Expression and Purification of G Protein-coupled Receptor from Polycystin-1 Using MBP Fusion in E. coli

Hala Salim Sonbol ¹*, Alaa Muqbil Al-sirhani ²

This study showed understanding of PC1's GPS domain, crucial for ADPKD, through successful expression and purification using MBP fusion.

Abstract

Background: Polycystin-1 (PC1), encoded by the PKD1 gene, is critical in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) when mutated. PC1 contains a unique proteolytic site, the G protein-coupled receptor (GPS) domain, suggesting a role in G protein signaling regulation. Disruption in the GPS region of PC1 leads to structural reorganization within renal cells, promoting aggressive renal cystogenesis. Understanding the process of GPS cleavage is crucial as it regulates the production and localization of PC1, essential for kidney growth and function. Methods: In this study, we amplified the GPS region of polycystin-1 from genomic DNA and cloned it into the pET-21a(+)-MBP(TEV) expression vector. The molecular chaperone maltose binding protein (MBP) was employed to ensure proper folding of the fusion proteins. The resulting fusion protein, MBP-His-GPS, was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). Results: The GPS region of PC1 was successfully amplified and cloned into the expression vector. The fusion protein, MBP-His-GPS, exhibited high levels of expression in E. coli. The purification process using IMAC resulted in a high yield of purified fusion protein. The use of MBP as a fusion partner enhanced the solubility and stability of the target protein, facilitating its purification. Conclusion: The study successfully developed a methodology for expressing and purifying the GPS region of PC1, utilizing MBP to improve protein solubility and stability. This technique is essential for the in-depth study and manipulation of PC1 in the laboratory. The approach underlines the potential of MBP fusion in enhancing protein expression and simplifying purification processes, thereby advancing molecular techniques for PC1 research and contributing to a better understanding of its role in ADPKD.

Keywords: Polycystin-1 (PC1), Autosomal dominant polycystic kidney disease (ADPKD), G protein-coupled receptor (GPS), Maltose binding protein (MBP), Protein expression and purification

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