Strategies and computational tools for improving randomized protein libraries

In the last decade, directed evolution has become a routine approach for engineering proteins with novel or altered properties. Concurrently, a trend away from purely 'blind' randomization strategies and towards more 'semi-rational' approaches has also become apparent. In this review, we discuss ways in which structural information and predictive computational tools are playing an increasingly important role in guiding the design of randomized libraries: web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, while algorithms such as GLUE, PEDEL and DRIVeR are useful for estimating library completeness and diversity. In addition, we review recent methodological developments that facilitate the construction of unbiased libraries, which are inherently more diverse than biased libraries and therefore more likely to yield improved variants.     

Mutant library construction in directed molecular evolution

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).     

A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 10⁹ sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an alpha-synuclein ep-PCR library and NNS synthetic oligonucleotide libraries are given as examples.     

Discovery and optimization of pseudopeptide leads towards peptidomimetic drugs

Summary Progress in the identification of primary leads is increasingly obtained by the production of molecular diversity via the synthesis of peptide and non-peptide libraries. In this review, statistical considerations are made about the feasibility and reliability of peptide libraries. It is shown that the number of beads per peptide engaged in synthesis controls the relative concentration of any two peptide types, while the total amount of resin determines the feasibility of the complete library in relation to peptide size. Molecular modelling is used to estimate the conformational diversity. A preliminary analysis of libraries by NMR, MS, MS/MS and capillary electrophoresis is advocated. Examples are given of optimized peptide leads in hirudin, neurokinin, bradykinin and angiopeptin series. Finally, the relative effect of hydrogen-bond potential and overall lipophilicity on oral absorption is evaluted on neurokinin-1 and endothelin-1 receptor antagonists.     

Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR

The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv(+)). dNTP alpha S and three degenerate bases (P, K and I) are used to control mutational bias flexibly. After quantifying incorporation rates of dPTP, dKTP and dITP by terminal transferase using a luciferase-based assay and investigating the read and/or write activities of eight DNA polymerases, a transversion-enriched protocol has been developed. In a mutant library generated using dGTP alpha S and dPTP, transversion frequencies of 16.22-22.58% (G-->T) and 6.38-9.69% (G-->C) were achieved. These mutational spectra are complementary and occur twice as frequently in comparison to standard epPCR methods employing Taq DNA polymerase. For generating more complex mutant libraries, the occurrence of consecutive nucleotide exchanges was increased by 10(5)-10(6)-fold compared to epPCR. Finally, 16.7% of all sequenced mutants contained consecutive nucleotide exchanges composed mainly of a transversion followed by a transition.     

Estimating and comparing the rates of gene discovery and expressed sequence tag (EST) frequencies in EST surveys

MOTIVATION: Expressed sequence tag (EST) surveys are an efficient way to characterize large numbers of genes from an organism. The rate of gene discovery in an EST survey depends on the degree of redundancy of the cDNA libraries from which sequences are obtained. However, few statistical methods have been developed to assess and compare redundancies of various libraries from preliminary EST surveys. RESULTS: We consider statistics for the comparison of EST libraries based upon the frequencies with which genes occur in subsamples of reads. These measures are useful in determining which one of several libraries is more likely to yield new genes in future reads and what proportion of additional reads one might want to take from the libraries in order to be likely to obtain new genes. One approach is to compare single sample measures that have been successfully used in species estimation problems, such as coverage of a library, defined as the proportion of the library that is represented in the given sample of reads. Another single library measure is an estimate of the expected number of additional genes that will be found in a new sample of reads. We also propose statistics that jointly use data from all the libraries. Analogous formulas for coverage and the expected numbers of new genes are presented. These measures consider coverage in a single library based upon reads from all libraries and similarly, the expected numbers of new genes that will be discovered by taking reads from all libraries with fixed proportions. Together, the statistics presented provide useful comparative measures for the libraries that can be used to guide sampling from each of the libraries to maximize the rate of gene discovery. Finally, we present tests for whether genes are equally represented or expressed in a set of libraries. Binomial and chi2 tests are presented for gene-by-gene comparisons of expression. Overall tests of the equality of proportional representation are presented and multiple comparisons issues are addressed. These methods can be used to evaluate changes in gene expression reflected in the composition of EST libraries prepared from different tissue types or cells exposed to different environmental conditions. AVAILABILITY: Software will be made available at http://www.mathstat.dal.ca/~tsusko     

Exploiting recombination in single bacteria to make large phage antibody libraries

The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.     

