Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.     

Rapid generation of incremental truncation libraries for protein engineering using α-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of α-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3′→5′ exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.     

A combinatorial approach to hybrid enzymes independent of DNA homology

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.     

Structure-based combinatorial protein engineering (SCOPE)

Presented here is the development a semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. The utility of structure-based combinatorial protein engineering (SCOPE) was demonstrated by its application to two distantly related members of the X-family of DNA polymerases: rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (Pol X). These proteins share similar folds but have low sequence identity, and differ greatly in both size and activity. "Equivalent" subdomain elements of structure were designed on the basis of the tertiary structure of Pol beta and the corresponding regions of Pol X were inferred from homology modeling and sequence alignment analysis. Libraries of chimeric genes with up to five crossovers were synthesized in a series of PCR reactions by employing hybrid oligonucleotides that code for variable connections between structural elements. Genetic complementation in Escherichia coli enabled identification of several novel DNA polymerases with enhanced phenotypes. Both the composition of structural elements and the manner in which they were linked were shown to be essential for this property, indicating the importance of these aspects of design.     

Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.     

Theoretical distribution of truncation lengths in incremental truncation libraries

Incremental truncation is a method for constructing libraries of every one base pair truncation of a segment of DNA. Incremental truncation libraries can be created using a time-dependent nuclease method or through the incorporation of alpha-phosphothioate dNTPs by PCR or by primer extension (THIO(pcr) truncation and THIO(extension) truncation, respectively). Libraries created by the fusion of two truncation libraries, known as ITCHY libraries, can be created using the above methods or by the incremental truncation-like method SHIPREC. Knowing and being able to tailor the distribution of truncations in incremental truncation, ITCHY and SHIPREC libraries would be beneficial for their use in protein engineering and other applications. However, the experimental determination of the distributions would require extensive, cost-prohibitive, DNA sequencing to obtain statistically relevant data. Instead, a theoretical prediction of the distributions was developed. Time-dependent incremental truncation libraries had the most uniform distribution of truncation lengths, but were biased against longer truncations. Essentially uniform distribution over the desired truncation range (from zero to N(max) base pairs) required that truncations be prepared up to at least 1.2-1.5 N(max). THIO(pcr) and THIO(extension) truncation libraries had a very nonuniform distribution of truncation lengths with a bias against longer truncations. Such nonuniformity could be significantly diminished by decreasing the incorporation rate of alphaS-dNTPs but at the expense of having a large fraction of the DNA truncated beyond the desired range or completely degraded. ITCHY libraries created using time-dependent truncation had the most uniform distribution of possible fusions and had the highest fraction of the library being parental-length fusions. However, the distribution of parental-length fusions was biased against fusions near the beginning/ends of genes unless the truncation libraries are prepared with a uniform distribution up to N(max). In contrast, SHIPREC libraries and THIO(pcr) ITCHY libraries, by the very nature of the nonuniform distributions of the truncated DNA, are ensured of having a uniform distribution of fusion points in parental-length fusions. This comes at the expense of having a smaller fraction of the library being parental-length fusions; however, this limitation can be overcome by performing size selection on the library.     

Random multi-recombinant PCR for the construction of combinatorial protein libraries

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries. For the construction of a ‘random shuffling library’, RM-PCR was used to shuffle six DNA fragments each encoding 25 amino acids; this affords many different fragment sequences whose every position has an equal probability to encode any of the six blocks. For the construction of the ‘alternative splicing library’, RM-PCR was used to perform different alternative splicings at the DNA level, which also yields different block sequences. DNA sequencing of the RM-PCR products in both libraries revealed that most of the sequences were quite different, and had a long open reading frame without a frame shift or stop codon. Furthermore, no distinct bias among blocks was observed. Here we describe how to use RM-PCR for the construction of combinatorial DNA libraries, which encode protein libraries that would be suitable for selection experiments in the global protein space.     

Tools for genome-wide strain design and construction

Advances in DNA sequencing and synthesis technologies concurrent with the development of new recombinant DNA approaches have enabled the extension of directed evolution algorithms to the genome-scale. It is now possible to simultaneously map the effect of mutation(s) in each and every gene in the genome onto almost any screenable or selectable phenotype in less than a week. Such maps can be used to direct the design and construction of libraries containing billions of rationally designed combinatorial mutations. Such combinatorial libraries can now also be created and evaluated in less than a week. The review presents and discusses these new technologies within the context of directed evolution and inverse metabolic engineering.     

Construction of hybrid gene libraries involving the circular permutation of DNA

The method of incremental truncation for the creation of hybrid enzymes (ITCHY) allows the creation of comprehensive fusion libraries between 5 and 3 fragments of two genes in a manner that is independent of DNA sequence homology. A methodology is presented for the creation of ITCHY libraries called circularly permuted ITCHY (CP-ITCHY) that allows the creation of ITCHY libraries in a manner that does not require extensive time point sampling. In addition, CP-ITCHY requires only a single vector and productively biases the library towards those fusions that are approximately the same size as the original genes. In the model system of creating fusions between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, the CP-ITCHY libraries are shown to contain a diverse set of active fusions including those in regions of low-homology. In addition, a high percentage of active fusions were temperature-sensitive as they complemented an auxotrophic strain of Escherichia coli at 22 °C but not at 37 °C.     

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

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

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.     

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins

In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.     

Generation of recombinant antibodies and means for increasing their affinity

Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.     

