Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis.
If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.
Wallace
C J
CJ
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Guillemette
J G
JG
Hibiya
Y
Y
Smith
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003488:Cyanogen Bromide; D003574:Cytochrome c Group / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D006736:Horses; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012694:Serine
D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012694:Serine; D008715:Methionine; D010088:Oxidoreductases; C022691:succinate oxidase; D003488:Cyanogen Bromide