Si Li-Gang, Liu Xi-Cheng, Lu You-Yong, Wang Gen-Yu, Li Wen-Mei
Beijing Pediatric Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Beijing, China.
Chin Med J (Engl). 2007 Apr 20;120(8):708-13.
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.
人β-防御素-3（HBD(3)）是一种上皮肽，已被证明具有对盐不敏感的广谱强效抗菌活性。在大肠杆菌（E. coli）中表达抗菌肽非常困难，因为这可能导致细菌宿主细胞死亡。我们的目的是建立一个表达可溶性HBD(3)蛋白的原核系统，并证明所表达蛋白的抗菌活性。然后我们研究宿主细胞是否会激活自杀途径。
我们首先克隆编码HBD(3)成熟链的互补DNA，将其插入载体PGEX-KG，然后用合适的重组质粒转化大肠杆菌BL21（DE3）。用0.5 mmol/L异丙基-1-硫代-β-D-半乳糖苷（IPTG）诱导后，转化的大肠杆菌产生重组谷胱甘肽S-转移酶和HBD(3)（GST-HBD(3)）融合蛋白。用凝血酶处理融合蛋白以产生纯HBD(3)蛋白，然后通过液体微量稀释试验评估HBD(3)的抗菌活性。
融合蛋白GST-HBD(3)被凝血酶有效切割，产生的HBD(3)对金黄色葡萄球菌具有抗菌活性，最低抑菌浓度水平为12.5 μg/ml。表达重组蛋白的大肠杆菌菌株生长速度不比空载体菌株慢。
通过表达重组蛋白GST-HBD(3)可在大肠杆菌中产生有活性的HBD(3)，且表达重组蛋白的大肠杆菌菌株未发生自杀现象。
