• Document: Chapter 2 Expanding the Synthetic Protein Universe by Guided Evolutionary Concepts
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Chapter 2 Expanding the Synthetic Protein Universe by Guided Evolutionary Concepts Abstract The genetic information content of a cell is maintained by the sequence composition of the DNA. The changes in the nucleotide content will potentially alter its transcriptional and translational events thus influencing the characteristics of the newly synthesized proteins. These nature’s alterations can be helpful in the evolution of proteins with novel/improved functionalities or they can contribute to the patho- genesis with loss of native functionalities. Unraveling the logistics of such a molecular evolutionary process is resourceful to strategically implement it for the benefit of the mankind through laboratory techniques. The laboratory process of synthesizing novel proteins in a constructive way through evolutionary guided principles is called “di- rected evolution”. This chapter will discuss various techniques, their strengths and pitfalls that are developed under the umbrella of directed evolution scheme. Keywords Directed evolution  Homologous recombination Random     mutagenesis Focused mutagenesis Gene and protein libraries Phage display methods 2.1 Directed Evolution Directed evolution (DE) has imitated the nature’s scheme of evolution for the cre- ation of new proteins. But there are several distinctions between the natural evo- lution and laboratory evolution. Mother Nature takes millions of years to evolve in contrast to directed evolution which takes only months or days to evolve proteins with novel characteristics. The distinguishing features of directed evolution methods are requiring no prior knowledge about protein structure and function gives an upper hand to this strategy in protein designing over the rational design approach. But it is leaned upon the two expectations from proteins, one is the tolerance power of proteins to a limited degree of amino acid residue substitutions without compro- mising its folding or stability and the other is, Mother nature has explored only small chunk of beneficial sequences but large unplumbed portion of sequence may unveil the admirable answers to peculiar biological disputes [1, 2]. Directed evolution © The Author(s) 2017 27 K.M. Poluri and K. Gulati, Protein Engineering Techniques, Springer Briefs in Forensic and Medical Bioinformatics, DOI 10.1007/978-981-10-2732-1_2 28 2 Expanding the Synthetic Protein Universe … strategy has gained victory to engineer ample proteins with desired activity, stability, selectivity, specificity, and affinity [3]. This approach has circumscribed the protein engineering era, as its chunks have been exploited to engineer operons, pathways, viruses, and whole organisms [4]. This method is an iterative two step method in which (a) libraries of protein variants are created followed by (b) high throughput screening process with the aim to select the variants with improved traits, which will then serve as template for the subsequent cycles and selection procedures. This process will continue until we get the variant whose properties are best tuned to the desired level. This implies that success rates of directed evolution are based on combination of two tools that is creation of diverse libraries and appropriate screening of these libraries [1, 5, 6]. With the aim to get the protein with desirable properties, it is essential to adopt the appropriate methodology for the creation of good libraries. Good libraries indicate the ones which are redundant and encompass large number of mutants with reno- vated properties. Such libraries can then be easily screened to get proteins with refined characteristics [7]. This implies that directed evolution is all in our hands, means a good start up will end up with excellent end results. To inaugurate the directed evolution process, several methods were developed for the creation of libraries early in 1990s by Arnold and co-workers which were asexual [8–10] but after that some sex was incorporated by stemmer in developing these libraries [11, 12]. Essentially, there are two major strategies for the creation of these libraries as depicted in Fig. 2.1. They comprise of: (a) Asexual methods (random mutagenesis and focused mutagenesis) and (b) sexual methods (homolo- gous and non-homologous recombination). Most of the popular methods under these categories are explained in detail in the following sections. However, the method of choice will depend on various factors like fraction of destined properties Fig. 2.1 Broad classification of direction evolution methods based on their principle methodologies 2.1 Directed Evolution 29 of protein to innovate, extent of innovation required, present structural and mechanistic status of destined protein properties, knowledge about the properti

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