Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein

Daniel Gonzalez (1) , Julien Hiblot (1) , Nune Darbinian (2) , Jernelle C. Miller (2) , Guillaume Gotthard (1) , Shohreh Amini (2) , Eric Chabriere (1) , Mikael Elias (3)

1 URMITE - Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes
2 Temple University [Philadelphia]
3 Weizmann Institute of Science [Rehovot, Israël]

Stable and soluble proteins are ideal candidates for functional and structural studies. Unfortunately, some proteins or enzymes can be difficult to isolate, being sometimes poorly expressed in heterologous systems, insoluble and / or unstable. Numerous methods have been developed to address these issues, from the screening of various expression systems to the modification of the target protein itself. Here we use a hydrophobic, aggregation-prone, phosphate-binding protein (HPBP) as a case study. We describe a simple and fast method that selectively uses ancestral mutations to generate a soluble, stable and functional variant of the target protein, here named sHPBP. This variant is highly expressed in Es-cherichia coli , is easily purified and its structure was solved at much higher resolution than its wild-type progenitor (1.3 versus 1.9 Å, respectively).

Mots clés: Hydrophobic proteins; Protein solubilization; Protein engineering; DING proteins; Phosphate-binding proteins; Ancestral librairies

Domaines: Biologie structurale [q-bio.BM]

Daniel Gonzalez, Julien Hiblot, Nune Darbinian, Jernelle C. Miller, Guillaume Gotthard, et al.. Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein. FEBS Open Bio, 2014, 4 (1), pp.121-127. ⟨10.1016/j.fob.2013.12.006⟩. ⟨hal-01772922⟩