Protein Engineering - Examples of Engineered Proteins

Examples of Engineered Proteins

Using computational methods, a protein with a novel fold has been designed, known as Top7, as well as sensors for unnatural molecules. The engineering of fusion proteins has yielded rilonacept, a pharmaceutical which has secured FDA approval for the treatment of cryopyrin-associated periodic syndrome.

Another computational method, IPRO, successfully engineered the switching of cofactor specificity of Candida boidinii xylose reductase. Iterative Protein Redesign and Optimization (IPRO) redesigns proteins to increase or give specificity to native or novel substrates and cofactors. This is done by repeatedly randomly perturbing the backbones of the proteins around specified design positions, identifying the lowest energy combination of rotamers, and determining if the new design has a lower binding energy than previous ones. The iterative nature of this process allows IPRO to make additive mutations to the protein sequence that collectively improve the specificity towards the desired substrates and/or cofactors. Details on how to download the software implemented in Python and experimental testing of predictions are outlined in the following paper.

Computation-aided design has also been used to engineer complex properties of a highly ordered nano-protein assembly. A protein cage, E. coli bacterioferritin (EcBfr), which naturally shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states is the model protein in this study. Through computational analysis and comparison to its homologs, it has been found that this protein has a smaller than average dimeric interface on its two-fold symmetry axis mainly due to the existence of an interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, a semi-empirical computational method is used to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to the wild-type EcBfr. This computational study also converges on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids results in proteins that fold into alpha-helical monomers and assemble into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirm that, all redesigned proteins, in agreement with the calculations, possesses increased stability. One of the three mutations shifts the population in favor of the higher order oligomerization state in solution as shown by both size exclusion chromatography and native gel electrophoresis.

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