Mathematical expressions useful in the construction, description and evaluation of protein libraries

The creation of protein libraries by random mutagenesis and cassette mutagenesis has proven to be a successful method of protein engineering. Appropriate statistical analysis is important for the proper construction of these libraries and even more important for the interpretation of data from these libraries. We present simple mathematical expressions useful in the creation and evaluation of such libraries. These equations are useful in estimating the distribution of mutations, the degeneracy of the library and the frequency of a particular clone in the library. In addition, general equations addressing the probability that a particular clone is in a library, the probability that a library is complete, and as the consequences of retransformation of the library on these probabilities are presented.     

Are Archaea inherently less diverse than Bacteria in the same environments?

Like Bacteria, Archaea occur in a wide variety of environments, only some of which can be considered 'extreme'. We compare archaeal diversity, as represented by 173 16S rRNA gene libraries described in published reports, to bacterial diversity in 79 libraries from the same source environments. An objective assessment indicated that 114 archaeal libraries and 45 bacterial libraries were large enough to yield stable estimates of total phylotype richness. Archaeal libraries were seldom as large or diverse as bacterial libraries from the same environments. However, a relatively larger proportion of libraries were large enough to effectively capture rare as well as dominant phylotypes in archaeal communities. In contrast to bacterial libraries, the number of phylotypes did not correlate with library size; thus, 'larger' may not necessarily be 'better' for determining diversity in archaeal libraries. Differences in diversity suggest possible differences in ecological roles of Archaea and Bacteria; however, information is lacking on relative abundances and metabolic activities within the sampled communities, as well as the possible existence of microhabitats. The significance of phylogenetic diversity as opposed to functional diversity remains unclear, and should be a high priority for continuing research.     

Primer extension mutagenesis powered by selective rolling circle amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material.     

Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries

How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.     

Bacterial Diversity in Mine Tailings Compared by Cultivation and Cultivation-independent Methods and their Resistance to Lead and Cadmium

To examine bacterial community composition in rhizosphere of plants colonizing on mine tailings and phylogenetic differences between subcommunities resistant to different metals, we constructed four clone libraries of 16S rDNA sequences. One was amplified directly from tailing microbial DNA (named as Ci library) and three from cultures on the plates containing of 0.5 mM CdCl(2) (Cd library), 2 mM Pb (NO(3))(2) (Pb library), and without any metals (Cw library). In total, nine bacterial divisions and two unclassified groups were identified from 352 clones of these libraries. Ci clones covered eight divisions, whereas all cultivable clones only covered four divisions. Thus, Ci library provided more phylogenetic diversity than cultivable libraries. However, the microbes represented by the cultivable clones were more similar to previously described bacteria than those represented by Ci clones. All Ci clones were not found in three cultivable libraries. Cd library were exclusively Gram-negative bacteria of Acinetobacter, Ralstonia, Comamonas, and Chryseobacterium. Meanwhile, dominant Gram-positive bacteria in Pb library, Paenibacillus and Bacillus, were also not found in Cd library. Our data indicate that phylogenetic structure was very different from those in acid mine drainage. Meanwhile, tailings harbored phylogenetically distinct subcommunities resistant to Pb and Cd.     

Polishing the craft of genetic diversity creation in directed evolution

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating “smart” libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.     

Glycan fingerprints: calculating diversity in glycan libraries

Carbohydrate libraries printed in glycan micorarray format have had a great impact on the high-throughput analysis of the specificity of a wide range of mammalian, plant, and bacterial lectins. Chemical and chemo-enzymatic synthesis allows the construction of diverse glycan libraries but requires substantial effort and resources. To leverage the synthetic effort, the ideal library would be a minimal subset of all structures that provides optimal diversity. Therefore, a measure of library diversity is needed. To this end, we developed a linear representation of glycans using standard chemoinformatic tools. This representation was applied to measure pairwise similarity and consequently diversity of glycan libraries in a single value. The diversities of four existing sialoside glycan arrays were compared. More diverse arrays are proposed reducing the number of glycans. This algorithm can be applied to diverse aspects of library design from target structure selection to the choice of building blocks for their synthesis.     

Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli

Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3′ primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.     

A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity

The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications.     

Statistics of protein library construction

SUMMARY: We have investigated the statistics associated with constructing and sampling large protein-encoding libraries. Using fairly simple statistics we have written algorithms for estimating the diversity in libraries generated by the most commonly used protocols, including error-prone PCR, DNA shuffling, StEP PCR, oligonucleotide-directed randomization, MAX randomization, synthetic shuffling, DHR, ADO and SISDC. AVAILABILITY: Web interface and C++ source code available at http://guinevere.otago.ac.nz/stats.html. SUPPLEMENTARY INFORMATION: Complete mathematical notes, model assumptions and justification, users' guide and worked examples at above website.     

Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.