Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution

Abstract

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.

Figure 1

Overview of methods for the randomization of DNA sequences. Random methods introduce changes at positions throughout the gene sequence. Directed methods will randomize only a specific position or positions. Recombination methods bring existing sequence diversity, either from point mutants or from different parental DNA sequences, together in novel combinations.

Figure 2

Random insertion/deletion mutagenesis (RID). The template DNA is converted to a covalently closed single-stranded circle which is cleaved at random sites by Ce(IV)-EDTA treatment. Linker fragment cassettes are then annealed to each end of the cleaved single-stranded DNA via a 10 nt random tail. The construct is amplified using primer sites in the cassettes to produce the second DNA strand. Finally the cassettes are cleaved off using a type II restriction enzyme (recognition site in the cassette) to leave the insertion or deletion behind. The remaining construct containing the modification is converted back to double-stranded circular DNA that can be cleaved with appropriate restriction sites to produce the gene library in a form ready for cloning. Adapted from Murakami et al. (26) with permission from Nature Publishing Group (http://www.nature.com/nbt/).

Figure 3

Approaches to randomizing synthetic DNA. Examples show randomization of one codon with mixed nucleotides (NNN, NNT/C, NNG/T or NNT/G/C) and with trinucleotide phosphoramidites. Synthesis in all three cases commences conventionally 3′ of the randomized codon. At the 3′-end of the randomized codon (A) all four nucleotides, (B) a mixture of T and C, (C) a mixture of G and T or (D) a mixture of T, G and C can be added. In each case a mixture of all four nucleotides is added at each of the remaining two positions. Having a mixture of G and C at the 3′-end of the codon will provide 32 codons, all 20 amino acids and one stop codon. (E) Conversely, the codon can be synthesized by the direct addition of a mixture of 20 trinucleotide phosphoramidites in one step. ALA–TRP represent 20 presynthesized 3-nt codons, one to code for each amino acid.

Figure 4

The MAX method of library generation. A template sequence contains the codons designated for randomization in the form of NNN triplets. A set of selection primers for each position contains those specific triplets that are desired in the final library. The selection primers are annealed to the template. Completely annealed selection primers can then be ligated to each other to form a full-length DNA fragment. Finally, the ligated selection primers are converted to double-stranded DNA for further manipulations.

Figure 5

Homology based methods for recombining DNA sequences. All methods commence with a series of parental DNA sequences. In DNA shuffling this parental DNA is cleaved into random small fragments by DNAse digestion. The fragments are then used in a self-priming reaction to reconstruct the full-length DNA. In StEP the DNA is not fragmented. Instead, small segments are added to the end of a growing DNA strand in a series of very short extension steps. When the strand is removed from an initial template it can reanneal to another to generate a crossover. In RACHITT one parental DNA is used as a template. One strand of this template containing dUTP is generated. Fragments of the opposite strand of the other parental DNAs are then produced and annealed to the template. Non-annealed flaps are then removed by exonuclease digestion and remaining gaps filled in with a DNA polymerase. These fragments are then ligated together and the template strand removed by endonuclease V digestion. The single strand is then converted to double-stranded DNA for further manipulations.