Directed evolution Protein engineering

in directed evolution, random mutagenesis, e.g. error-prone pcr or sequence saturation mutagenesis, applied protein, , selection regime used select variants having desired traits. further rounds of mutation , selection applied. method mimics natural evolution and, in general, produces superior results rational design. added process, termed dna shuffling, mixes , matches pieces of successful variants produce better results. such processes mimic recombination occurs naturally during sexual reproduction. advantages of directed evolution requires no prior structural knowledge of protein, nor necessary able predict effect given mutation have. indeed, results of directed evolution experiments surprising in desired changes caused mutations not expected have effect. drawback require high-throughput screening, not feasible proteins. large amounts of recombinant dna must mutated , products screened desired traits. large number of variants requires expensive robotic equipment automate process. further, not desired activities can screened easily.

natural darwinian evolution can imitated in lab toward tailoring protein properties diverse applications, including catalysis. many experimental technologies exist produce large , diverse protein libraries , screening or selecting folded, functional variants. folded proteins arise surprisingly in random sequence space, occurrence exploitable in evolving selective binders , catalysts. while more conservative direct selection deep sequence space, redesign of existing proteins random mutagenesis , selection/screening particularly robust method optimizing or altering extant properties. represents excellent starting point achieving more ambitious engineering goals. allying experimental evolution modern computational methods broadest, fruitful strategy generating functional macromolecules unknown nature.

the main challenges of designing high quality mutant libraries have shown significant progress in recent past. progress has been in form of better descriptions of effects of mutational loads on protein traits. computational approaches have showed large advances in innumerably large sequence space more manageable screenable sizes, creating smart libraries of mutants. library size has been reduced more screenable sizes identification of key beneficial residues using algorithms systematic recombination. significant step forward toward efficient reengineering of enzymes has been made development of more accurate statistical models , algorithms quantifying , predicting coupled mutational effects on protein functions.

generally, directed evolution may summarized iterative 2 step process involves generation of protein mutant libraries, , high throughput screening processes select variants improved traits. technique not require prior knowledge of protein structure , function relationship. directed evolution utilizes random or focused mutagenesis generate libraries of mutant proteins. random mutations can introduced using either error prone pcr, or site saturation mutagenesis. mutants may generated using recombination of multiple homologous genes. nature has evolved limited number of beneficial sequences. directed evolution makes possible identify undiscovered protein sequences have novel functions. ability contingent on proteins ability tolerant amino acid residue substitutions without compromising folding or stability.

directed evolution methods can broadly categorized 2 strategies, asexual , sexual methods.