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.     

Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.     

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.     

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.     

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.     

Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A(1) prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.     

Incremental truncation of PHA synthases results in altered product specificity

PHA synthase is the key enzyme involved in the biosynthesis of microbial polymers, polyhydroxyalkanoates (PHA). In this study, we created a hybrid library of PHA synthase gene with different crossover points by an incremental truncation method between the C-terminal fragments of the phaC(Cn) (phaC from Cupriavidus necator) and the N-terminal fragments of the phaC1(Pa) (phaC from Pseudomonas aeruginosa). As the truncation of the hybrid enzyme increased, the in vivo PHB synthesis ability of the hybrids declined gradually. PHA synthase PhaC(Cn) with a deletion on N-terminal up to 83 amino acid residues showed no synthase activity. While with the removal of up to 270 amino acids from the N-terminus, the activity of the truncated PhaC(Cn) could be complemented by the N-terminus of PhaC1(Pa). Three of the hybrid enzymes W188, W235 and W272 (named by the deleted nucleic acid number) were found to have altered product specificities.     

Construction of rice mini-chromosomes by telomere-mediated chromosomal truncation

Telomere truncation has been shown to be an efficient technology for the creation of mini-chromosomes that can be used as artificial chromosome platforms for genetic engineering. Artificial chromosome-based genetic engineering is considered to be superior to the existing techniques of randomized gene integration by Agrobacterium or biolistic-mediated genetic transformation. It organizes multiple transgenes as a unique genetic linkage block for subsequent manipulations in breeding. Telomere truncation technology relies on three components: the telomere sequence that mediates chromosomal truncation, a selection marker that allows the selection of transgenic events, and a site-specific recombination system that can be used to accept future genes into the mini-chromosome by gene targeting. These elements are usually pre-assembled before transformation, a process that is both time and labor consuming. We found in this research that the three elements could be mixed to transform plant cells in a biolistic transformation, and produced efficient chromosomal truncations and mini-chromosomes in rice. This system will allow rapid construction of mini-chromosomes with a flexible selection of resistant markers, site-specific recombination systems and other desirable elements. In addition, a rice telotrisomic line was used as the starting material for chromosomal truncations. Mini-chromosomes from the truncations of both the telocentric chromosome and other chromosomes were recovered. The mini-chromosomes remained stable during 2 years of subculture. The construction of mini-chromosomes in rice, an economically important crop, will provide a platform for future artificial chromosome-based genetic engineering of rice for stacking multiple genes.     

The self-perpetuating tau truncation circle

Pathological truncations of human brain proteins represent the common feature of many neurodegenerative disorders including AD (Alzheimer's disease), Parkinson's disease and Huntington's disease. Protein truncations significantly change the structure and function of these proteins and thus can engender their pathological metamorphosis. We have shown previously that truncated forms of tau protein are contained in the core of the paired helical filaments that represent the main constituent of neurofibrillary pathology. Recently, we have identified truncated tau species of a different molecular signature. We have found that tau truncation is not produced by a random process, but rather by highly specific proteolytic cleavage and/or non-enzymatic fragmentation. In order to characterize the pathophysiology of AD-specific truncated tau species, we have used a transgenic rat model for AD expressing human truncated tau. Expression of the tau protein induces the formation of novel truncated tau species that originate from both transgenic human tau and endogenous rat tau proteins. Moreover, these truncated tau proteins are found exclusively in the misfolded fraction of tau, suggesting that they actively participate in the tau misfolding process. These findings corroborate further the idea that the appearance of truncated tau species starts a self-perpetuating cycle of further tau protein truncation leading to and accelerating tau misfolding and formation of neurofibrillary pathology.     

Libraries of hybrid proteins from distantly related sequences

We introduce a method for sequence homology-independent protein recombination (SHIPREC) that can create libraries of single-crossover hybrids of unrelated or distantly related proteins. The method maintains the proper sequence alignment between the parents and introduces crossovers mainly at structurally related sites distributed over the aligned sequences. We used SHIPREC to create a library of interspecies hybrids of a membrane-associated human cytochrome P450 (1A2) and the heme domain of a soluble bacterial P450 (BM3). By fusing the hybrid gene library to the gene for chloramphenicol acetyl transferase (CAT), we were able to select for soluble and properly folded protein variants. Screening for 1A2 activity (deethylation of 7-ethoxyresorufin) identified two functional P450 hybrids that were more soluble in the bacterial cytoplasm than the wild-type 1A2 enzyme.     

