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High-throughput screening method of signal peptide library based on fluorescent probe Rho-IDA-CoII

A technology of rho-ida-coii and fluorescent probes, applied in the field of high-throughput screening, to achieve the effect of ensuring diversity and avoiding loss

Pending Publication Date: 2022-03-25
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no example of secreted expression of active nitrile hydratase disclosed in any invention
At the same time, there is no specific method for fluorescent high-throughput screening of protein secretion and expression signal peptide libraries disclosed in any invention

Method used

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  • High-throughput screening method of signal peptide library based on fluorescent probe Rho-IDA-CoII
  • High-throughput screening method of signal peptide library based on fluorescent probe Rho-IDA-CoII
  • High-throughput screening method of signal peptide library based on fluorescent probe Rho-IDA-CoII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation and plasmid transformation of Bacillus subtilis WB800 competent cells

[0045] 1) Streak the strain of Bacillus subtilis WB800 frozen at -80 °C on an antibiotic-free LB plate, and incubate at a constant temperature of 37 °C for 2 days to activate the strain;

[0046] 2) Pick a single colony and inoculate it in 5 mL of GMI solution, shake it at 30 °C and 100 rpm overnight;

[0047] 3) Transfer 2 mL of the overnight culture to 18 mL of GMI solution, incubate at 37˚C, 200 rpm for 3.5 h (determine OD every 30 min 600 , draw the curve of OD value versus time, culture to late logarithmic period);

[0048] 4) Take 10 mL of Bacillus subtilis culture medium in the late logarithmic period and transfer it into 90 mL of GM II solution, incubate at 37 °C and 100 rpm for 90 min, and then collect the cells (cultivate to the late logarithmic period);

[0049] 5) Use 2 mL of GM II solution (containing 10% glycerol) to suspend the bacteria to prepare Bacillus subti...

Embodiment 2

[0053] Example 2: Construction of a signal peptide library for secretion and expression of nitrile hydratase

[0054] 1) According to the instructions of Axygen's plasmid mini-extraction kit, from recombinant Escherichia coli Escherichia coli The recombinant plasmid pBE-S-HBA was extracted from DH5α-pBE-S-HBA. The nucleotide sequence of wild-type nitrile hydratase HBA is shown in SEQ ID NO. 2.

[0055] 2) Design PCR vector linearization primers 5'-CGCGTCCCTCTCCCTTTTGCTTAAGTTCAGAGTAG-3' (SEQ ID NO. 3) and 5'- GGCCGGTGCACATATGAAGGACAACAACAAAGTT-3' (SEQ ID NO. 4) Using the plasmid pBE-S as a template, pre-denature at 95°C for 3 minutes according to the program; 30 cycles (denaturation at 95°C for 15 s, annealing at 68°C for 15 s, extension at 72°C for 3 min); final extension at 72°C for 5 min for PCR amplification Preparation of linearized plasmid pBE-S-HBA containing specific ends L ;

[0056] 3) According to the instructions of the ClonExpress Ultra One Step Cloning Kit kit...

Embodiment 3

[0061] Embodiment 3: the chemical synthesis of fluorescent probe Rho-IDA

[0062] 1) In a 50 mL reaction flask equipped with a magnetic stirrer, add 0.1 mmol of rhodamine B and 10 mL of dichloromethane, and fully dissolve.

[0063] 2) Slowly add 0.15 mmol N-hydroxysuccinimide and 0.15 mmol N, N'-dicyclohexylcarbodiimide, react at room temperature overnight, and filter to remove the precipitate.

[0064] 3) Slowly add 0.2 mmol of triethylamine and mix well.

[0065] 4) Add 0.15 mmol of iminodiacetic acid and react overnight at room temperature.

[0066] 5) Evaporate the solvent under reduced pressure, dissolve the residue with 30 mL of dichloromethane, continue to add 30 mL of saturated saline for extraction, and separate the dichloromethane layer.

[0067] 6) Filter to remove impurities, use silica gel column chromatography, the eluent is petroleum ether:ethyl acetate=5:1, and obtain rose bengal powder with a total yield of 64%.

[0068] The synthetic product obtained above...

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Abstract

The invention discloses a high-throughput screening method of a signal peptide library based on a fluorescent probe Rho-IDA-CoII. Compared with a traditional screening method, the method has the advantages that the histidine tag fusion protein in the range of 50-500 ng can be accurately measured by using a fluorescent probe labeling quantitative method, high background fluorescence caused by complex samples and environmental pollution is avoided, and the application range is wide. The method comprises the following steps: screening bacillus subtilis recombinants capable of secreting and expressing active nitrile hydratase from a bacillus subtilis expression nitrile hydratase signal peptide library by virtue of a fluorescent high-throughput screening method, and detecting that the nitrile hydratase activity is 12.97 + / -0.77 U / mL in culture supernate of the bacillus subtilis recombinants. The recombinant capable of secreting and expressing the nitrile hydratase can be applied to industrial production of the nitrile hydratase and downstream application of the nitrile hydratase.

Description

(1) Technical field [0001] The invention relates to a high-throughput screening method of a signal peptide library based on a fluorescent probe Rho-IDA-CoII. (2) Background technology [0002] Nitrile hydratase is a metalloenzyme that biocatalyzes the conversion of organic nitriles to the corresponding amides. Nitrile hydratase has the characteristics of mild reaction conditions, high yield, and environmental friendliness, and has been successfully used in the production of amide chemicals. Although many nitrile hydratase-producing strains have been used for the industrial production of amide compounds, during the production process, the amide compounds will be hydrolyzed by amidases in the cells of the strains to generate the corresponding carboxylic acid by-products. Therefore, the use of heterologous hosts to express recombinant nitrile hydratase can eliminate by-products produced by homologous amidase and nitrilase hydrolysis. At the same time, in order to further impr...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/10C12N9/88C07K14/245G01N21/64
CPCC12N15/75C12N15/1086C12N9/88C12Y402/01084C07K14/245G01N21/64G01N2021/6419C12Q2531/113
Inventor 柳志强沈骥冬蔡雪金利群郑裕国
Owner ZHEJIANG UNIV OF TECH
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