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.

Proteins from Thermophilic Thermus thermophilus Often Do Not Fold Correctly in a Mesophilic Expression System Such as Escherichia coli

“…Naturally, protein crowding may occur in both E. coli and T. thermophilus expression systems, as well as other systems, but recombinant expression systems are more likely to have this effect. , E. coli natural cellular concentration could be unsuitable for T. thermophilus protein folding and may lead to increased crowding and inclusion body formation . In addition, chaperone-assisted misassemble prevention mechanisms may be compromised in recombinants as they would lack the required chaperones. , …”

“…26 In addition, chaperone-assisted misassemble prevention mechanisms may be compromised in recombinants as they would lack the required chaperones. 52,54 4.5. Significance.…”

Techniques for Enzyme Purification

“…Next to being a suitable purification step [28,29,35,40,42], SEC is extremely useful to get information about the molecular weight of the native protein and its possible subunit composition [43]. By using this technique, we established that 4-hydroxybenzoate 3-hydroxylase from P. fluorescens is a homodimer, both in its holo and apo form [19,44].…”

“…The hyperthermostable laminarinase from Pyrococcus furiosus (LamA) was also purified using the His-tag technique. The cell-fee extract, obtained after breaking overexpressing E. coli cells and subsequent centrifugation, was subjected to Ni-NTA affinity chromatography, in which the LamA was eluted with a linear gradient of 0-0.5 M imidazole [42]. Next to highly active monomers, a minor part of the soluble enzyme constituted less active native-like oligomers and non-native monomers.…”

A set of simple methods for detection and extraction of laminarinase

“…Consequently, a large bulk of literature describe AS precipitation as the preliminary step in protein purification. In the recent times, there is a predictable shift towards cloning of laminarinase gene in recombinant hosts 1,[22][23][24][25] . Based on a survey of the literature, the strategies for the extraction and purification of laminarinase can broadly be classified into the following categories:(i) AS precipitation of the supernatant followed by chromatographic methods of purification (gel filtration, ion exchange, affinity and hydrophobic interaction) 8,17-22 .…”