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Engineered escherichia coli for rapid and efficient induction of expression of foreign proteins, and preparation method and application of engineered escherichia coli

A technology of Escherichia coli and foreign proteins, applied in the field of bioengineering, can solve problems such as unfavorable stable expression of foreign proteins

Pending Publication Date: 2021-10-22
YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the original TOP10 strain used with the pBAD expression vector also has its shortcomings. Because the host strain TOP10 has not deleted the proteolytic enzyme Ion and OmpT outer membrane proteolytic enzyme genes, the Ion and OmpT gene products will cause the hydrolysis of foreign proteins. , tends to degrade exogenous proteins in cells, which is not conducive to the stable expression of exogenous proteins
Although the E. coli strain B834 genome does not contain Ion and OmpT genes, the E. coli strain B834 has its obvious defects: because the complete arabinose operon is retained in the strain genome, the L-arabinose added in the system can be processed. Metabolism, so protein expression systems such as pBAD that use L-arabinose as an inducer cannot use this strain as a host for long-term induction (>5 hours)

Method used

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  • Engineered escherichia coli for rapid and efficient induction of expression of foreign proteins, and preparation method and application of engineered escherichia coli
  • Engineered escherichia coli for rapid and efficient induction of expression of foreign proteins, and preparation method and application of engineered escherichia coli
  • Engineered escherichia coli for rapid and efficient induction of expression of foreign proteins, and preparation method and application of engineered escherichia coli

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Embodiment 1

[0025] Example 1 Construction of Escherichia coli expression strain E.coli B834 (ΔaraBAD)

[0026] A kind of Escherichia coli expression strain E.coli B834 (ΔaraBAD) was constructed, and the genome of the strain had deleted the araBAD coding region (the nucleotide sequence of the araBAD coding region is shown in SEQ ID NO: 1), which can be applied to arabinose-induced The rapid and efficient induction protein expression system greatly expands the application range of the strain.

[0027] The construction of Escherichia coli expression strain E.coli B834 (ΔaraBAD) is first to replace the araBAD gene in the Escherichia coli strain B834 genome with FRT-Kan cassette-FRT (the nucleotide sequence of FRT is shown in SEQID NO: 2 by homologous recombination) The nucleotide sequence of FRT-Kan cassette-FRT is shown in SEQ ID NO: 3), and then the Kan cassette between the FRTs is deleted by Flpe-mediated site-specific recombination, leaving only one FRT site , the residue of this site wi...

Embodiment 2

[0053] Example 2 Functional verification of Escherichia coli expression strain E.coli B834 (ΔaraBAD)

[0054] In order to verify that the transformed strain E.coli B834 (ΔaraBAD) has a rapid and high-efficiency expression effect on the foreign protein based on the arabinose inducible carrier, we added the gene of the EGFP protein (67.88kDa, the amino acid sequence is shown in SEQ ID NO: 8) Cloned into the pBAD (MBP / TEV) vector to obtain the pBAD-MBP / TEV / EGFP prokaryotic expression vector, and then transformed into the E.coli B834 (ΔaraBAD) strain, and the BL21 and TOP10 commonly used protein expression strains transformed with the same plasmid were used as controls , adding L-arabinose for induction for 1-4 hours, and detecting the expression level of EGFP by SDS-PAGE electrophoresis. Specific steps are as follows:

[0055] 1) Construction of pBAD-MBP / TEV / EGFP prokaryotic expression vector

[0056] The pBAD (MBP / TEV) vector is obtained by adding the MBP and TEV tags to the v...

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Abstract

The invention relates to the technical field of bioengineering, in particular to engineered escherichia coli for rapid and efficient induction of foreign proteins, and a preparation method and application of the engineered escherichia coli. According to the invention, an arabinose operon araBAD coding region of an escherichia coli strain B834 is knocked out, and an E. coli B834 (delta araBAD) strain is constructed, so an arabinose-induced prokaryotic expression system is successfully applied to the E. coli B834 (delta araBAD) strain, and a set of novel escherichia coli expression system for rapid and efficient induction of the stable expression of foreign proteins. According to the invention, a pBAD vector can be applied to the E. coli B834 (delta araBAD) strain, and a target protein can be stably expressed at a high level.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Escherichia coli engineering bacterium for rapid and efficient induction and expression of foreign proteins, a preparation method and application thereof. Background technique [0002] Escherichia coli engineering bacteria is one of the most widely used prokaryotic expression hosts for the production of foreign proteins, and its genetic characteristics are far superior to other microorganisms. In recent years, as Escherichia coli has made great progress in basic research on transcription, translation and protein folding, as well as the use of successively discovered and improved genetic tools, the prokaryotic expression system of Escherichia coli has been able to express complex eukaryotic proteins Aspects have shown stronger vitality than ever before. Compared with other exogenous protein expression systems, the E. coli prokaryotic expression system has the following ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/67C12N15/52C12R1/19
CPCC12N15/52C12N15/70C12N2800/30
Inventor 施金秀罗燕刘泽宇林映君蒙伟能
Owner YUNZHOU BIOSCIENCES (GUANGZHOU) INC