Method for improving exocytosis level of recombinant protein of escherichia coli based on dacA

Abstract

The invention discloses a method for improving the exocytosis level of a recombinant protein of escherichia coli based on dacA, relating to the fields of gene engineering and fermentation engineering. By virtue of overexpression of a D-alanyl-D-alanine carboxypeptidase gene, the recombinant escherichia coli with the recombinant protein with the exocytosis level in overexpression is obtained. By virtue of the method, the secretion capacity of the recombinant protein of the escherichia coli can be remarkably improved. By utilizing amylase as a mode recombinant protein, the content of extracellular recombinant amylase of the escherichia coli is increased from 8.54% of a control group to 51.77%. The method has important guiding significance to the efficient secretion and production of the recombinant protein in the escherichia coli.

Description

technical field

[0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a dacA-based method for increasing the extracellular secretion level of Escherichia coli recombinant proteins and its application. technical background

[0002] The Escherichia coli expression system is currently the most commonly used recombinant protein expression system in genetic engineering. It has the advantages of simple operation and low cost, and can quickly and large-scale produce the target protein. However, in the heterologous expression of E. Signal peptide-guided secretion into the periplasmic space will lead to a large accumulation of intermediates, hindering protein production.

[0003] Peptidoglycan is a multi-layer network macromolecular structure composed of disaccharide units, tetrapeptide tails and peptide bridges. The skeleton of the peptidoglycan layer is composed of N-acetylglucosamine and N-acetylmuramic acid connected by ...

Examples

Embodiment 1

[0025] Example 1 This example illustrates the effect of overexpression of dacA on the extracellular secretion of recombinant amylase by E.coli

[0026] After double-enzyme digestion verification of the successfully constructed recombinant plasmid pRSFDuet-dacA ( figure 1 ), after being transformed into E.coliBL21(DE3) containing amylase recombinant plasmid pETDuet-AmyK, the level of extracellular secreted recombinant amylase in E.coli overexpressing carboxypeptidase dacA was significantly increased when the fermentation was induced for 24 hours, and the extracellular starch Enzyme activity reached 6.77U / mL ( figure 2 ). However, after induction and fermentation of the control strain without overexpressing any carboxypeptidase for 24 hours, the enzyme activity of the recombinant amylase measured outside the E.coli cell was only 1.74U / mL.

Embodiment 2

[0027] Example 2 This example illustrates the effect of overexpression of dacA on the distribution of recombinant amylase in E.coli cells

[0028] When carboxypeptidase dacA was overexpressed in E.coli, the intracellular content of recombinant amylase decreased from 91.46% of the control group to 48.23% after induction for 24 hours. The content of extracellular recombinant amylase was increased from 8.54% of the control group to 51.77% ( image 3 ).

Embodiment 3

[0029] Example 3 This example illustrates the effect of overexpression of dacA on soluble peptidoglycan in E.coli cells

[0030] When the carboxypeptidase dacA was overexpressed in E.coli, the content of soluble peptidoglycan in E.coli cells was significantly increased after induction for 6 hours, and the content of intracellular soluble peptidoglycan reached 92.79 mg / g ( Figure 4 ). However, the content of soluble peptidoglycan in E.coli cells was only 29.17mg / g after induction and fermentation of the control strain without any carboxypeptidase overexpression for 6h.

[0031]

technical field

[0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a dacA-based method for increasing the extracellular secretion level of Escherichia coli recombinant proteins and its application. technical background

[0002] The Escherichia coli expression system is currently the most commonly used recombinant protein expression system in genetic engineering. It has the advantages of simple operation and low cost, and can quickly and large-scale produce the target protein. However, in the heterologous expression of E. Signal peptide-guided secretion into the periplasmic space will lead to a large accumulation of intermediates, hindering protein production.

[0003] Peptidoglycan is a multi-layer network macromolecular structure composed of disaccharide units, tetrapeptide tails and peptide bridges. The skeleton of the peptidoglycan layer is composed of N-acetylglucosamine and N-acetylmuramic acid connected by ...

Examples

Embodiment 1

[0025] Example 1 This example illustrates the effect of overexpression of dacA on the extracellular secretion of recombinant amylase by E.coli

[0026] After double-enzyme digestion verification of the successfully constructed recombinant plasmid pRSFDuet-dacA ( figure 1 ), after being transformed into E.coliBL21(DE3) containing amylase recombinant plasmid pETDuet-AmyK, the level of extracellular secreted recombinant amylase in E.coli overexpressing carboxypeptidase dacA was significantly increased when the fermentation was induced for 24 hours, and the extracellular starch Enzyme activity reached 6.77U / mL ( figure 2 ). However, after induction and fermentation of the control strain without overexpressing any carboxypeptidase for 24 hours, the enzyme activity of the recombinant amylase measured outside the E.coli cell was only 1.74U / mL.

Embodiment 2

[0027] Example 2 This example illustrates the effect of overexpression of dacA on the distribution of recombinant amylase in E.coli cells

[0028] When carboxypeptidase dacA was overexpressed in E.coli, the intracellular content of recombinant amylase decreased from 91.46% of the control group to 48.23% after induction for 24 hours. The content of extracellular recombinant amylase was increased from 8.54% of the control group to 51.77% ( image 3 ).

Embodiment 3

[0029] Example 3 This example illustrates the effect of overexpression of dacA on soluble peptidoglycan in E.coli cells

[0030] When the carboxypeptidase dacA was overexpressed in E.coli, the content of soluble peptidoglycan in E.coli cells was significantly increased after induction for 6 hours, and the content of intracellular soluble peptidoglycan reached 92.79 mg / g ( Figure 4 ). However, the content of soluble peptidoglycan in E.coli cells was only 29.17mg / g after induction and fermentation of the control strain without any carboxypeptidase overexpression for 6h.

[0031]

Images