Rezaei Sadegh, Hadadian Shahin, Khavari-Nejad Ramazan Ali, Norouzian Dariush
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Nano-Biotechnology Department, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran.
Rep Biochem Mol Biol. 2020 Oct;9(3):348-356. doi: 10.29252/rbmb.9.3.348.
Antimicrobial peptides (AMPs) are promising candidates for new generations of antibiotics to overcome the threats of multidrug-resistant infections as well as other industrial applications. Recombinant expression of small peptides is challenging due to low expression rates and high sensitivity to proteases. However, recombinant multimeric or fusion expression of AMPs facilitates cost-effective large-scale production of AMPs. In This project, S3 and SΔ3 AMPs were expressed as fusion partners. S3 peptide is a 34 amino acid linear antimicrobial peptide derived from lipopolysaccharide (LPS) binding site of factor C of horseshoe crab hemolymph and SΔ3 is a modified variant of S3 possessing more positive charges.
Two copy tandem repeat of the fusion protein (named as SΔ3S3-2mer-GS using glycine- serine linker was expressed in . BL21 (DE3). After cell disruption and solubilization of inclusion bodies, the protein was purified by Ni -NTA affinity chromatography. Antimicrobial activity and cytotoxic properties of purified SΔ3S3-2mer-GS were compared with a previously produced tetramer of S3 with the same glycine- serine linker (S3-4mer-GS) and each of monomeric blocks of S3 and SΔ3.
SΔ3S3-2mer-GS was successfully expressed with an expression rate of 26%. The geometric average of minimum inhibitory concentration (MIC GM) of SΔ3S3-2mer-GS was 28%, 34%, and 57% lower than SΔ3, S3-4mer-GS, and S3, respectively. SΔ3S3-2mer-GS had no toxic effect on eukaryotes human embryonic kidney cells at its MIC concentration.
tandem repeated fusion expression strategy could be employed as an effective technique for recombinant production of AMPs.
抗菌肽是新一代抗生素的有前景的候选者，可用于克服多重耐药感染的威胁以及其他工业应用。由于表达率低和对蛋白酶高度敏感，小肽的重组表达具有挑战性。然而，抗菌肽的重组多聚体或融合表达有助于实现抗菌肽的经济高效大规模生产。在本项目中，S3和SΔ3抗菌肽作为融合伴侣进行表达。S3肽是一种由鲎血淋巴因子C的脂多糖（LPS）结合位点衍生而来的34个氨基酸的线性抗菌肽，SΔ3是S3的一种修饰变体，带有更多正电荷。
使用甘氨酸 - 丝氨酸接头的融合蛋白双拷贝串联重复序列（命名为SΔ3S3 - 2聚体 - GS）在大肠杆菌BL21（DE3）中表达。细胞破碎和包涵体溶解后，通过镍 - 氮三乙酸亲和层析纯化该蛋白。将纯化的SΔ3S3 - 2聚体 - GS的抗菌活性和细胞毒性特性与先前生产的具有相同甘氨酸 - 丝氨酸接头的S3四聚体（S3 - 4聚体 - GS）以及S3和SΔ3的每个单体片段进行比较。
SΔ3S3 - 2聚体 - GS成功表达，表达率为26%。SΔ3S3 - 2聚体 - GS的最低抑菌浓度几何平均值（MIC GM）分别比SΔ3、S3 - 4聚体 - GS和S3低28%、34%和57%。在其MIC浓度下，SΔ3S3 - 2聚体 - GS对真核生物人胚肾细胞没有毒性作用。
串联重复融合表达策略可作为抗菌肽重组生产的有效技术。
