An approach for protein to be completely reversible to thermal denaturation even at autoclave temperatures

Abstract

Reversibility of protein denaturation is a prerequisite for all applications that depend on reliable enzyme catalysis, particularly, for using steam to sterilize enzyme reactors or enzyme sensor tips, and for developing protein-based devices that perform on-off switching of the protein function such as enzymatic activity, ligand binding and so on. In this study, we have successfully constructed an immobilized protein that retains full enzymatic activity even after thermal treatments as high as 120 degrees C. The key for the complete reversibility was the development of a new reaction that allowed a protein to be covalently attached to a surface through its C-terminus and the protein engineering approach that was used to make the protein compatible with the new attachment chemistry.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

“…Therefore, sulfur-free proteins should be resistant to oxidative damage and could be used in highly oxidizing environments such as those found in pollution treatment facilities. Additionally, the availability of a sulfur-free protein allows one to selectively introduce a cysteine or methionine residue to specific positions for the purposes of immobilization, modification, and fragmentation (57)(58)(59). Because almost all naturally occurring proteins contain cysteine and methionine residues, it would be quite useful to develop a reliable strategy to create a sulfur-free protein that is as active as the wild type.…”

Stabilization of Hyperactive Dihydrofolate Reductase by Cyanocysteine-mediated Backbone Cyclization

“…First, the extension attached to the C terminus should not be long in order to avoid nonspecific ligation reactions, whereas the extension attached to the N terminus should be long enough to reach the Cys residue in the C-terminal extension. Second, the residue subsequent to the Cys residue in the C-terminal extension should be Ala, because larger side chains at this position reduced the efficiency of the cyanocysteine-mediated reaction (51). Third, the addition of negative charges at the C-terminal extension would increase the efficiency of the cyclization.…”

“…Third, the addition of negative charges at the C-terminal extension would increase the efficiency of the cyclization. Indeed, the cyanocysteine-mediated protein immobilization on the solid phase having the surface of primary amine proceeded efficiently when several Asp residues were introduced after the Cys-Ala residues of the C-terminal extension (51).…”

“…Alternatively, the cyanocysteine residue can be converted to a dehydroalanine residue by a ␤-elimination reaction (route 2). The use of a primary amine as a nucleophile results in the formation of an amide bond (aminolysis) both intramolecularly (route 3) and intermolecularly (route 4) (45,50,51). The route 3 reaction is quite appealing, because it allows the formation of an amide bond between the C terminus of the protein and the N-terminal ␣-amino group, if the N and C termini are in close proximity in the native structure (Fig.…”

“…2). Such head-totail cyclization by the cyanocysteine-mediated ␣-carbon activation reaction is promising because of the success in the intermolecular aminolysis using the route 4 reaction (45,50,51). It is noteworthy that this method can remove the cysteine residue, whereas one nonnative residue, mostly Cys, remains in the sequence of the final product in the intein-mediated cyclization method.…”