Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein

• Published in: Chembiochem : a European journal of chemical biology 2015-11, Vol.16 (16), p.2392-2402
• Main Authors: Pierre, Brennal, Labonte, Jason W., Xiong, Tina, Aoraha, Edwin, Williams, Asher, Shah, Vandan, Chau, Edward, Helal, Kazi Yasin, Gray, Jeffrey J., Kim, Jin Ryoun
• Format: Article
• Language: English
• Subjects: Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; beta-Lactamases - genetics; beta-Lactamases - metabolism; Calorimetry, Differential Scanning; Chromatography, Gel; Circular Dichroism; Entropy; Kinetics; protein aggregation; protein engineering; protein fusion; Protein Stability; Protein Unfolding; protein-protein interactions; Proteins; Pyrococcus furiosus - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201500310

A universal method that improves protein stability and evolution has thus far eluded discovery. Recently, however, studies have shown that insertional fusion to a protein chaperone stabilized various target proteins with minimal negative effects. The improved stability was derived from insertion into a hyperthermophilic protein, Pyrococcus furiosus maltodextrin‐binding protein (PfMBP), rather than from changes to the target protein sequence. In this report, by evaluating the thermodynamic and kinetic stability of various inserted β‐lactamase (BLA) homologues, we were able to examine the molecular determinants of stability realized by insertional fusion to PfMBP. Results indicated that enhanced stability and suppressed aggregation of BLA stemmed from enthalpic and entropic mechanisms. This report also suggests that insertional fusion to a stable protein scaffold has the potential to be a useful method for improving protein stability, as well as functional protein evolution. Determining determinants: Insertional fusion to Pyrococcus furiosus maltodextrin‐binding protein (PfMBP) increases thermodynamic and kinetic stability of β‐lactamase (BLA) homologues and suppresses their aggregation. The improved stability is derived from enthalpic and entropic mechanisms. Our results demonstrate that insertional fusion to PfMBP might be a generally applicable means to improve protein stability and benefit protein evolution.