Application of Lpp or mutant thereof as molecular chaperone to secretory expression of recombinant protein in escherichia coli

A technology of Escherichia coli, secreted expression, applied in the field of biomedicine, which can solve the problems of low proportion of target protein, increased probability of miscutting, and more impurities, and achieve the effect of high proportion, increased resistance, and increased production

Pending Publication Date: 2022-07-01
ZHUHAI UNITED LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the effect on the growth of E. coli is small, the molecular weight is relatively large (mostly above 20KDa), and the proportion of the target protein in the fusion protein is low, resulting in low yield; and the molecular chaperone with large molecular weight has a longer sequence, which is difficult to digest when separating the target gene. The increased probability of miscutting will generate more impurities; in addition, longer sequences will also form more complex spatial structures, which are easy to covalently bind to the target protein, block the enzyme cleavage site, and increase the difficulty of separation

Method used

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  • Application of Lpp or mutant thereof as molecular chaperone to secretory expression of recombinant protein in escherichia coli
  • Application of Lpp or mutant thereof as molecular chaperone to secretory expression of recombinant protein in escherichia coli
  • Application of Lpp or mutant thereof as molecular chaperone to secretory expression of recombinant protein in escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The technical service company synthesized the gene sequence, and its translated amino acid sequence had the following characteristics as shown in Table 1:

[0051] Table 1

[0052]

[0053]

[0054] Transformation of pET-28a(+) vector: PCR amplification of the tac promoter region of pFLAG-CTC vector (Sigma), adding BlpI and SphI restriction sites at both ends of the primers, BlpI and SphI double restriction PCR products and pET-28a (+) The vector was ligated with T4 DNA ligase and transformed into E. coli top10 competent cells, and positive clones were identified by PCR. After the plasmid was extracted, a promoter-replaced pET-28a(+) vector was obtained, which was named pETFLAG-CTC.

[0055] The gene sequence encoding the above-mentioned amino acid sequence was synthesized with reference to the codon bias of Escherichia coli (as shown below, synthesized by a technical service company). Taking SEQ ID NO.4 as an example, BamHI and NdeI restriction sites were added...

Embodiment 2

[0062] Example 2 Escherichia coli transformation and screening

[0063] The recombinant expression vector obtained in Example 1 was transformed into Escherichia coli for replication and amplification, and the specific process was as follows: according to the calcium chloride method (refer to the third edition of "Molecular Cloning Experiment Guide"), Escherichia coli top10 competent state was prepared, and 1 μL of the recombinant expression vector was taken. Add to top10 competent, ice bath for 30min, heat shock at 42°C for 90s, ice bath for 5min, add 1ml liquid SOC medium (2%w / v tryptone, 0.5%w / v yeast extract, 0.05%w / v NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 20 mM glucose), incubate at 37 °C for 1 h after shaking in a shaker, spread on LB solid medium (containing 50 mg / L kanamycin kan), and cultivate overnight in a 37 °C incubator until colonies are visible to the naked eye. Pick bacteria into LB liquid medium (10g / L peptone, 5g / L yeast extract, 5g / L sodium chloride, pH7.0~7.5, co...

Embodiment 3

[0065] Example 3. Escherichia coli fermentation and purification

[0066] The bacterial classification that embodiment 2 preserves prepares seed culture and 20L fermentor fermentation process:

[0067] ① Preparation of seed culture

[0068] Take 20 μL of the strains BL21(DE3) / pETFLAG-CTC-Lpp-GLP-1(X) and W3110 / pETFLAG-CTC-Lpp-GLP-1(X) cryopreserved at -70℃, respectively, inoculate to 50 mL with added The LB liquid medium of kanamycin (final concentration of 50 μg / mL) was cultured at 28° C. and 250 rpm in a shaker for 16 hours to activate the strains. Then inoculate 50 mL of activated strains into 400 mL of LB liquid medium supplemented with 50 μg / mL kanamycin, and continue to cultivate for 3 hours at 28 °C and 250 rpm to obtain a seed culture and control its bacterial concentration. OD600 is between 0.8 and 1.2.

[0069] ②Fermentation culture in 20L fermenter

[0070] A 20L stirring fermenter (Nanjing Hualong Company) was used, and feeding was carried out according to the ...

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Abstract

The invention discloses application of Lpp or a mutant thereof as a molecular chaperone in secretory expression of recombinant protein in escherichia coli. The inventor accidentally finds that as the molecular chaperone of the target protein, the Lpp is beneficial to secretory expression of the recombinant protein containing the molecular chaperone and the target protein, the yield is high, the influence on the growth of escherichia coli thalli is small, no obvious thallus cracking condition exists, high-density fermentation is easy to realize, and industrial production is facilitated. Through the application, the invention also provides a fusion protein for secretory expression of escherichia coli and a method for secretory expression of the fusion protein by escherichia coli.

Description

technical field [0001] The invention belongs to the field of biomedicine, and particularly relates to the application of Lpp or its mutants as molecular chaperones for secreting and expressing recombinant proteins in Escherichia coli. Background technique [0002] Novo Nordisk patent US20090156478 introduces a preparation method of a GLP-1 receptor agonist (liraglutide, Victoza), 34-position lysine is mutated to arginine to avoid modification, and 26-position lysine Site acylation connects fatty acid chains, which can be non-covalently bound to human serum albumin (HSA), which can extend the half-life to about 1 day, only needs to be injected once a day, and has cardiovascular protection and weight loss effects. [0003] Novo Nordisk's patent US9732137 introduces the preparation method of another new GLP-1 receptor agonist (semaglutide, Ozempic), 34-position lysine is mutated to arginine to avoid modification, DPP-IV The eighth amino acid of the recognition site was mutated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12P21/02C07K19/00
CPCC12N15/70C07K14/245C07K14/605C12N2800/22C07K2319/35C07K2319/02C07K2319/50
Inventor 黄国周刘合栋曹春来梁雄基杨晓纯李张万金樊昌李素雯周翠何秀仪
Owner ZHUHAI UNITED LAB
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