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首页> 外文期刊>Journal of Biotechnology >Expression and purification of a nanostructure-forming peptide
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Expression and purification of a nanostructure-forming peptide

机译：纳米结构形成肽的表达和纯化

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摘要

Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, Pd1d1-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.
机译：最近，肽作为组装新型功能材料的基础材料引起了人们的兴趣，这些功能材料包括可转换的表面活性剂，纳米涂层，水凝胶和水性囊泡。我们表达了一种形成β-折叠肽，该肽已在自组装过程中被广泛研究，Pd1d1-2是与单体，二聚体，四聚体和九聚体融合在一起的不溶性表达伴侣酮固醇异构酶的最小化介质。表达之后是全细胞提取，并通过单个固定的金属亲和色谱（IMAC）步骤将融合蛋白分离到纯度大于90％。彼此化学裂解肽，并从融合伴侣化学裂解肽，然后丙酮沉淀污染的蛋白片段。通过反相HPLC回收纯的肽。融合蛋白的表达水平随着肽前体数目的增加而降低，化学裂解的效率也随之降低，从而使单肽过程成为最有效的方法。尽管如此，将该实验室方法应用于单肽融合蛋白仍导致纯肽产率大于表达的肽的30％。

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