Repetitive Protein Research Articles

Preparing Genes for Repetitive Elastin-Like Polypeptides Using Gibson Assembly.

Elastin-like polypeptides (ELPs) are stimuli-responsive polymers consisting of peptide repeat units from the elastin protein. Most commonly these materials are biosynthesized in E. coli using genes that have been built by standard molecular biology techniques. The inherent repetitive nature of these polypeptides makes the construction of genes that encode them more challenging than for typical recombinant proteins. Described here is a robust technique to produce genes encoding long chains of ELP through successive rounds of Gibson assembly that nearly double the ELP length with each cloning iteration. Using Gibson assembly to produce the genes for high molecular weight ELPs requires fewer steps and significantly less time than current techniques. This approach is modular and allows for the flexible assembly of ELPs with various compositions. The procedure can be adapted for preparing genes encoding other repetitive proteins.

Research Update: Programmable tandem repeat proteins inspired by squid ring teeth

Cephalopods have evolved many interesting features that can serve as inspiration. Repetitive squid ring teeth (SRT) proteins from cephalopods exhibit properties such as strength, self-healing, and biocompatibility. These proteins have been engineered to design novel adhesives, self-healing textiles, and the assembly of 2d-layered materials. Compared to conventional polymers, repetitive proteins are easy to modify and can assemble in various morphologies and molecular architectures. This research update discusses the molecular biology and materials science of polypeptides inspired by SRT proteins, their properties, and perspectives for future applications.

Efficient enrichment cloning of TAL effector genes from Xanthomonas

Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars of Xanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL effectors in their genomes. Some of the TAL effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL effector genes remains a challenge. In order to isolate complete TAL effector gene repertoires, we developed an enrichment cloning strategy based on•genome-informed in silico optimization of restriction digestions,•selective restriction digestion of genomic DNA, and•size fractionation of DNA fragments.Our rapid, cheap and powerful method allows efficient cloning of TAL effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains of Xanthomonas oryzae from Africa.

Intrinsic Disorder in Proteins with Pathogenic Repeat Expansions.

Intrinsically disordered proteins and proteins with intrinsically disordered regions have been shown to be highly prevalent in disease. Furthermore, disease-causing expansions of the regions containing tandem amino acid repeats often push repetitive proteins towards formation of irreversible aggregates. In fact, in disease-relevant proteins, the increased repeat length often positively correlates with the increased aggregation efficiency and the increased disease severity and penetrance, being negatively correlated with the age of disease onset. The major categories of repeat extensions involved in disease include poly-glutamine and poly-alanine homorepeats, which are often times located in the intrinsically disordered regions, as well as repeats in non-coding regions of genes typically encoding proteins with ordered structures. Repeats in such non-coding regions of genes can be expressed at the mRNA level. Although they can affect the expression levels of encoded proteins, they are not translated as parts of an affected protein and have no effect on its structure. However, in some cases, the repetitive mRNAs can be translated in a non-canonical manner, generating highly repetitive peptides of different length and amino acid composition. The repeat extension-caused aggregation of a repetitive protein may represent a pivotal step for its transformation into a proteotoxic entity that can lead to pathology. The goals of this article are to systematically analyze molecular mechanisms of the proteinopathies caused by the poly-glutamine and poly-alanine homorepeat expansion, as well as by the polypeptides generated as a result of the microsatellite expansions in non-coding gene regions and to examine the related proteins. We also present results of the analysis of the prevalence and functional roles of intrinsic disorder in proteins associated with pathological repeat expansions.

Diminishing Returns on Intragenic Repeat Number Expansion in the Production of Signaling Peptides.

Signaling peptides enable communication between cells, both within and between individuals, and are therefore key to the control of complex physiological and behavioral responses. Since their small sizes prevent direct transmission to secretory pathways, these peptides are often produced as part of a larger polyprotein comprising precursors for multiple related or identical peptides; the physiological and behavioral consequences of this unusual gene structure are not understood. Here, we show that the number of mature-pheromone-encoding repeats in the yeast α-mating-factor gene MFα1 varies considerably between closely related isolates of both Saccharomyces cerevisiae and its sister species Saccharomyces paradoxus. Variation in repeat number has important phenotypic consequences: Increasing repeat number caused higher pheromone production and greater competitive mating success. However, the magnitude of the improvement decreased with increasing repeat number such that repeat amplification beyond that observed in natural isolates failed to generate more pheromone, and could actually reduce sexual fitness. We investigate multiple explanations for this pattern of diminishing returns and find that our results are most consistent with a translational trade-off: Increasing the number of encoded repeats results in more mature pheromone per translation event, but also generates longer transcripts thereby reducing the rate of translation—a phenomenon known as length-dependent translation. Length-dependent translation may be a powerful constraint on the evolution of genes encoding repetitive or modular proteins, with important physiological and behavioral consequences across eukaryotes.

