A robust osmotic shock method for extraction of periplasmic recombinant protein from E. coli

Abstract

The selection of a simple, robust and efficient extraction method for periplasmic or soluble recombinant proteins from Escherichia coli is critical for maximizing recovery, preserving protein quality and reducing downstream processing costs. Present study proposes an optimized osmotic shock method (OSM) using phosphate buffer saline (PBS) for extraction of periplasmic recombinant protein expressed in E. coli. The effectiveness of the PBS‐based OSM with and without sucrose and EDTA has been successfully demonstrated for two different periplasmic proteins (nanobody and alpha synuclein). In case of nanobody and alpha synuclein products, the highest extraction yields of 89.33 ± 0.9 mg L −1 and 139.3 ± 0.7 mg L −1 of cell culture were obtained, respectively. The PBS‐based OSM with and without sucrose and EDTA also exhibited 87–90% reduction in host cell proteins with purity levels of ~50% for both the proteins without needing excessive downstream processing steps. Analytical characterization via reversed‐phase high‐performance liquid chromatography (RP‐HPLC), intact mass and peptide mapping for both the proteins has assessed the product quality. The purification yield of the nanobody product using PBS‐based OSM was ~67% higher with purity levels 21% better as compared to conventional method ( P  < 0.05). Likewise, alpha synuclein protein exhibited 50–55 mg L −1 of purification yield with ~89% purity levels. The proposed PBS‐based OSM would be interest to manufacturers, engaged in production of periplasmic or soluble recombinant proteins expressed in E. coli , as it provides a simple and efficient approach for protein extraction while maintaining product quality and reducing downstream processing complexity. © 2026 Society of Chemical Industry (SCI).