Wang Jue, Gao Qiurong, Liu Qiang, Xiang Linping, Wang Yuqin, Wang Dun
Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, China.
Wei Sheng Wu Xue Bao. 2010 Sep;50(9):1185-93.
Bradysia hygida salivary glands antimicrobial peptide 1 (BhSGAMP-1) is one of the antimicrobial peptides involved in a preventive mechanism of defense of the fly Bradysia hygida. To know better about the molecular characterization of this antimicrobial peptide, we expressed and purified the modified BhSGAMP-1-S and investigated its antimicrobial activity.
We synthesized the gene of BhSGAMP-1-S designed with preferred codons of Escherichia coli and expressed it as a fusion protein in E. coli TB1 by using pMAL-c2X as vector. We purified the fusion protein using amylase resin affinity chromatography. In addition, we cleaved the fusion protein by enterokinase and the released recombinant BhSGAMP-1-S was separated by size exclusion chromatography, then followed by reversed-phase high performance liquid chromatography. We analyzed the antimicrobial activities of the purified recombinant BhSGAMP-1-S by bioassays.
The fusion protein was mostly expressed in soluble form under the optimized conditions. The recombinant BhSGAMP-1-S was produced with a pure yield of 0.38 mg/100 mL culture medium. Antimicrobial assays demonstrated that the recombinant BhSGAMP-1-S was active against several Gram-positive and Gram-negative bacteria and fungi.
It appears to be the first successful production of the recombinant BhSGAMP-1-S from fly Bradysia hygida. Data presented here confirm that the recombinant BhSGAMP-1-S is now ready for further studying and characterizing their antimicrobial properties.
潮湿地种蝇唾液腺抗菌肽1（BhSGAMP-1）是参与潮湿地种蝇防御机制的抗菌肽之一。为了更好地了解这种抗菌肽的分子特征，我们表达并纯化了修饰后的BhSGAMP-1-S，并研究了其抗菌活性。
我们合成了用大肠杆菌偏爱密码子设计的BhSGAMP-1-S基因，并以pMAL-c2X为载体在大肠杆菌TB1中作为融合蛋白进行表达。我们使用淀粉酶树脂亲和色谱法纯化融合蛋白。此外，我们用肠激酶切割融合蛋白，释放的重组BhSGAMP-1-S通过尺寸排阻色谱法分离，然后进行反相高效液相色谱法。我们通过生物测定分析纯化的重组BhSGAMP-1-S的抗菌活性。
在优化条件下，融合蛋白大多以可溶形式表达。重组BhSGAMP-1-S的产量为0.38 mg/100 mL培养基。抗菌试验表明，重组BhSGAMP-1-S对几种革兰氏阳性菌、革兰氏阴性菌和真菌具有活性。
这似乎是首次成功从潮湿地种蝇中生产出重组BhSGAMP-1-S。此处提供的数据证实，重组BhSGAMP-1-S现已准备好进一步研究和表征其抗菌特性。