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a "Y. ruckeri invasin-like molecule", (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.

Trans-Sialidase Overcomes Many Antigens to be Used as a Vaccine Candidate Against Trypanosoma Cruzi

The development of vaccines against Trypanosoma cruzi remains in an exploratory stage. Despite several antigen candidates have been evaluated, a comparison among the performance of the immunogens cannot be carried out because the available reports differ in formulations and infection model. In this work, we compared the protective capacity of seven T. cruzi antigens in the same model of five new antigens and two well-established candidates. Materials& methods: We evaluated highly immunogenic proteins that contain tandem repeats (FRA [flagelar repetitive protein], Tc3, Tc6); enzymes involved in metabolic pathways critical for parasite survival (cytosolic tryparedoxin peroxidase and tryparedoxin peroxidase); and enzymes involved in parasite invasion (trans-sialidase [TS] and cruzipain). All these antigens were formulated with Freund's adjuvant and protection against the parasite infection was assessed in BALB/c mice. Tc3, cytosolic tryparedoxin peroxidase, cruzipain and TS showed the best outcome after infection in survival level and parasitemia. According to these data, these groups were also assessed using the ISCOMATRIX™ adjuvant which is being used in clinical trials. Taken together, our results showed that the TS overcomes the performance of other antigens when the same model is employed, confirming that TS is a promising antigen that could be used as a vaccine against T. cruzi.

Transcriptome analysis and identification of induced genes in the response of Harmonia axyridis to cold hardiness

Harmonia axyridis is an important predatory lady beetle that is a natural enemy of agricultural and forestry pests. In this research, the cold hardiness induced genes and their expression changes in H. axyridis were screened and detected by the way of the transcriptome and qualitative real-time PCR under normal and low temperatures, using high-throughput transcriptome and digital gene-expression-tag technologies. We obtained a 10Gb transcriptome and an 8Mb gene expression tag pool using Illumina deep sequencing technology and RNA-Seq analysis (accession number SRX540102). Of the 46,980 non-redundant unigenes identified, 28,037 (59.7%) were matched to known genes in GenBank, 21,604 (46.0%) in Swiss-Prot, 19,482 (41.5%) in Kyoto Encyclopedia of Genes and Genomes and 13,193 (28.1%) in Gene Ontology databases. Seventy-five percent of the unigene sequences had top matches with gene sequences from Tribolium castaneum. Results indicated that 60 genes regulated the entire cold-acclimation response, and, of these, seven genes were always up-regulated and five genes always down-regulated. Further screening revealed that six cold-resistant genes, E3 ubiquitin-protein ligase, transketolase, trehalase, serine/arginine repetitive matrix protein 2, glycerol kinase and sugar transporter SWEET1-like, play key roles in the response. Expression from a number of the differentially expressed genes was confirmed with quantitative real-time PCR (HaCS_Trans). The paper attempted to identify cold-resistance response genes, and study the potential mechanism by which cold acclimation enhances the insect's cold endurance. Information on these cold-resistance response genes will improve the development of low-temperature storage technology of natural enemy insects for future use in biological control.

Reconstruction of Primordial P-loop NTPase Precursors

In collaboration with: Agnes Toth-Petroczy, Alexander Goncearenco and Igor Berezovsky (inference of the ancestral P-loop sequence) and Lin Yu-Ru and David Baker (computational protein design).Short functional peptides are likely to have served as crucial intermediates between a primordial RNA world and the extant protein world. Our working hypothesis is that relics of these ancestral peptides still exist in the form of key motifs in active sites of present-day proteins. One such motif, probably the most obvious one, is the P-loop (Walker A motif) that typically binds the transferred phosphate moiety of ATP. The P-loop's extended motif comprises a β-strand and an α-helix connected by the P-loop – a glycine-rich phosphate-binding loop. The P-loop containing nucleoside triphosphate hydrolase fold includes this motif, and is one of the most ancient folds and by far the most abundant (10-18% of all ORFs). In the present work, the sequence of a primordial β-Ploop-α motif was inferred. We further showed that this β-Ploop-α motif (∼25 amino acids) could have served as a building block for the creation of ancestral globular domains by means of duplication and fusion. Computational designed proteins were generated consisting of 2 repetitions of the ancestral motif grafted into an ideal β/α- repeat protein. Our results indicate that these simple, repetitive proteins avidly bind ssDNA and RNA (possibly a crucial step in the transition from the RNA world to a protein world). Further, a β-α-β fragment of this protein (< 40 amino acids) was found to self-assemble and thereby confer ssDNA binding. Overall, our results show that P-loop NTPases could have emerged from a relatively short P-loop containing peptide and that self-assembly played a key role in endowing biochemical function despite limited size and complexity.

