Bi Chongpeng, Feng Xingjun, Shan Anshan, Guo Jiayin
Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Jul;25(7):975-81.
In order to get new antibacterial peptide, we designed a hybrid peptide LfcinB(1-15)-Melittin(5-12), composed of 1-15 amino acid residues of bovine Lactoferricin and 5-12 amino acid residues of Melittin. According to the bias of codon utilization of Escherichia coli, We synthesized the gene encoding the hybrid peptide. We inserted the gene between the sites of Nco I and Sal I of pET-32a and obtained the recombinant expression vector for heterologous expression of LfcinB(1-15)-Melittin(5-12) in Escherichia coli. We used Escherichia coli BL21(DE3) as expression host for the recombinant plasmid. After induced by isopropyl-beta-D-thiogalactoside (IPTG) under the optimized conditions, we realized the fusion protein was successfully expressed. The fusion protein was expressed in soluble form and the level was more than 35% of the total proteins. With (His)6 x Tag, the fusion protein was easily purified by His x Bind Purification Kit. After purification, we obtained 35 mg of fusion protein from 1 L of culture medium. At last, we accomplished that the peptide LfcinB(1-15)-Melittin(5-12) was released from the fusion protein cleaved by enterokinase. The recombinant LfcinB(1-15)-Melittin(5-12) showed antimicrobial activity assayed by agar diffusion test. This is the first report on the heterologous expression of the hybrid antibacterial peptide LfcinB(1-15)-Melittin(5-12) in Escherichia coli and also provides basis for next cost-effective expression of other antimicrobial peptides in genetic engineering.
为了获得新的抗菌肽，我们设计了一种杂合肽LfcinB(1 - 15)-蜂毒肽(5 - 12)，它由牛乳铁蛋白的1 - 15个氨基酸残基和蜂毒肽的5 - 12个氨基酸残基组成。根据大肠杆菌密码子使用偏好，我们合成了编码该杂合肽的基因。我们将该基因插入到pET - 32a的Nco I和Sal I位点之间，获得了用于在大肠杆菌中异源表达LfcinB(1 - 15)-蜂毒肽(5 - 12)的重组表达载体。我们使用大肠杆菌BL21(DE3)作为重组质粒的表达宿主。在优化条件下用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后，我们实现了融合蛋白的成功表达。融合蛋白以可溶形式表达，其水平超过总蛋白的35%。带有(His)6 x标签，融合蛋白很容易通过His x Bind纯化试剂盒进行纯化。纯化后，我们从1升培养基中获得了35毫克融合蛋白。最后，我们完成了通过肠激酶切割融合蛋白释放出肽LfcinB(1 - 15)-蜂毒肽(5 - 12)。通过琼脂扩散试验测定，重组LfcinB(1 - 15)-蜂毒肽(5 - 12)显示出抗菌活性。这是关于杂合抗菌肽LfcinB(1 - 15)-蜂毒肽(5 - 12)在大肠杆菌中异源表达的首次报道，也为下一步在基因工程中经济高效地表达其他抗菌肽提供了基础。
