fermentation is used to produce 2′, 3′-cNMPs. E. coli has a known genetic background and a fast growing speed, which is beneficial for further increasing the yield of 2′, 3′-cNMPs via bioengineering.
The following comprises a list of illustrative embodiments according to the present invention which represent various embodiments of the present invention. These examples are offered to illustrate, but not to limit the present invention. E. coli K12 was purchased from China Center of Industrial Culture Collection (CICC) in these examples. The address is: the 6th building, 24th courtyard of the Jiuxianqiao middle road, Chaoyang district, Beijing, China. The accession number is CICC 10424.
E. coli BL21 (DE3) competent cell in these examples was purchased from Beijing TransGen Biotech Co., Ltd. The address is: the 4th floor of the B-3 building, Dongsheng Science Park of Zhongguancun, 66th of Xixiaokou Road, Haidian District, Beijing, China.
The method to construct the recombinant strain includes the following steps:
1. Cloning Gene If3 from E. coli K12.
1.1 Extraction of Genomic DNA from E. coli K12
The genomic DNA was extracted according to the instructions of genome extraction kit from Bioteke Corporation, China.
1.2 Design and Synthesis of the Primers
Primers were designed according to the If3 gene sequence. The primer sequences were as follows:
Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd, China.
1.3 Amplification of Gene If3 by PCR and its Recovery
(1) Gene If3 was amplified with primers F and R, using the genomic DNA as a template. Conditions for PCR reaction were as follows: pre-denaturing at 95° C. for 5 min; denaturing at 95° C. for 30 sec; annealing at 55° C. for 30 sec; extending at 72° C. for 30 sec; 30 cycles; finally extending at 72° C. for 5 min.
The PCR amplification system (50 μl) was as follows:
(2) The PCR products were separated by 1% agarose gel electrophoresis. The DNA fragment of gene If3 was recovered from the gel with DNA purification kit from Omega.
2. Construction of the Recombinant Expression Vector and Recombinant Expression Strain.
(1) Digestion of the Gene and Expression Vector
The If3 gene obtained from step 1 and vector pET-22b were digested with restriction enzymes Nde I and Xho I. The reaction system for digestion was as follows:
The sample was mixed smoothly, centrifuged for 2 sec and then incubated at 37° C. for 30 min. The digested products were separated by 1% agarose gel electrophoresis, and the target DNA segments of gene If3 and vector pET-22b were recovered from the gel using DNA purification kit from Omega.
(2) Ligation of Gene If3 and Vector pET-22b to Construct the Recombinant pET-22b-If3 Vector
The reaction system was as follows:
The sample was mixed smoothly, centrifuged for 2 sec and incubated at 16° C. overnight. Then the recombinant pET-22b-If3 vector was constructed.
(4) The recombinant pET-22b-If3 vector was transformed into E. coli BL21 (DE3) according to the method described in Molecular Cloning Manual, and transformed E. coli cells were obtained.
(5) The transformed E. coli cells were spread on LB agar plate containing 100 μg/ml ampicilin and cultivated at 37° C. overnight.
(6) The recombinant strain was screened from the plate and verified by recombinant plasmid extraction and sequencing in Sangon Biotech (Shanghai) Co., Ltd.
1. Fermentation of the Recombinant Strain
(1) Cultivation of Inoculum
The recombinant E. coli strain was inoculated in liquid LB medium containing 100 μg/ml ampicillin and incubated at 180 rpm and 37° C. overnight to obtain inoculum.
(2) The inoculum was inoculated in 1 L fermentation medium with 1% inoculum size and cultivated at 180 rpm and 35-38° C. until the OD600 reached 0.8. Then, 0.1 mM isopropul-β-D-thiogalactopyranoside (IPTG) was added in the culture, which was further cultivated at 120 rpm and 20° C. for 24 hours.
2. Purification of Recombinant Protein IF3
(1) Buffers used for Purification
Lysis buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 8.0
Washing buffer: 50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8.0
Elution buffer: 50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0
(2) The cells of the fermented recombinant E. coli strain from step (1) were collected by centrifugation at 10,000 rpm, resuspended in 50 ml lysis buffer (for 1 liter broth), and broken at the pressure of 1,000 bar.
(3) The solution from step (2) was centrifuged at 12,000 rpm and 4° C. for 50 min.
(4) The supernatant from step (3) was loaded on a nickel column containing 2 ml nickel gel. After the supernatant flowed through the nickel column, equilibrate the column with 20 ml lysis buffer. Wash the column with washing buffer 20 ml, and elute the recombinant protein IF3 from the column with 10 ml elution buffer. Collect the eluent, which was the recombinant protein IF3 solution.
1. Extraction of 2′, 3′-cNMPs
(1) The recombinant protein IF3 solution obtained in example 2 was incubated at 0° C. for 3 days to release 2′, 3′-cNMPs from IF3, then centrifuged at 12,000 rpm for 20 min, and the supernatant was collected.
(2) The supernatant obtained from step (1) was ultrafiltrated by using an ultrafiltration tube with a molecular weight cut-off of 3,000 Da to remove proteins. The filtrate containing four kinds of 2′, 3′-cNMPs was collected.
2. Purification of Four Kinds of 2′, 3′-cNMPs (2′, 3′-cAMP, 2′, 3′-cGMP, 2′, 3′-cCMP and 2′, 3′-cUMP).
(1) Four kinds of 2′, 3′-cNMPs in the filtrate were separated by high-performance liquid chromatographic (HPLC) on a C18 reversed-phase column and collected separately.
The mobile phases used in the gradient program were as follows: buffer A (10 mM ammonium acetate, pH 5.0) and buffer B (75% (v/v) buffer A, 25% (v/v) methanol).
(2) The protocol used for purification was as follows (the values are times in minutes and percentage of buffer B used): 0.0, 0; 2.5, 0; 5.0, 30; 10.0, 60; 14.0, 100; 21.0, 100; 22.0, 50 and 23.0, 0 at a flow rate of 10 ml/min. The detection wavelength was 254 nm.
The result of HPLC separation of four kinds of 2′, 3′-cNMPs was shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610112458.5 | Feb 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/103058 | 10/24/2016 | WO | 00 |
