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

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 membraneassociated peptide at neutral pH and an alpha-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP acute accent 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 pHresponsive 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 Stransferase-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 similar to 26 mu g with an apparent GSH-bead saturation and a total production of similar to 82 mu g 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.