Enzyme mutant
a technology of enzymolysis and mutants, applied in the field of enzymolysis mutants, can solve the problems of slow and expensive technology of existing
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1 Materials and Methods
1.1 Overview
[0368]One method of sequencing nucleic acids using stochastic sensing involves the use of an exonuclease attached to the α-HL pore. This can be done by introducing a chemical linkage between the two proteins. The chemical linkage joins one cysteine on the exonuclease to one cysteine on the hemolysin, either directly (a disulphide bond), or via a linker molecule between the two cysteine residues.
[0369]In order to introduce the chemical linkage in a controlled and directed approach it was necessary to replace 5 naturally occurring cysteine residues found within Exonuclease I with other residues. An additional mutation would then be made in order to introduce the cysteine residue attachment point. Computer modeling was used to identify suitable attachment positions (see below).
1.2 Modelling Exo I CYS Knockout Mutations
[0370]Molecular modelling has been used in an attempt to predict which mutations of the 5 wild type cysteine residues of E. coli Exo I ...
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