Testing goodness of fit of a uniform truncation model

Several goodness-of-fit tests of a lifetime distribution have been suggested in the literature; many take into account censoring and/or truncation of event times. In some contexts, a goodness-of-fit test for the truncation distribution is of interest. In particular, better estimates of the lifetime distribution can be obtained when knowledge of the truncation law is exploited. In cross-sectional sampling, for example, there are theoretical justifications for the assumption of a uniform truncation distribution, and several studies have used it to improve the efficiency of their survival estimates. The duality of lifetime and truncation in the absence of censoring enables methods for testing goodness of fit of the lifetime distribution to be used for testing goodness of fit of the truncation distribution. However, under random censoring, this duality does not hold and different tests are required. In this article, we introduce several goodness-of-fit tests for the truncation distribution and investigate their performance in the presence of censored event times using simulation. We demonstrate the use of our tests on two data sets.     

Build-up mechanisms determining the topology of mutualistic networks

The frequency distribution of the number of interactions per species (i.e., degree distribution) within plant-animal mutualistic assemblages often decays as a power-law with an exponential truncation. Such a truncation suggests that there are ecological factors limiting the frequency of supergeneralist species. However, it is not clear whether these patterns can emerge from intrinsic features of the interacting assemblages, such as differences between plant and animal species richness (richness ratio). Here, we show that high richness ratios often characterize plant-animal mutualisms. Then, we demonstrate that exponential truncations are expected in bipartite networks generated by a simple model that incorporates build-up mechanisms that lead to a high richness ratio. Our results provide a simple interpretation for the truncations commonly observed in the degree distributions of mutualistic networks that complements previous ones based on biological effects.     

C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger

When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein.     

Epitope mapping of recombinant antigens by transposon mutagenesis

We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies. The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure. Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion. One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary.     

High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries

DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.     

Telomere-mediated truncation of barley chromosomes

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

A universal,vector-based system for nucleic acid reading-frame selection

The identification of a nucleic acid sequence's correct reading frame has important implications for homology-independent protein engineering techniques such as incremental truncation and SCRATCHY. We report the development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest. This dual selection approach overcomes inconsistencies observed with traditional C-terminally fused reporter proteins. In the pSALect vector, sequences of interest are positioned between an N-terminal Tat-signal sequence and a C-terminal beta-lactamase reporter. In-frame selection of the resulting three-domain protein is performed by growing colonies on ampicillin-containing plates, requiring full-length translation in order to link covalently the signal sequence to the lactamase for export into the periplasm. This dual selection scheme has been validated successfully using defined sequences of glycinamide ribonucleotide formyltransferases (GARTs) from Escherichia coli and human and, in contrast to C-terminal fusion systems, proved effective when applied towards the selection of in-frame constructs in an incremental truncation library.     

Infrared imaging of 2-D temperature distribution during cryogen spray cooling

Cryogen spray cooling (CSC) is used in conjunction with pulsed laser irradiation for treatment of dermatologic indications. The main goal of this study was to determine the radial temperature distribution created by CSC and evaluate the importance of radial temperature gradients upon the subsequent analysis of tissue cooling throughout the skin. Since direct measurement of surface temperatures during CSC are hindered by the formation of a liquid cryogen layer, temperature distributions were estimated using a thin, black aluminum sheet. An infrared focal plane array camera was used to determine the 2-D backside temperature distribution during a cryogen spurt, which preliminary measurements have shown is a good indicator of the front-side temperature distribution. The measured temperature distribution was approximately gaussian in shape. Next, the transient temperature distributions in skin were calculated for two cases: 1) the standard 1-D solution which assumes a uniform cooling temperature distribution, and 2) a 2-D solution using a nonuniform surface cooling temperature distribution based upon the back-side infrared temperature measurements. At the end of a 100-ms cryogen spurt, calculations showed that, for the two cases, large discrepancies in temperatures at the surface and at a 60-micron depth were found at radii greater than 2.5 mm. These results suggest that it is necessary to consider radial temperature gradients during cryogen spray cooling of tissue.