Protein engineering combinatorial methods

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Progress in utilizing domain swapping for protein engineering has been partially hindered by the lack of combinatorial methods for per-... 

Youvan, D. C. Goldman, E. Delagrave, S. Yang, M. M. Recursive ensemble mutagenesis a combinatorial optimization technique for protein engineering Methods Enzymol. 1995, 246, 732-749. 

In order to actually make alterations to proteins, two main approaches have been developed and described (1) rational mutagenesis, and (2) combinatorial methods (Figure 14.1). In rational mutagenesis, a top-down approach is taken, where a hypothesis is made about mutations at a specific location, which is often guided by 3-D structural information, and the hypothesis is tested through the mutation of specific amino acids and assays of the subsequent mutant proteins. This is in contrast to the combinatorial paradigms, which are described in the next section, where a bottom-up approach is taken. In this approach, a library of different mutant proteins is produced. A method is then developed to screen or select members of the library that have an improved trait, and then the mutations that caused the improvement are determined later. Both of these methods have been extensively used in the literature for the successful engineering of a wide variety of important proteins. 

Site-directed mutagenesis has been a valuable method for the engineering of many proteins, but a significant limitation on this technique is that it can be difficult to know what mutations should be made in order to obtain a desired functionality. For example, in order to increase the thermostability of a protein, it is not clear by looking at a 3-D structure which amino acid side chains will affect this trait. In addition, improvements made in the thermostability of the enzyme may adversely affect other properties of the protein, such as enzymatic activity. Therefore, there has been a good deal of interest in combinatorial methods for protein engineering, which can be used to sample a large area of the protein solution space, and thus rapidly identify proteins with desired functionalities. 

Once mutant proteins are produced, either rationally or combinatoriaUy, the new proteins need to be characterized in order to determine the full effects of the underlying mutations. It has been repeatedly shown that improvements in one trait can come at the cost of another trait. This can be especially true in combinatorial methods, where you get what you screen for. Once the effects of the mutations are understood, the process is repeated in order to produce further improvements in the protein of interest. In some cases, it may also be useful to combine the methods or protein engineering. For example, combinatorial methods may suggest an area in the protein that should be further investigated using site-directed mutagenesis. 

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