title: Enhancing the stability and solubility of TEV protease using in silico design.
abstract: The ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. Poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. Rational design is achieved here by means of the PoPMuSiC program, which performs in silico predictions of stability changes upon single-site mutations. We have used this program to increase the stability of the tobacco etch virus (TEV) protein. TEV is a 27-kDa nuclear inclusion protease with stringent specificity that is commonly used for the removal of solubility tags during protein purification protocols. However, while recombinant TEV can be produced in large quantities, a limitation is its relatively poor solubility (generally approximately 1 mg/mL), which means that large volumes and often long incubation times are required for efficient cleavage. Following PoPMuSiC analysis of TEV, five variants predicted to be more stable than the wild type were selected for experimental analysis of their stability, solubility, and activity. Of these, two were found to enhance the solubility of TEV without compromising its functional activity. In addition, a fully active double mutant was found to remain soluble at concentrations in excess of 40 mg/mL. This modified TEV appears thus as an interesting candidate to be used in recombinant protein technology.
authors: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Australia.
authors: Lisa D
authors: LD
authors: Cabrita
authors: Dimitri
authors: D
authors: Gilis
authors: Amy L
authors: AL
authors: Robertson
authors: Yves
authors: Y
authors: Dehouck
authors: Marianne
authors: M
authors: Rooman
authors: Stephen P
authors: SP
authors: Bottomley
mesh_terms: D010450:Endopeptidases / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D010450:Endopeptidases; C099488:TEV protease; D019791:Guanidine