BCH210H1 Lecture Notes - Lecture 17: Protein Engineering, Expression Vector, Nonsense Mutation

• School: UTSG
• Department: Biochemistry
• Course: BCH210H1
• Professor: Stavroula Andreopoulos, Roman Melnyk, Sian Patterson, Reinhart Reithmeier

Document Summary

To check for protein stability if there is no green fluorescence, then the protein is probably not stable. In the early 1970s methods emerged to manipulate dna. Introduce point mutations into proteins or truncations (elimination of the c- or n-terminus portion of the protein or the introduction of a nonsense mutation) Truncations for post-translational modifications, binding interactions, membrane insertions, cellular trafficking etc. Ability to stitch dna together to make chimeric proteins. Ability to express large quantities of these proteins we need protein in order of mg; we can overexpress these proteins in bacteria or mammalian cells. Dna enzymes & dna ligase cut, rearrange and ligate pieces of dna together. Restriction endonucleases cleave dna at specific palindromic dna sequences. Pcr typically thought to amplify dna but can be used to introduce mutations. Plasmids customized to make expression vectors for protein engineering.