Mutagenesis 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. 

All these techniques create genetic diversity by recombination and point mutations and are well developed. However, insertions and deletions (indels) are also important types of mutation which are probably underrepresented in many conventional mutagenesis strategies. Methods for incorporation of indels in predefined positions in a combinatorial manner have been developed.Although there are some published studies on their use in the directed evolution of biocatalysts,the full potential of these newer methods of gene mutation for enzyme improvement are yet to be demonstrated. 

The following new trends in enzymatic synthesis can be delineated the development of new enzymatic reactions enzyme immobilization and stabilization the use of organic solvents and two phase systems site-directed mutagenesis chemical modification of enzymes antibody catalysis catalysis by RNA and DNA de novo design ofbiocatalists employment of recombinant DNA for production of enzymes and use computational and combinatorial methods... 

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. 

Merino E, Osuna F, Bolivar F, Soberon X, A general PCR-based method for single or combinatorial oligonucleotide-directed mutagenesis on pUC-M13 vectors, Bio techniques, 12 508-510, 1992. 

The starting point for further exploration of protein sequence space was the conjecture that Stemmer s method of Combinatorial Multiple Cassette Mutagenesis (CMCM) [74] can be applied in appropriately modified form. It is a special type of DNA-shuffling which can be used to generate mutant gene libraries in which cassettes composed of random or defined sequences and the wild-type are incorporated randomly. CMCM had been developed for use in the area of functional antibodies [74],... 

Selected Methods Error-prone PCR Mutator strains Chemical mutagens Combinatorial cassette mutagenesis Recursive ensemble mutagenesis Scanning saturation mutagenesis... 

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. 

New combinatorial mutagenesis methods in molecular genetics make it possible to generate simultaneously millions of altered proteins in a single library of mutants. In the study of pigment-protein complexes, this advance in biology necessitated the invention of an instrument capable... 

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