High-yield production and purification of the fusion pH-responsive peptide GST-pHLIP in Escherichia coli BL21

IF 0.7 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Oscar Cienfuegos-Jiménez, Abril Morales-Hernández, Olivia A. Robles‐Rodríguez, Sergio Bustos-Montes, Kevin A. Bañuelos-Alduncin, Aurora R. Cortés-Castillo, Hugo D. Barreto-Hurtado, Luis Carrete-Salgado, I. Marino-Martínez
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引用次数: 0

Abstract

The pH Low Insertion Peptide (pHLIP) has versatile applications in several diseases due to its differential behavior at slightly different pH values. pHLIP is an unstructured and peripheral membrane-associated peptide at neutral pH and an α-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP´s expanding applications require high-yield production to further scale-up its usefulness. To date, synthesis of the pHLIP has not been reported in a prokaryotic platform, mainly relying on solid-phase synthesis. Bacterial production arises as an option for high-amount peptide generation and larger pHLIP fusion protein-synthesis; however, cell-based pH-responsive peptide production could be challenging due to intracellular peptide interactions or degradation due to unstructured conformations. An Escherichia coli (E. coli)-BL21 cell culture was induced with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) in order to produce a Glutathione S-transferase-pHLIP (GST-pHLIP) fusion construct. Purification was done with Glutathione (GSH)-decorated magnetic beads using 4 ml of the induced cell culture. The production was quantified with Bradford reagent and characterized with SDS-PAGE and Western blot, contrasting Bradford results with densitometry analysis to obtain production approximate absolute values. A purified approximate total yield of ~26 µg with an apparent GSH-bead saturation and a total production of ~82 µg was obtained. Our Western Blot assay confirmed the presence of the GST-pHLIP construct in all the IPTG-induced fractions. Conclusion: A high-yield pHLIP production irrespective of its membrane affinity in acidic environments or its unstructured nature was achieved. Our study could be useful to scale up pHLIP synthesis for future applications.
大肠杆菌BL21融合ph响应肽gst - phillip的高产制备与纯化
pH低插入肽(pHLIP)由于其在轻微不同的pH值下的不同行为,在几种疾病中具有广泛的应用。philip是中性pH下的非结构化外周膜相关肽,酸性pH下是α-螺旋跨膜肽。与胰岛素和生长激素类似,pHLIP的应用范围不断扩大,需要高产量的产品来进一步扩大其用途。迄今为止,pHLIP的合成尚未在原核平台上报道,主要依赖于固相合成。细菌生产出现作为一种选择,以产生大量的肽和更大的philips融合蛋白合成;然而,由于细胞内肽相互作用或非结构化构象导致的降解,基于细胞的ph响应肽生产可能具有挑战性。用异丙基ß- d -1-硫代半乳糖苷(IPTG)诱导大肠杆菌(E. coli)-BL21细胞培养,产生谷胱甘肽s -转移酶- phillip (gst - phillip)融合构建体。用4ml诱导细胞培养物用谷胱甘肽(GSH)修饰的磁珠进行纯化。用Bradford试剂定量,SDS-PAGE和Western blot对产量进行表征,将Bradford结果与密度分析进行对比,以获得产量的近似绝对值。纯化后的总产率约为~26µg, GSH-bead明显饱和,总产量约为~82µg。我们的Western Blot检测证实在所有iptg诱导的部分中都存在gst - phillip构建体。结论:无论其在酸性环境中的膜亲和性或非结构化性质如何,均可获得高产率的pHLIP。我们的研究可能有助于扩大philips合成的规模,以用于未来的应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
AIMS Molecular Science
AIMS Molecular Science BIOCHEMISTRY & MOLECULAR BIOLOGY-
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