BCH3052 Lecture Notes - Lecture 20: Protein Engineering, Protein Tag, Elution
25 May 2018
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Lecture 20 – Protein Engineering and its Applications
• Protein engineering is the design, construction and characterisation of non-
natural proteins
Why Engineer Proteins?
• To facilitate studies of natural proteins
o Facilitate purification in folded form (affinity tags)
o Identify binding sites (scanning mutagenesis, cross-linking)
o Understand mechanism (enzyme action, folding, etc)
o Obtain structural insights
o Identify binding partners and cellular function (FRET)
o Image location and movement in tissues/cells
• To develop new proteins with new functions
o Change specificity
o Incorporate new chemical functionalities
o Increase shelf-life
o Modulate pharmacokinetics (protein absorption)
o Obtain new binders/therapeutics (antibodies)
o Develop new materials
Rational Protein Engineering
• Use understanding of molecules to make modifications
• Mixture of human input and molecular modelling calculations
• Human input based on
o Creative ideas
o Structure visualization
o Chemical knowledge and intuition
o Wild guesswork
• E.g. Protein Engineering to facilitate purification
o Fuse vector with DNA encoding protein gene tagged with His-Tag
gene
o Clone and express protein (PCR) → purify by binding to Ni2+ coated
beads (column), elute protein from nickel → gel
▪ See that protein is overexpressed – 90% purity
• E.g. Protein Engineering to Map Binding Sites
o CD2 binding surface of adhesion receptor CD58 mapped by
▪ NMR – collect 2D NMR spectrum (peaks)
• Large and small shifts
▪ Ala-scanning mutagenesis
• Use alanine because has CH3 – side chain
o Don’t use glycine – too flexible (affects
structure)
• Measuring effect it has on binding partner
• Red: affects binding
• Green: no affect on binding
▪ Give complimentary information
o If you make a mutation on residue but doesn’t affect binding
▪ Is on binding interface but just has very small effect
• E.g. Insulin Analogues
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