Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles.
There are two general strategies for protein engineering, 'rational' protein design and directed evolution. These techniques are not mutually exclusive; researchers will often apply both. In the future, more detailed knowledge of protein structure and function, as well as advancements in high-throughput technology, may greatly expand the capabilities of protein engineering. Eventually, even unnatural amino acids may be incorporated, thanks to a new method that allows the inclusion of novel amino acids in the genetic code.
In rational protein design, the scientist uses detailed knowledge of the structure and function of the protein to make desired changes. In general, this has the advantage of being inexpensive and technically easy, since site-directed mutagenesis techniques are well-developed. However, its major drawback is that detailed structural knowledge of a protein is often unavailable, and, even when it is available, it can be extremely difficult to predict the effects of various mutations.
Computational protein design algorithms seek to identify novel amino acid sequences that are low in energy when folded to the pre-specified target structure. While the sequence-conformation space that needs to be searched is large, the most challenging requirement for computational protein design is a fast, yet accurate, energy function that can distinguish optimal sequences from similar suboptimal ones.
In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used to pick out variants that have the desired qualities. Further rounds of mutation and selection are then applied. This method mimics natural evolution and, in general, produces superior results to rational design. An additional technique known as DNA shuffling mixes and matches pieces of successful variants in order to produce better results. This process mimics the recombination that occurs naturally during sexual reproduction. The advantage of directed evolution is that it requires no prior structural knowledge of a protein, nor is it necessary to be able to predict what effect a given mutation will have. Indeed, the results of directed evolution experiments are often surprising in that desired changes are often caused by mutations that were not expected to have that effect. The drawback is that they require high-throughput, which is not feasible for all proteins. Large amounts of recombinant DNA must be mutated and the products screened for desired qualities. The sheer number of variants often requires expensive robotic equipment to automate the process. Furthermore, not all desired activities can be easily screened for.
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 that 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 structure of the proteins around specified design positions, identifying the lowest energy combination of rotamers, and determining whether the new design has a lower binding energy than previous ones.
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 due mainly 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, possess 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.
Enzyme engineering is the application of modifying an enzyme's structure (and, thus, its function) or modifying the catalytic activity of isolated enzymes to produce new metabolites, to allow new (catalyzed) pathways for reactions to occur, or to convert from some certain compounds into others (biotransformation). These products will be useful as chemicals, pharmaceuticals, fuel, food, or agricultural additives.
An enzyme reactor  consists of a vessel containing a reactional medium that is used to perform a desired conversion by enzymatic means. Enzymes used in this process are free in the solution.
- Biomolecular engineering
- Expanded genetic code
- Fast parallel proteolysis (FASTpp)
- Gene synthesis
- Nucleic acid analogues
- Protein folding
- Protein design
- Protein structure prediction software
- SCOPE (protein engineering)
- Structural biology
- Synthetic biology
- Kuhlman, Brian; Dantas, Gautam; Ireton, Gregory C.; Varani, Gabriele; Stoddard, Barry L. & Baker, David (2003), "Design of a Novel Globular Protein Fold with Atomic-Level Accuracy", Science 302 (5649): 1364–1368, Bibcode:2003Sci...302.1364K, doi:10.1126/science.1089427, PMID 14631033
- Looger, Loren L.; Dwyer, Mary A.; Smith, James J. & Hellinga, Homme W. (2003), "Computational design of receptor and sensor proteins with novel functions", Nature 423 (6936): 185–190, Bibcode:2003Natur.423..185L, doi:10.1038/nature01556, PMID 12736688
- Khoury, GA; Fazelinia, H; Chin, JW; Pantazes, RJ; Cirino, PC; Maranas, CD (October 2009), "Computational design of Candida boidinii xylose reductase for altered cofactor specificity", Protein Science 18 (10): 2125–38, doi:10.1002/pro.227, PMC 2786976, PMID 19693930
- The iterative nature of this process allows IPRO to make additive mutations to the protein sequence that collectively improve the specificity toward 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: Khoury, GA; Fazelinia, H; Chin, JW; Pantazes, RJ; Cirino, PC; Maranas, CD (October 2009), "Computational design of Candida boidinii xylose reductase for altered cofactor specificity", Protein Science 18 (10): 2125–38, doi:10.1002/pro.227, PMC 2786976, PMID 19693930
- Ardejani, MS; Li, NX; Orner, BP (April 2011), "Stabilization of a Protein Nanocage through the Plugging of a Protein–Protein Interfacial Water Pocket", Biochemistry 50 (19): 4029–4037, doi:10.1021/bi200207w, PMID 21488690
- ['Designer Enzymes' at http://www.medicalnewstoday.com/articles/101236.php] Accessed 22 May 2009.
- [Enzyme reactors at http://www.lsbu.ac.uk/biology/enztech/reactors.html] Accessed 22 May 2009.
- EGAD; a free and open-source program for automated protein design
- servers for protein engineering and related topics based on the WHAT IF software
- DNA2.0 Protein Engineering
- Enzymes Built from Scratch - Researchers engineer never-before-seen catalysts using a new computational technique, Technology Review, March 10, 2008