title: Chaotropic heat treatment resolves native-like aggregation of a heterologously produced hyperthermostable laminarinase.
abstract: Production of hyperthermostable enzymes in mesophilic hosts frequently causes undesired aggregation of these proteins. During production of Pyrococcus furiosus endo-β-1,3 glucanase (LamA) in Escherichia coli, soluble and insoluble species form. Here, the authors address the composition of this mixture, including the nature of LamA conformers, and establish a method to increase the yield of native monomer. With gel electrophoresis, size-exclusion chromatography, light scattering, circular dichroism and enzyme kinetics the authors show that approximately 50 % of heterologously produced LamA is soluble, and that 40 % of this fraction constitutes native-like oligomers and non-native monomers. Soluble oligomers display, like native LamA monomer, substrate inhibition, although with poor activity. Treatment of soluble oligomers with 3 M guanidinium hydrochloride at 80 °C yields up to 75 % properly active monomer. Non-native monomer shows low specific activity without substrate inhibition. Incubating non-native monomer with 3 M guanidinium hydrochloride at 80 °C causes formation of 25 % native LamA. Also, a large amount of insoluble LamA aggregates can be converted into soluble native monomer by application of this procedure. Thus, chaotropic heat treatment can improve the yield and quality of hyperthermostable proteins that form aberrant species during production in E. coli.
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Wageningen, The Netherlands.
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Wageningen, The Netherlands.|Present address: Technology & Support, Aspen Oss B.V., Oss, The Netherlands.
authors.forename: Astrid A
authors.initials: AA
authors.lastname: Geerke-Volmer
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Wageningen, The Netherlands.
authors.forename: Carlo P M
authors.initials: CPM
authors.lastname: van Mierlo
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Wageningen, The Netherlands.
authors.forename: Willem J H
authors.initials: WJH
authors.lastname: van Berkel
mesh_terms: D055162:Biocatalysis; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006146:Guanidines; D006358:Hot Temperature; D066329:Protein Aggregates; D055550:Protein Stability; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D013861:Thiocyanates
chemical_list: D006146:Guanidines; D066329:Protein Aggregates; D013861:Thiocyanates; C054436:guanidine thiocyanate; D044602:Cellulases
keywords: Biocatalysis; Endoglucanase; Glycosyl hydrolase; Protein expression; Protein stability