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Bacterial expression vector for enhanced protein secretion

A technology of expression vectors and bacteria, applied in the field of bacterial expression vectors, can solve the problems of incorrect output of functional status, output, misfolding, etc.

Pending Publication Date: 2021-09-10
昂科斯密斯生物技术私人有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although large quantities of proteins have been successfully produced by controlled secretion methods in E. coli, they cannot be exported correctly or in a functional state due to aggregation in the cytoplasm, cell lysis, misfolding, restriction on translocation, or protein degradation. output, production volume remains a limiting factor

Method used

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  • Bacterial expression vector for enhanced protein secretion
  • Bacterial expression vector for enhanced protein secretion
  • Bacterial expression vector for enhanced protein secretion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Secretion efficiency of different combinations of signal sequence and carrier peptide

[0074] A. Luciferase Assay:

[0075] E. coli strains NEB 5-alpha and BL21(DE3) were used for transformation and luciferase assays.

[0076] Different combinations of secretion signals were constructed as follows:

[0077] a) the signal sequence of Seq.ID 1 and the carrier protein represented by Seq.ID 5,

[0078] b) the signal sequence of Seq.ID 2 and the carrier protein represented by Seq.ID 5,

[0079] c) the signal sequence of Seq.ID 3 and the carrier protein represented by Seq.ID 5,

[0080] d) Signal sequence of Seq.ID 4 and carrier protein represented by Seq.ID 5.

[0081] Gaussian luciferase was used as a reporter system to detect secretory activity of E. coli. Gaussian luciferase assays were performed using the Pierce Gaussian luciferase flash assay kit. Medium was collected from the cultures at the indicated time intervals after induction and luciferase activity was me...

Embodiment 2

[0093] Secretion efficiency of Seq.ID 6 compared to Seq.ID 5

[0094] The DNA sequence of the recombinant protein was cloned into the pBacSec-LC vector with a secretion signal sequence comprising the following combination: Seq.ID 4 and Seq.ID 5, or Seq.ID 4 and Seq.ID 6.

[0095] Seq.ID 6 was synthesized by mutating Seq.ID 5, wherein the TGC codon at position 40 of Seq.ID 5 was mutated into the GCG codon of Seq.ID 6, making the 14th position of Seq.ID 7 Cys is mutated to Ala at position 14 of Seq.ID 8. The Cys residue at position 14 of the peptide enables dimerization and increases the chance of inclusion body formation. Mutation of Cys to Ala abolishes the dimerizing properties of the peptide, thereby reducing the chance of inclusion body formation, which in turn enhances the secretion of the peptide and the recombinant peptide.

[0096] E. coli cells were transformed with the respective vectors, and two sets of cultures were prepared under reducing and non-reducing conditi...

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Abstract

The invention provides a bacterial expression vector (100) comprising of a secretory signal sequence in tandem with DNA sequence encoding recombinant protein (103), wherein, the secretory signal sequence is a combination comprising of: a) at least one DNA sequence encoding a signal sequence (101) of gene selected from the group consisting of pelB, ompA, yebF, and ompF, and b) at least one DNA sequence encoding a carrier peptide (102) selected from the group consisting of Seq. ID 5 and 6 encoding truncated yebF.

Description

technical field [0001] The present invention relates to bacterial expression vectors capable of increasing secretion of recombinant proteins in the periplasmic space or extracellularly. More specifically, the present invention relates to expression vectors for expressing and secreting recombinant proteins from Escherichia coli. Background technique [0002] Heterologous expression and purification of recombinant proteins in vitro and in vivo represent routine applications of modern molecular biology. Expression of recombinant proteins is usually carried out in prokaryotic host cells, and the microorganism Escherichia coli is often used for the large-scale production of recombinant proteins relevant to the pharmaceutical and industrial industries. In view of this, considerable modifications have been made in host cells to increase the production of the protein of interest in these host cells, using several known methods and mechanisms, thereby maximizing their utility in ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70
CPCC12N15/70C07K2319/02C07K2319/20C07K2319/61C12P21/02Y02A50/30C12N2840/002
Inventor S·洛基雷迪
Owner 昂科斯密斯生物技术私人有限公司