Protein Structure and Function Lecture 5 Notes: Protein Engineering Protein Engineering - Proteins have evolved in nature to fulfil a defined in vivo function. However, they have not evolved for use outside of their natural environment (e.g. as drugs or in technology), which present unique challenges such as the need for high stability (e.g. to store for long periods,) and expression levels (e.g. for large-scale manufacturing). Protein properties can be changed through protein engineering. - Protein engineering is used to: (a) Study protein functions (change/modify structure of native proteins to study their function, most frequently used approach to study protein structure/function) (b) Improve protein stability/solubility and overcome expression limitations (c) Improve protein functions (e.g. enzymes) (d) Create new functional proteins (e.g. therapeutic proteins/antibodies, regulatory proteins, new biosensors/diagnostics) - This technique can be used to improve enzymes and enzyme inhibitors so that they are more stable (e.g. thermostable DNA polymerases for PCR) and efficient (better catalytic function for use in biotechnology) - New proteins/enzymes can be fabricated (e.g. higher processivity, new metabolic pathways) to be used as drugs for therapeutic applications or in biotechnology - For example, this can be achieved by combining different known protein domains. Because domains can fold independently, they are not affected by the presence of another domain, so the combination of different functional units creates a new functional protein. - Examples of therapeutic proteins and antibodies created using protein engineering include unconjugated antibodies (e.g. Fc attracts macrophages), enzyme- or toxin-conjugated antibodies (recombinant) and radioactively tagged antibodies (chemical modification). - Regulatory proteins (e.g. transcription factors) can be engineered to regulate expression of genes (up/down) which may be useful in genetic research and to cure diseases (e.g. zinc- finger proteins, alternative to small drugs). - New biosensors and diagnostics can be created using protein engineering by devising proteins which fit existing technologies - The best-selling therapeutic medicine (based on worldwide sales) is the Human monoclonal antibody Humira (adalimumab). This is used for the treatment of severe autoimmune disease (e.g. rheumatoid arthritis, psoriatic arthritis, Chron's disease, etc.) as it inactivates the tumour necrosis factor alpha TNF. This is the first fully in vitro human monoclonal antibody ever engineered and is estimated to be worth $19.9 billion. - To improve the stability of proteins, rational engineering aims to optimise structure and folding. This can include introducing disulphide bonds (+22º with three new S-S bridges), stabilising dipoles (and other modifications) of a-helices, filling hydrophobic cavities in their core hydrophobic regions and modifying ß-sheets. - For example, in the phage T4 lysozyme, two cysteines were introduced very close together to allow a spontaneous reaction to occur, forming a disulphide bridge between residues 21-142 (i.e. spanning the active site cleft). This substantially enhanced the stability of the protein
(increases the melting temperature by 11?), without making the folded structure more rigid or affecting the functional properties of the enzyme. Furthermore, the triple-disulphide variant unfolds at 23.4? higher than the wild-type lysozyme. The effect of the disulphide bond is presumed to be due to