Guillén-Chable Francisco, Arenas-Sosa Iván, Islas-Flores Ignacio, Corzo Gerardo, Martinez-Liu Cynthia, Estrada Georgina
Unidad de Bioquímica y Biología Molecular de Plantas. Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán 97205, México.
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM. Apartado Postal 510-3, Cuernavaca, Morelos, 61500, México.
Protein Expr Purif. 2017 Aug;136:45-51. doi: 10.1016/j.pep.2017.06.007. Epub 2017 Jun 15.
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.
将来自辣椒的四硫键桥联防御素J1-1基因克隆到含有6His标签的表达载体pQE30中，作为融合蛋白进行表达。将该构建体转染到大肠杆菌的Origami菌株中，并用异丙基硫代半乳糖苷（IPTG）诱导表达。表达水平为4mg/L培养基，带有His标签的重组防御素（HisXarJ1-1）仅在包涵体中表达。溶解后，通过亲和色谱和疏水相互作用色谱对HisXarJ1-1进行纯化。从亲和色谱步骤获得的HisXarJ1-1产物的反相高效液相色谱图谱显示，主要肽段分子量为7050.6Da，用二硫苏糖醇（DTT）处理后，观察到对应于还原肽的单一7042.6Da肽段。HisXarJ1-1的体外折叠步骤产生了该肽氧化形式的独特图谱，这种氧化肽能够在体外结合磷脂酸。在凝胶电泳中可见HisXarJ1-1可能的二聚体和寡聚体，并用抗His抗体进行免疫检测。纯重组防御素HisXarJ1-1对铜绿假单胞菌具有抗菌活性。
