BCH210H1 Lecture Notes - Lecture 8: Wild Type, Methionine, Cysteine

• School: UTSG
• Department: Biochemistry
• Course: BCH210H1
• Professor: Reinhart Reithmeier

Document Summary

Technique to separate polypeptides by molecular weights. Uses sds: denaturant dissolves membranes & solubilizes proteins, long hydrophobic carbon chain, hydrophilic sulphate region (negatively charged) Start at the (-) cathode gel runs towards the (+) anode. Transfer proteins from (-) membrane (+) membrane: creates attraction binding b/w protein & matrix. Use primary antibody specific to the target protein: antibody recognize the epitope or natural antigen. Use enzyme-attached (enzyme-conjugated) secondary antibody: enzyme substrate elicits signal. Secreted protein: but doesn"t have di-sulphide bonds! contains both and secondary structures. Genetically unrelated to serine proteases, but has similar active site structure. Important residues: ser 221 (active site residue ie catalytic residue, asp 32, his 64. All elements of the catalytic triad are necessary: mutation to any one (asp 32, his 64, ser 221) no reaction. Method: random mutagenesis to a codon in the gene. Goal: to engineer an oxidative-resistant version of subtilisin, that is also stable to denaturants.