Expression and purification of the antimicrobial peptide Bin1b in Escherichia coli tagged with the fusion proteins CusF3H+ and SmbP
| Autor: | Jorge M Montfort-Gardeazabal, Xristo Zarate, Néstor Casillas-Vega, Isaías Balderas-Rentería |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Pore Forming Cytotoxic Proteins
0106 biological sciences Enteropeptidase Staphylococcus aureus Recombinant Fusion Proteins Peptide medicine.disease_cause 01 natural sciences Inclusion bodies law.invention 03 medical and health sciences Plasmid law 010608 biotechnology Escherichia coli medicine 030304 developmental biology chemistry.chemical_classification 0303 health sciences Chemistry Antimicrobial Fusion protein Biochemistry Recombinant DNA Biotechnology |
| Zdroj: | Protein Expression and Purification. 178:105784 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2020.105784 |
| Popis: | We have previously shown that the small metal-binding proteins CusF3H+ and SmbP can be used as fusion proteins for the expression and purification of recombinant proteins in Escherichia coli. Because of their small size, both around 10 kDa, they are suitable for the production of peptides to avoid meager yields after the final purification step of tag removal. Bin1b is a beta-defensin found in the epididymis of rats that has shown to have antimicrobial activity. Previous methodologies used to express this antimicrobial peptide in E. coli involve the expression of the peptide as inclusion bodies followed by in vitro refolding or the supplementation of the proteins necessary for proper folding of the peptide in the cytoplasm via a second plasmid. Here, we developed a methodology that forgoes these approaches and instead uses the fusion proteins CusF3H+ or SmbP and the E. coli strain SHuffle to obtain a soluble recombinant protein that contains the mature Bin1b peptide. The recombinant protein is purified using IMAC chromatography and is subsequently cleaved with enterokinase to separate the fusion protein from Bin1b. The purified peptide displays antimicrobial activity against E. coli, as previously shown. Furthermore, we also tested its antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and found that Bin1b is also capable of inhibiting the growth of this bacterium. In conclusion, we developed a practical methodology for the expression and purification of the bioactive Bin1b peptide in E. coli using the fusion proteins CusF3H+ and SmbP. This approach could be further applied for the production of more biologically active peptides. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect