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Escherichia coli recombinant strain as well as preparation method and application thereof

A technology of recombinant strains and Escherichia coli, which is applied in the field of biomedicine, can solve the problems of ompF protein excretion outside the cell, ompC potential function or absence, micF regulatory function loss, etc., to achieve the effect of increasing expression and secretion efficiency

Active Publication Date: 2022-07-12
BEIJING QL BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the insertion elements (insertion elements) insA / B-17 belonging to the transposable element in the B strain were inserted into the ompC-micF site, resulting in the lack of potential functions of ompC in the B strain, the complete loss of the regulatory function of micF, and ompF The expression regulation fails, resulting in the overexpression of ompF protein in the B strain, and even a large amount of ompF protein is directly excreted outside the cell
Energy waste for recombinant protein expression and production at the cellular level

Method used

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  • Escherichia coli recombinant strain as well as preparation method and application thereof
  • Escherichia coli recombinant strain as well as preparation method and application thereof
  • Escherichia coli recombinant strain as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Example 1 Construction of Escherichia coli Recombinant Strain

[0057] In this study, through the sequence alignment of strain K and strain B, it was found that insA / B-17 was inserted into the ompC-micF site in strain B, resulting in the deletion of 56 amino acid coding sequences from the N-terminal of ompC gene. The promoter and downstream rcsD and rcsB genes are deleted, and the 56 amino acid sequence deleted from the N-terminus of the ompC protein contains a signal peptide (signal peptide) and a transmembrane βstrand that is crucial for anchoring ompC to the outer membrane, K strain ompC The protein expression sequence is shown in SEQ ID NO: 16, and the B strain ompC protein expression sequence is shown in SEQ ID NO: 17. Based on the above findings, the present invention transforms Escherichia coli B strain by scarless homologous recombination of I-sceI DNA endonuclease to obtain a new strain with ompC-micF gene locus repaired, and the method includes: 1) Knock out ...

Embodiment 2

[0092] Example 2 Application of the modified Escherichia coli B strain in the expression of foreign proteins

[0093] Recombinant protein S (GLP1(7-37) linked to an expressive fusion tag), recombinant protein P (PTH (teriparatide) linked to an expressive fusion tag) and recombinant protein F (Fc fragment of immunoglobulin IgG) will be expressed ) expression plasmids were transferred into SQLa (control group) and SQLb (recombinant strain) competent cells by heat shock method, and cultured in TB medium at 37°C to OD 600 was about 2.0, and IPTG (final concentration of 1 mM) was added to induce recombinant protein expression. After induction at 37°C overnight, the cells were collected by centrifugation, and the walls were broken by ultrasonic waves. SDS-PAGE electrophoresis analysis of whole protein, soluble protein and insoluble protein, the results are as follows Figure 6A As shown, the molecular weight of Protein S is 5154 Da, the molecular weight of Protein P is 5888 Da, an...

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Abstract

The invention provides an escherichia coli recombinant strain as well as a preparation method and application thereof, and particularly relates to modification of an ompC site of an escherichia coli B strain, recovery of the integrity of ompC protein and an expression regulation mechanism of two membrane channel proteins of ompC and ompF. The modified new strain can improve the expression quantity and secretion efficiency of recombinant protein.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant strain of Escherichia coli, a preparation method and application thereof. Background technique [0002] Escherichia coli B strain and K strain are the most widely used Escherichia coli host strains for the expression of recombinant polypeptide proteins at industrial production level. Through the analysis of the whole genome sequences of the B and K strains, it was found that the average nucleotide identity of the genome regions of the B strain REL606 strain [GenBank: NC_012967] and the K strain MG1655 strain [GenBank: NC_000913] was > 99.1%. [0003] A comparison of the B and K genomes showed that more than half of the 3793 proteins in their basic genomes were predicted to be identical, and about 310 appeared to be functional only in either B or K. [0004] The analysis of the expressed protein group showed that the expression difference of membrane channel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/113C12R1/19
CPCC07K14/245C12N15/70C12N2830/002Y02A50/30
Inventor 陈旭张旭家张媛媛翟鹏王作斌曾伶俐
Owner BEIJING QL BIOPHARMACEUTICAL CO LTD
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