Authors
Marcia Brady
Summary
The Escherichia coli expression system is the most common among all the methods of genetically engineered bacteria to produce antimicrobial peptides. The clear genetic background, mature expression technology and fast growth of bacteria make it widely used. For example, the compound antibacterial peptide AL32-P113 and the recombinant antibacterial peptide UBI 18-35 realizes expression in E. coli, and obtained considerable expression levels of 12.1 mg/L and 6 mg/L. Moreover, the fusion expression of Fowlicidin-3 in E. coli BL21 Rosetta produces antimicrobial peptides Trx-Fowlicidin-3a and Trx-Fowlicidin-3b with expression levels of 0.84 mg/mL and 0.71 mg/mL. At present, the method of using Escherichia coli to produce antimicrobial peptides has been very mature and has been widely used. It is an effective way for the large-scale production of antimicrobial peptides.
Introduction
Factors affecting the production of antimicrobial peptides by the E. coli expression system:
1) Since the E. coli expression system can not modify proteins in the post-translational stage like eukaryotes, it leads to excessive accumulation of intermediate products and easy to form inclusion body. The target peptide is generally not directly controllable in the process of inclusion body formation. The inclusion body expression is expected to be carried out during the fusion expression process to solve the problem mentioned above. Due to the weak translation ability in the late stage, the stability and activity of the secreted antimicrobial peptides are weaker.
2) The expression system is easy to produce endotoxins, which will cause the antimicrobial peptides to kill the host bacteria itself during the late expression process.
3) The choice of expression vector is complicated. Different types of expression vectors will have different effects on the production of antimicrobial peptides.
Procedure
Pichia pastoris is the most widely used expression system among the yeasts. But there is no stable episomal plasmid when expressing foreign antimicrobial peptide genes, almost all expression vectors, such as pPICZ and pIPC3k, can be directly integrated into the yeast genome. The host strains used in the expression system to produce antimicrobial peptides are derived from the original wild-type strain NRRL-Y11430. A variety of antibacterial peptides have been expressed in yeast, and have considerable expression levels. The gene of the antimicrobial peptide Moricin has been amplified and then introduced into Pichia pastoris. The expression level is high. 248.98 mg/L of recombinant protein can be obtained in the supernatant after fermentation, with obvious antibacterial activity. The recombinant expression vector pPICZαA-GST-Apidaecin has also been expressed in Pichia pastoris. The system has successfully expressed Apidaecin with potent antibacterial activity, and the expression amount was 1.76 g/L. Compared with E. coli, Pichia pastoris has more advantages as a eukaryotic expression system.
When foreign antimicrobial peptide genes are expressed in Pichia pastoris, many factors will affect the production of antimicrobial peptides.
1) The internal factors of the exogenous antimicrobial peptide genes. Choosing the bias codons of Pichia pastoris can improve the expression efficiency of antimicrobial peptides to a certain extent.
2) Keeping the composition length of the untranslated region at the 5' end of mRNA consistent with AOX1/MOX can also increase the production of antimicrobial peptides.
3) UTR sequence, A+T content and gene copies will limit the production of antimicrobial peptides.
4) Pichia pastoris secretes protease by itself, which can dissolve foreign antimicrobial peptides. This is the main problem that the system faces.
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