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
{"title":"High-yield production and purification of the fusion pH-responsive peptide GST-pHLIP in Escherichia coli BL21","authors":"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","doi":"10.3934/molsci.2022008","DOIUrl":null,"url":null,"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.","PeriodicalId":44217,"journal":{"name":"AIMS Molecular Science","volume":null,"pages":null},"PeriodicalIF":0.7000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"AIMS Molecular Science","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3934/molsci.2022008","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
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.