A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor

Abstract

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.

GFP‐visualized immobilized enzymes: Degradation of paraoxon via organophosphorus hydrolase in a packed column

“…This can lead to an unfavorable decrease in entropy when these sidechains are exposed to the aqueous solution. Thus, the protein stability is increased in their presence (Ru et al, 2000;Stempfer et al, 1996aStempfer et al, , 1996bVoet and Voet, 1995). This increased stability can also enhance the adsorption to the IMAC matrix surface (Wong et al, 1991).…”

Recent advances in biomolecular process intensification

“…Interactions between charged molecules and oppositely charged IEC matrix are controlled reversibly in order to achieve selective capture of target protein with subsequent refolding and elution. Selective binding of denatured target proteins to IEC matrices were facilitated via the fused purification handles such as Z basic (Graslund et al, 2002), polycationic tags (Kweon et al, 2004;Stempfer et al, 1996). However, not all the proteins tested could be successfully recovered in native form through IEC.…”

Matrix-assisted refolding of oligomeric small heat-shock protein Hsp26

“…Third, the refolded protein is eluted at rather high concentrations and can easily be processed. Typically, this method is used for proteins fused to affinity-tags such as the HIS-tag, the cellulose binding domain from Clostridium thermocellum or a glutamic acid tag [12][13][14][15] refolding was used to refold a cysteine variant of Hsp26, an oligomeric small heat-shock protein from Saccharomyces cerevisiae. In contrast to the Hsp26 wild type protein, this cysteine variant was detected in the insoluble fraction after protein synthesis in E. coli BL21 (DE3) cells and in vitro refolding involving anion-exchange (IEX) and size exclusion chromatography (SEC) was required.…”