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Small molecular protein for efficiently mediating recombinant polypeptide to form inclusion body

A technology for small molecular proteins and recombinant polypeptides, applied in the field of bioengineering, can solve problems such as cumbersome operations, and achieve the effect of simplifying the separation and purification process, with significant effects, large market value and application prospects.

Active Publication Date: 2021-06-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that an acidic environment or an alkaline environment can effectively promote the dissolution of inclusion bodies. The reported classical inclusion body dissolution methods include guanidine hydrochloride dissolution, Tris-HCl solution combined with organic solvent dissolution, urea dissolution, and strong anion washing. Reagent SDS, strong pH environment dissolution, and the use of physical methods to help dissolve, follow-up needs to combine affinity chromatography purification, metal-catalyzed reaction, desalination, solution concentration dilution, etc., the operation is cumbersome, and it is difficult to have a suitable enzymatic digestion environment to remove the fusion It is one of the current research hotspots and difficulties to explore the appropriate reaction conditions for enzymatic digestion of inclusion bodies and simplify the operation of protein separation and purification.

Method used

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  • Small molecular protein for efficiently mediating recombinant polypeptide to form inclusion body
  • Small molecular protein for efficiently mediating recombinant polypeptide to form inclusion body
  • Small molecular protein for efficiently mediating recombinant polypeptide to form inclusion body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The construction of embodiment 1 pET28b-Am-Metch expression vector

[0046] (1) In vitro synthesis of genes encoding small molecular proteins (Am) that efficiently mediate the formation of inclusion bodies by recombinant polypeptides

[0047] According to the hydrophobic and hydrophilic properties of different amino acids and their tendency to form β sheets, the C-terminal sequence (100-161 amino acids) of Escherichia coli palmitoylphospholipid transferase (PagP) was optimized and transformed (PagP-100), and the design was easy The fusion tag (Am) of the inclusion body was formed, and the nucleotide sequence of the gene encoding Am was designed according to the codon bias of E. coli. Wherein, the nucleotide sequence of the gene encoding Am was synthesized by Beijing Ruibo Biotechnology Co., Ltd. Guangzhou Branch.

[0048] Nucleotide sequence of PagP-100:

[0049] AATTTTCATTTAGGTCTGGGATTCACCGCTGGCGTAACGGCACGCGATAACTGGAATTACATCCCTCTCCCGGTTCTACTGCCATTGGCCTCCGTGGGTTATGGCC...

Embodiment 2

[0075] Example 2 Induced expression of recombinant polypeptides in the form of inclusion bodies in Escherichia coli

[0076] The pET28b-Am-Metch expression vector constructed above and the control pET28b-PagP-100-Metch expression vector (PagP-100 replacing Am) were transformed into Escherichia coli BL21 (DE3) competent cells, and a single clone was picked and inoculated into liquid LB Cultivate overnight in the culture medium, expand the culture to 0D at a volume ratio of 1:100 600 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.6mM, and induce protein expression at 37°C for 24h. The bacterial cells were collected by centrifugation under the conditions of 5000 g, 10 min, and 4° C. to obtain the bacterial cells expressing the recombinant polypeptide.

[0077] Detect the expression of the recombinant polypeptide by 18% SDS-PAGE, the results are as follows figure 2 As shown, the expression level of Am-Metch was significantly better than th...

Embodiment 3

[0080] Example 3 Dissolution of inclusion bodies and separation and purification of target polypeptide Metch

[0081] (1) Take 1×Lysis Buffer (50mmol NaH 2 PO 4 , 300mmol NaCl, 20mmol imidazole, pH8.0) Suspend the bacterium expressing the recombinant polypeptide collected in Example 2, sonicate it, and centrifuge at 5000g, 30min, 4°C to obtain the inclusion body expressing the recombinant polypeptide ;

[0082] (2) Mix the inclusion body with 100 or 200 mmol acetic acid solution at a ratio of 10 mg: 1 ml to achieve dissolution of the inclusion body. SDS-PAGE was used to detect the dissolution of inclusion bodies in the dissolved samples, such as Figure 5 As shown, the Am-Metch inclusion body dissolution effect is good after the 200mmol acetic acid solution is dissolved.

[0083] (3) Add Tween-20 at 2% volume percentage to the dissolved sample, shake at 37°C for 2h to further promote dissolution; then use NaOH to adjust the pH to 4.5, add TEV enzyme at 10% volume percentag...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a small molecular protein for efficiently mediating recombinant polypeptide to form an inclusion body. The amino acid sequence of the micromolecular protein for efficiently mediating the recombinant polypeptide to form the inclusion body is as shown in SEQ ID NO: 1. The micromolecular protein can be used as a fusion tag to improve the inclusion body expression level of recombinant polypeptide, has a remarkable effect in the aspect of mediating the expression of antibacterial polypeptide and the like, and has relatively high market value and application prospect. The invention further provides a method for separating and purifying the inclusion body, the method is very simple and convenient to operate, the biological activity of the target polypeptide is not influenced, and the technology can be widely applied to industrial production, purification and scientific research work of the polypeptide.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a small molecular protein that efficiently mediates the formation of inclusion bodies of recombinant polypeptides. Background technique [0002] Escherichia coli expression system is currently the most widely used recombinant protein expression system. The culture period of this system is short, the culture conditions are easy to meet, and the ability of different gene expression products to form inclusion bodies is different. Inclusion bodies (IBs) are protein aggregates with non-natural conformation. The presence of recombinant proteins in the form of inclusion bodies has many advantages, such as the stability of recombinant proteins, enhanced resistance to protease degradation, simplified subsequent extraction and purification processes, and easy expression of toxic proteins, etc. . Some short peptides are easily degraded by proteases during recombinant expression. Us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/70C12N15/62C12N1/21C12P21/02C12P21/06C07K14/435C12R1/19
CPCC07K14/001C12N15/70C12P21/06C07K14/43581C07K2319/35C07K2319/50C12N2800/22
Inventor 王声斌李雪凤徐蔚腾王媛郑玉娇
Owner SOUTH CHINA AGRI UNIV
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