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A novel fusion protein nscr5 and its preparation method

A technology of fusion protein and protein, applied in the field of biopolymer research, can solve the problems of few application research reports and so on

Inactive Publication Date: 2019-04-23
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long peptides and proteins cannot be obtained by chemical methods, so in the case of limited access methods, there are few reports on the biomedical applications of long peptides and proteins

Method used

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  • A novel fusion protein nscr5 and its preparation method
  • A novel fusion protein nscr5 and its preparation method
  • A novel fusion protein nscr5 and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Step 1: Entrust the NSCR5 expression cassette sequence to Biotechnology Co., Ltd. for whole gene synthesis ( figure 2 , SEQ NO:2), and inserted into the EcoRⅤ site on PUC57 by restriction enzyme ligation method, and then introduced into Escherichia coli DH5α strain by chemical method or electric shock method, and was screened by carbenicillin-resistant medium and verified by DNA sequencing to obtain Fusion protein NSCR5 expression strain.

[0038] The restriction enzyme ligation method is as follows: digest the PUC57 plasmid with EcoRⅤ, use agarose gel electrophoresis to separate the linear fragment of PUC57 digested by EcoRⅤ, and then recover the linearized PUC57 plasmid with an agarose gel recovery kit. The artificially synthesized NSCR5 expression frame sequence was amplified with high-fidelity DNA polymerase, the NSCR5 expression frame fragment was also separated by agarose gel electrophoresis, and the NSCR5 expression frame fragment was recovered with an agarose ge...

Embodiment 2

[0047] Step 1: Refer to the method of Step 1 in Example 1 to obtain a strain expressing fusion protein NSCR5.

[0048] Step 2: Inoculate the fusion protein NSCR5 expressing strain clone into 200mL self-induction medium, culture at 40°C for 24h at a shaking speed of 220 rpm, and then centrifuge at 6000×g for 5min at room temperature to collect the bacteria. The self-induction medium formula is: yeast extract 5.0g / L, tryptone 10.0g / L, casein peptone 15.0g / L, glucose 1.5g / L, dipotassium hydrogen phosphate 10.0g / L, potassium dihydrogen phosphate 9.0g / L, ammonium phosphate 6.0g / L, magnesium sulfate 1.6g / L, calcium chloride 5.0mg / L, cobalt chloride 3.5mg / L, copper chloride 1.5mg / L, manganese sulfate 5.0mg / L, Sodium molybdate 8.0mg / L, boric acid 0.5mg / L, ferric chloride 8.0mg / L, zinc chloride 3.0mg / L, glycerin 10.0g / L, serine 2.0g / L, glycine 10.0g / L, tyrosine Acid 1.0g / L, isopropylthio-β-D-galactoside (IPTG) 300mg / L.

[0049] Step 3: The cells collected by centrifugation in step 2 ...

Embodiment 3

[0054] Step 1: Refer to the method of Step 1 in Example 1 to obtain a strain expressing fusion protein NSCR5.

[0055] Step 2: Inoculate the fusion protein NSCR5 expressing strain clone into 200mL self-induction medium, culture at 28°C for 18h at a shaking speed of 220 rpm, and then centrifuge at 6000×g for 5min at room temperature to collect the bacteria. The self-induction medium formula is: yeast extract 3.0g / L, tryptone 7.5g / L, casein peptone 10.0g / L, glucose 0.75g / L, dipotassium hydrogen phosphate 7.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium phosphate 4.0g / L, magnesium sulfate 1.0g / L, calcium chloride 3.5mg / L, cobalt chloride 2.0mg / L, copper chloride 1.0mg / L, manganese sulfate 3.1mg / L, Sodium molybdate 5.6mg / L, boric acid 0.3mg / L, ferric chloride 4.5mg / L, zinc chloride 1.7mg / L, glycerin 5.0g / L, serine 1.25g / L, glycine 6.0g / L, tyrosine Acid 0.5g / L, isopropylthio-β-D-galactoside (IPTG) 160mg / L.

[0056] Step 3: The cells collected by centrifugation in step 2 we...

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Abstract

The invention provides novel fusion protein NSCR5 and a preparation method thereof. The novel fusion protein NSCR5 is characterized in that the fusion protein NSCR5 is formed by sequential connection of four sections including 6 poly histidine labels (6X His), a spider silk protein amino terminal sequence (NT), drosophila Resilin, a spider silk protein carbon terminal sequence (CT) and diatom silicon deposition peptide (R5) and the like; the novel fusion protein NSCR5 is as long as 602 amino acid residues and is obtained by a whole-genome synthesis method, and codon compositions are optimized. The fusion protein NSCR5 can be used for producing 70-1200mg / L of fermentation liquor with purity ranging from 82-95%. The novel fusion protein NSCR5 has photocatalysis crosslinking, highly elastic, self-assembling and silicon depositing characteristics and has a broad application prospect in the biomedical field.

Description

technical field [0001] The invention relates to the field of biopolymer research, and mainly relates to a novel fusion protein NSCR5 and a preparation method thereof. Background technique [0002] Polypeptides (or proteins) are new carrier materials with superior performance, and have achieved good research results in the targeted delivery of drugs, especially the targeted delivery and fixed-point release of anti-tumor drugs. Due to the short polypeptide molecular chain and weak single-molecule drug-loading capacity, existing polypeptide drug-loading is achieved through multi-stage assembly of polypeptides to form larger drug-loading complexes. Long peptides or proteins have longer molecular chains than polypeptides, have higher single-molecule drug loading capacity than polypeptides, and have more abundant and varied assembly forms, and their material properties are superior. However, long peptides and proteins cannot be obtained by chemical methods, so there are few repor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
Inventor 柳永汤江武王新孙宏姚晓红吴逸飞
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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