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Recombinant protein purification using complementary peptides as affinity tags

机译：使用互补肽作为亲和标签的重组蛋白纯化

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Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile- Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn- Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20 mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1 M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.
机译：亲和标签已成为通过亲和色谱从粗提物中纯化重组蛋白的高度流行的工具。此外，短肽是亲和色谱的极佳配体，因为它们在泄漏到产品中时不太可能引起免疫反应，它们比洗脱和清洁条件抗体更稳定，并且通常具有非常可接受的选择性。从头设计的亲水互补肽具有足够的选择性，可以成功用作从粗提取物中纯化蛋白质的肽配体。互补肽对His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu和Lys-Asn-Tyr-Pro-Lys-Lys-Lys-之间相互作用的识别特异性和选择性Met-Glu-Lys-Arg-Phe已被其他作者证明。在这项工作中，我们设计了一种使用肽亲和标签的重组蛋白纯化方法，该亲和标签与固定在色谱基质上的肽结合伴侣结合。以大肠杆菌中增强的绿色荧光蛋白表达（EGFP）为模型。肽Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu通过固相合成，使用Fmoc化学方法固定在NHS-Sepharose（PC-Sepharose）中）。 Gly残基作为间隔臂在N末端添加。 EGFP在C末端的融合标签Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe（EGFP-CPTag）上表达或不表达任何融合标签。细胞破裂后，将提取物直接加到用20 mM磷酸钠缓冲液（pH 7.0）平衡的PC-Sepharose柱上。然后用1 M Tris洗脱吸附的EGFP-CPTag。产率为98％，纯化系数为4.6。相反，没有标签的EGFP可以通过而不会与PC-Sepharose色谱柱相互作用。设计的方法可用于纯化其他重组蛋白。

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