Soluble expression of active human beta-defensin-3 in Escherichia coli and its effects on the growth of host cells
| Autor: | Li-Gang, Si, Xi-Cheng, Liu, You-Yong, Lu, Gen-Yu, Wang, Wen-Mei, Li |
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| Rok vydání: | 2007 |
| Předmět: |
Staphylococcus aureus
DNA Complementary beta-Defensins Reverse Transcriptase Polymerase Chain Reaction Recombinant Fusion Proteins Molecular Sequence Data Thrombin Sequence Analysis DNA Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Escherichia coli Humans Electrophoresis Polyacrylamide Gel Amino Acid Sequence Glutathione Transferase Plasmids |
| Zdroj: | Chinese medical journal. 120(8) |
| ISSN: | 0366-6999 |
| Popis: | Human beta-defensin-3 (HBD(3)) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD(3) protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways.We first cloned the complementary DNA coding for the mature chain of HBD(3), inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-beta-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD(3) (GST-HBD(3)) fusion protein. The fusion protein was treated with thrombin to produce pure HBD(3) protein then the antimicrobial activity of HBD(3) was evaluated in a liquid microdilution assay.The fusion protein GST-HBD(3) was efficiently cleaved by thrombin and yielded HBD(3) that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 microg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain.Active HBD(3) in E. coli by expressing the recombinant protein GST-HBD(3) could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein. |
| Databáze: | OpenAIRE |
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