Proof-of-concept validation of p2GUS, a modular expression system for enhanced Ni-binding purification of functional recombinant proteins in Escherichia coli

Abstract

Abstract Protein expression and purification remain critical steps in basic research, biotechnology, and industrial applications. Here, p2GUS, a modular Escherichia coli expression system generated through targeted modification of Precursor 1, a parental pBAD-derived vector, is described. The principal feature is a tandem His₁₀–GST–His₁₀ architecture designed to enhance interaction with Ni–NTA matrices while retaining compatibility with post-expression processing strategies. The GST module serves primarily as a spacer between the two polyhistidine tags while also contributing solubility-enhancing properties. Engineered PreScission and Enterokinase cleavage sites, enabling controlled post-expression processing and generation of alternative protein forms derived from the same multitagged recombinant protein. As a proof of concept, p2GUS was evaluated using a truncated mitochondrial transcription factor A [mTFAM(p.1_41del)]. Recombinant protein production, purification, protease-mediated processing, and DNA-binding activity were assessed using multitagged mTFAM(p.1_41del) as a model substrate. Purification outcomes were compared with those obtained using the parental construct Precursor 1, encoding a single-His 10 –mTFAM(p.1_41del). Under the experimental conditions examined, the p2GUS-derived construct, purified using a simple single-microcentrifuge tube Ni–NTA batch procedure, showed improved recovery and yielded a processed His 10 –EK–mTFAM(p.1_41del) of substantially higher purity than the corresponding His 10 –mTFAM(p.1_41del) produced by Precursor 1. The purified recombinant protein retained DNA-binding activity, supporting preservation of its functional properties following expression, purification and storage. These results support proof-of-concept validation of p2GUS as a modular system for recombinant protein production, purification, and controlled post-expression processing. Although validated here using a DNA-binding protein, broader applicability remains to be established through evaluation with additional recombinant proteins. Key points • Proof-of-concept validation of the modular p2GUS expression system • Tandem His₁₀–GST–His₁₀ architecture improves Ni-NTA retention and purification • Multitagged p2GUS-derived mTFAM retained DNA-binding activity