Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus

作者：Guillen Chable Francisco; Arenas Sosa Ivan; Islas Flores Ignacio; Corzo Gerardo; Martinez Liu Cynthia; Estrada Georgina^*

来源：Protein Expression and Purification, 2017, 136: 45-51.

DOI：10.1016/j.pep.2017.06.007

• Capsicum
• Expression
• Protein folding
• Recombinant
• AMP
• Defensin
• Antibacterial
• Phospholipid

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa.

• 出版日期2017-8