title: Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield.
abstract: Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.
authors.affiliation: Ultrasensitive Protein Detection Unit, Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Simeonov
authors.forename: Renate
authors.initials: R
authors.lastname: Berger-Hoffmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
authors.forename: Norbert
authors.initials: N
authors.lastname: Sträter
authors.forename: Thole
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D055672:Static Electricity
chemical_list: D021122:Protein Subunits; D004765:Enteropeptidase