Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II

Boda Ferenc-András, Szabó Zoltán-István, Szőcs Erika, S. Pál, Orbán Csongor, Székely Edit
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引用次数: 1

Abstract

Abstract Antimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the prevention of infections, through their direct antimicrobial and immunomodulatory properties. While the amino acid composition and spatial structure vary, most antibacterial peptides have a positive surface charge, which allows them to bind to the negative bacterial membranes. Buforin II is a widely studied antimicrobial peptide first obtained through the structural modification of buforin I, a peptide isolated from Bufo gargarizans. The peptide showed significant antibacterial activity against Gram-positive and Gram-negative bacterial strains. The mechanism of action of buforin II differs from that of other antimicrobial peptides, as it binds directly to bacterial DNA and RNA. The aim of our study was to obtain recombinant buforin II with a ubiquitin fusion partner, through heterologous expression in Escherichia coli Rosetta™ (DE3)pLysS cells, using a laboratory scale bioreactor. The incubation of expression host cells in a bioreactor allowed the constant monitoring and control of the process parameters, leading to high biomass levels and an increased production rate of the peptide. The parameters used during incubation were: 37°C, pH=6.9 and dissolved oxygen level above 40%. Purification of the recombinant protein was accomplished by affinity chromatography using a Ni-chelate solid phase to which the 10xHistag of our construct showed affinity. Method optimisation consisted in the use of gradient and linear elution, of which the latter was found to be more effective. Digestion of the fusion partner from the target peptide was performed with ubiquitin carboxyl-terminal hydrolase enzyme. The expression and purification protocols developed in our experiment allow the production of a significant amount of buforin II, allowing its use for further research. Furthermore, the presented methods could be suitable for industrial production of the recombinant peptide..
抗菌肽Buforin II的异种表达和纯化
抗菌肽是一种天然物质,在适应性免疫系统的发展中发挥作用,目前通过其直接的抗菌和免疫调节特性参与感染的预防。虽然氨基酸组成和空间结构各不相同,但大多数抗菌肽具有正表面电荷,这使它们能够与负细菌膜结合。Buforin II是一种被广泛研究的抗菌肽,最初是通过对Buforin I进行结构修饰而获得的,Buforin I是一种从Bufo gargarizans中分离出来的肽。该肽对革兰氏阳性和革兰氏阴性菌株均有明显的抑菌活性。buforin II的作用机制不同于其他抗菌肽,因为它直接与细菌DNA和RNA结合。本研究的目的是利用实验室规模的生物反应器,通过在大肠杆菌Rosetta™(DE3)pLysS细胞中异源表达,获得具有泛素融合伙伴的重组丁福林II。在生物反应器中孵育表达宿主细胞,可以对过程参数进行持续监测和控制,从而提高生物量水平和多肽的生产率。孵育过程中使用的参数为:37℃,pH=6.9,溶解氧浓度大于40%。重组蛋白的纯化是通过ni -螯合固相亲和层析完成的,我们构建的10xHistag具有亲和性。方法优化包括使用梯度和线性洗脱,其中后者被发现更有效。用泛素羧基末端水解酶从目标肽中消化融合伙伴。在我们的实验中开发的表达和纯化协议允许生产大量的buforin II,允许其用于进一步的研究。此外,该方法适合于重组肽的工业化生产。
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