Patent ID: 9062296
Filing Date: 2015-06-23
Classification: C12N,C12Y,G16B

Abstract:
1. A method of making a recombinant meso-active thermo-stable protein by transplanting an active surface from a donor protein to a recipient protein, said method comprising the steps of: a. superimposing a representation of three dimensional structures of the donor protein and the recipient protein and estimating a degree of superimposibility of their structures at a polypeptide backbone level of the donor protein and the recipient protein to a root mean square deviation (RMSD) of between about 0.5-2.5 Angstroms, b. generating structural analogy-based sequence alignments of protein sequences and DNA sequences encoding the protein sequences of the recipient protein and donor protein based upon step (a); c. identifying specific amino acid residues in the protein sequences of the recipient protein and the donor protein that constitute a beta sheet-based substrate-binding and catalytically active surface of the recipient protein and donor protein, respectively, d. determining specific amino acid residues that are identical in the recipient protein and donor protein, and eliminating identical amino acid residues that represent thermophile functional characteristics of the recipient protein to obtain a set of corresponding non-identical residues that represent mesophile functional characteristics of the donor protein, at structurally-equivalent positions in the donor protein and recipient protein, e. synthesizing a polypeptide having an amino acid sequence wherein a non-contiguous set of amino acid residues comprising said beta sheet-based substrate-binding and catalytically active surface of the recipient protein are replaced with the corresponding non-contiguous set of non-identical residues of the donor protein at the structurally equivalent position through splicing by overlap extension (SOE) polymerase chain reaction (PCR) based synthesis to produce a recombinant meso-active thermo stable protein, f. purifying said recombinant meso-active thermo-stable protein obtained in step (e), and g. measuring activity and stability of said recombinant meso-active thermo-stable protein.