Discovery and engineering of enhanced SUMO protease enzymes

Yue-Ting K Lau,Vladimir Baytshtok,Tessa A Howard,Brooke M Fiala,Jaylee M Johnson,Lauren P Carter,David Baker,Christopher D Lima,Christopher D Bahl

doi:10.1074/jbc.ra118.004146

Abstract

Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.

Highlights

Genetic fusion to the C terminus of small ubiquitin-like modifier (SUMO)5 can chaperone folding and increase the soluble yield obtained from heterologous protein expression [1,2,3,4]
Ulp1 is a 621-amino acid protein, and residues 403– 621 constitute a conserved protease domain [2]. This fragment can be purified without the N-terminal residues, is constitutively active, and can be used for removing genetically fused SUMO domains from recombinant protein in vitro in reactions that are analogous to the processing of pre-Smt3 [2]; we will refer to this construct as Ulp1_WT
Using Rosetta, we developed a generally applicable computational method that identifies hydrophobic residue positions on the surface of a protein and determines amino acid substitutions to polar residues that yield low-energy solutions

Summary

Introduction

Genetic fusion to the C terminus of small ubiquitin-like modifier (SUMO) can chaperone folding and increase the soluble yield obtained from heterologous protein expression [1,2,3,4]. Ulp is a 621-amino acid protein, and residues 403– 621 constitute a conserved protease domain [2] This fragment can be purified without the N-terminal residues, is constitutively active, and can be used for removing genetically fused SUMO domains from recombinant protein in vitro in reactions that are analogous to the processing of pre-Smt3 [2]; we will refer to this construct as Ulp1_WT. Precipitation of the enzyme can lead to incomplete digestion of SUMO fusion constructs as well as potentially nucleate

Objectives

Results

Conclusion