Construction of a random circular permutation library using an engineered transposon

Circular permutation is an important protein engineering tool used to create sequence diversity of a protein by changing its linear order of amino acid sequence. Circular permutation has proven to be effective in the evolution of proteins for desired properties while maintaining similar three-dimensional structures. Due to the lack of a robust design principle guiding the selection of new termini, construction of a combinatorial library is much preferred for comprehensive evaluation of circular permutation. Unfortunately, the conventional methods used to create random circular permutation libraries cause significant sequence modification at new termini of circular permutants. In addition, these methods impose additional limitations by requiring either relatively inefficient blunt-end ligation during library construction or redesign of transposons for tailored expression of circular permutants. In this study, we present the development of an engineered transposon for facile construction of random circular permutation libraries. We provide evidence that minimal modification at the new termini of the random circular permutants is possible with our engineered transposon. In addition, our method enables the use of sticky-end ligation during library construction and provides external tunability for expression of random circular permutants.     

Unbiased libraries in protein directed evolution

Directed evolution is a powerful approach to study the molecular basis of protein evolution and to engineer proteins for a wide range of applications in synthetic organic chemistry and biotechnology. There are many methods based on random or focused mutagenesis to engineer successfully any protein trait. Focused approaches such as site-directed and saturation mutagenesis have become methods of choice for improving protein activity, selectivity, stability and many other traits because the screening step can be practically handled (bottleneck in directed evolution). Although novel mutagenesis methods based on CRISPR or solid-phase gene synthesis can eliminate bias when creating protein libraries, traditional PCR approaches, although imperfect, remain widely used due to their ease and low cost. One of the most common approaches in focused mutagenesis relies on NNK mutagenesis, however, the primer-based 22c-trick and small-intelligent methods have emerged as key tools for constructing less biased and unbiased libraries when all 20 canonical amino acids are needed for various reasons. In this minireview, we assess studies employing such methods for library creation and their areas of application. We also discuss the advantages and disadvantages of both methods and provide a perspective for creating smarter libraries.     

Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes

Water borne cercaria(ae) of the trematode genus Schistosoma rapidly penetrate host skin. A single serine protease activity, cercarial elastase, is deposited in advance of the invading parasite by holocytosis of vesicles from ten large acetabular gland cells. Cercarial elastase activity is a composite of multiple isoforms. Genes coding for the isoforms can be divided into two classes by amino acid and promoter sequence homology. Two of the five genes identified in Schistosoma mansoni account for over 90% of the activity and protein released. The remaining genes produce little protein or are silent. Positional scanning synthetic combinatorial substrate libraries demonstrate that the two major isoforms have similar substrate specificities and are, therefore, isoenzymes. The closely related Schistosoma hematobium and the distantly related Schistosomatium douthitti also contain multiple orthologous cercarial elastase genes suggesting that gene duplication may have occurred after speciation in Schistosoma evolution and that this duplication has been conserved.     

The keratin BIIIB gene family: isolation of cDNA clones and structure of a gene and a related pseudogene

The nucleotide sequence of cDNA clones encoding the three major BIIIB high-sulfur wool keratin proteins (BIIIB2, 3, and 4) and the structure of a BIIIB4 gene and a BIIIB3 pseudogene are reported. Although Southern blot analysis indicates that the BIIIB genes comprise a multigene family in the sheep genome, they are poorly represented in genomic DNA libraries. The family sequence homology of the coding region extends into the 5' and 3' untranslated regions and the near 5' flanking region of the BIIIB3 and 4 genes. These homologies suggest that the BIIIB3 and 4 genes represent the latest gene duplication event in the evolution of the BIIIB multigene family. Like the genes coding for other wool keratin matrix protein components, the BIIIB genes have the conserved 18-bp sequence immediately 5' to the initiation codon and also appear to lack introns.     

DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs.

A number of eukaryotic DNA binding proteins have been isolated by screening phage expression libraries with DNA probes containing the binding site of the DNA-binding protein. This methodology was employed here to isolate clones of the factor that interacts with the W box element of the human major histocompatibility complex HLA-DQB gene. Surprisingly, several cDNA clones of YB-1, a cDNA clone that was previously isolated with a CCAAT element-containing sequence were found. Independently, the screening of phage expression libraries with depurinated DNA resulted in the isolation of YB-1 and dbpA, a previously isolated cDNA that has homology to YB-1. Additional characterization of YB-1 showed that it bound a wide variety of DNA sequences and suggested that the binding of this protein is promiscuous. Furthermore, we show that both YB-1 and dbpA bind to depurinated DNA better than undamaged DNA and that the extent of specificity of binding is influenced by Mg2+. Due to the lack of sequence specificity and high degree of binding to depurinated DNA, we suggest that these proteins might be involved in chromosome functions such as maintenance of chromatin structure or DNA repair that do not require sequence-specific binding.     

Combinatorial biosynthesis for new drug discovery

Combinatorial biosynthesis involves interchanging secondary metabolism genes between antibiotic-producing microorganisms to create unnatural gene combinations or hybrid genes if only part of a gene is exchanged. Novel metabolites can be made by both approaches, due to the effect of a new enzyme on a metabolic pathway or to the formation of proteins with new enzymatic properties. The method has been particularly successful with polyketide synthase (PKS) genes: derivatives of medically important macrolide antibiotics and unusual polycyclic aromatic compounds have been produced by novel combinations of the type I and type II PKS genes, respectively. Recent extensions of the approach to include deoxysugar biosynthesis genes have expanded the possibilities for making new microbial metabolites and discovering valuable drugs through the genetic engineering of bacteria.