Directed evolution, protein

Combinatorial methods are often referred to as in vitro or directed evolution techniques. In nature, the random DNA mutations that lead to changes in protein sequences occur rarely and so evolution is usually a slow... 

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

These initial systematic studies regarding the directed evolution of PAL allowed several conclusions to be made. Protein sequence space can be explored successfully by applying the following strategies [8c,33j ... 

Lee, PC. et al., Alteration of product specificity of Aeropyrum pemix famesylgeranyl diphosphate synthase (fgs) by directed evolution. Protein Eng. Design Sel. 17, 771, 2004. 

Ai HW, Henderson JN, Remington SJ, Campbell RE (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein structural characterization and applications in fluorescence imaging. Biochem J 400 531-540... 

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Roodveldt, C., Aharoni, A. and Tawfik, D.S. (2005) Directed evolution of proteins for heterologous expression and stability. Current Opinion in Structural Biology, 15, 50-56. 

Hamamatsu, N., Aita, T., Nomiya, Y. et al. (2005) Biased mutation-assembling an efficient method for rapid directed evolution through simultaneous mutation accumulation. Protein Engineering Design Selection, 18, 265-271. 

Kolkman, J.A. and Stemmer, W.P. (2001) Directed evolution of proteins by exon shuffling. Nature Biotechnology, 19, 423-428. 

Bittker, J.A., Le, B.V., Liu, J.M. and Liu, D.R. (2004) Directed evolution of protein enzymes using nonhomologous random recombination. Proceedings of the National Academy of Sciences of the United States of America, 101, 7011-7016. 

Our discussion indicates that, in the light of this clearly directional evolution, a re-evaluation of the role and functioning of the genetic machinery (not just of the coded molecules, DNA, RNA, proteins) is necessary. How does chance mutation lead to directional change when DNA is both conservative and changes of its sequence are undeniably linked only to chance mutation There is growing evidence of occurrence... 

Reetz MT, Wang LW, Bocola M (2006) Directed evolution of enantioselective enzymes iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed 45 1236-1241... 

There have been many attempts to improve protein stability and protein properties, utilizing methods such as random mutagenesis, directed evolution, and rational protein design approaches. In general, these methods are far from straightforward and can be time-consuming. In addition, the stabilization of proteins without loss of function is not a trivial problem. 

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