BCH210H1 Lecture Notes - Lecture 8: Sodium Dodecyl Sulfate, Subtilisin, Fusion Protein

• School: UTSG
• Department: Biochemical Engineering and Applied Chemistry
• Course: BCH210H1
• Professor: Wright

Document Summary

Proteins unfold and are separated by size. Denaturing detergent and dissolves membranes and solubilizes proteins. The purified expressed (his-tagged) fusion protein eluted from the column with 400 mm imidazole. Same as lane 1-3 but blotted with anti fusion protein antibody. Genetically unrelated to mammalian serine proteases, but have similar active site structure. Secreted in large amounts from many bacillus species. X-ray structures have been determined (globular protein, helices and sheets) Determining effect of disulfide bonds on secretion, structure and stability. Subtilisin is stable to denaturants but susceptible to chemical oxidation. Peptide mapping studies showed that oxidizing met-222 90% loss in enzyme activity. Met-222 is conserved residue in all subtilisins. Proved by the fact that the enzyme is easily inactivated by oxidizing met-222. All 19 aa"s were produced at subtilisin codon 222 to see if they"re expressed. All 19 subtilisin mutants were purified and specific mutants were purified. All worse than wild type but cys.