Origin of a folded repeat protein from an intrinsically disordered ancestor.

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin.

Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension

The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops.

A Novel Analytical Strategy to Identify Fusion Transcripts between Repetitive Elements and Protein Coding-Exons Using RNA-Seq.

Repetitive elements (REs) comprise 40–60% of the mammalian genome and have been shown to epigenetically influence the expression of genes through the formation of fusion transcript (FTs). We previously showed that an intracisternal A particle forms an FT with the agouti gene in mice, causing obesity/type 2 diabetes. To determine the frequency of FTs genome-wide, we developed a TopHat-Fusion-based analytical pipeline to identify FTs with high specificity. We applied it to an RNA-seq dataset from the nucleus accumbens (NAc) of mice repeatedly exposed to cocaine. Cocaine was previously shown to increase the expression of certain REs in this brain region. Using this pipeline that can be applied to single- or paired-end reads, we identified 438 genes expressing 813 different FTs in the NAc. Although all types of studied repeats were present in FTs, simple sequence repeats were underrepresented. Most importantly, reverse-transcription and quantitative PCR validated the expression of selected FTs in an independent cohort of animals, which also revealed that some FTs are the prominent isoforms expressed in the NAc by some genes. In other RNA-seq datasets, developmental expression as well as tissue specificity of some FTs differed from their corresponding non-fusion counterparts. Finally, in silico analysis predicted changes in the structure of proteins encoded by some FTs, potentially resulting in gain or loss of function. Collectively, these results indicate the robustness of our pipeline in detecting these new isoforms of genes, which we believe provides a valuable tool to aid in better understanding the broad role of REs in mammalian cellular biology.

ING4 Inhibits Proliferation and Induces Apoptosis in Human Melanoma A375 Cells via the Fas/Caspase-8 Apoptosis Pathway

Background: Inhibitor of growth 4 (ING4) plays a role in regulating the cell cycle, apoptosis, cell invasion and migration, but the mechanisms involved remain to be elucidated. Objective: To explore how ING4 affects human malignant melanoma A375 cells. Methods: Recombinant lentiviral vectors (A375/pLenO-GTP-ING4) were constructed and transfected into A375 cells (experimental group). The impact of ING4 on the proliferation and apoptosis of A375 cells was investigated in in vitro and in vivo experiments in mice using the MTT assay and flow cytometry. Results: In the experimental group, optical density was lower and apoptotic cells were more frequent from days 2-5 (p = 0.000 and p < 0.01); there were smaller xenografts and more apoptotic cells in mice (all p < 0.05); moreover, increased levels of Fas, cleaved caspase-8 and caspase-3, and decreased levels of FasL and procyclic acidic repetitive protein were observed in vitro and in vivo. Conclusion: ING4 might suppress proliferation and enhance apoptosis in human malignant melanoma cells by activating Fas-induced apoptosis in a caspase-8-dependent pathway.

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

Tally: a scoring tool for boundary determination between repetitive and non-repetitive protein sequences.

Tandem Repeats (TRs) are abundant in proteins, having a variety of fundamental functions. In many cases, evolution has blurred their repetitive patterns. This leads to the problem of distinguishing between sequences that contain highly imperfect TRs, and the sequences without TRs. The 3D structure of proteins can be used as a benchmarking criterion for TR detection in sequences, because the vast majority of proteins having TRs in sequences are built of repetitive 3D structural blocks. According to our benchmark, none of the existing scoring methods are able to clearly distinguish, based on the sequence analysis, between structures with and without 3D TRs. We developed a scoring tool called Tally, which is based on a machine learning approach. Tally is able to achieve a better separation between sequences with structural TRs and sequences of aperiodic structures, than existing scoring procedures. It performs at a level of 81% sensitivity, while achieving a high specificity of 74% and an Area Under the Receiver Operating Characteristic Curve of 86%. Tally can be used to select a set of structurally and functionally meaningful TRs from all TRs detected in proteomes. The generated dataset is available for benchmarking purposes. Source code is available upon request. Tool and dataset can be accessed through our website: http://bioinfo.montp.cnrs.fr/?r=Tally andrey.kajava@crbm.cnrs.fr Supplementary data are available at Bioinformatics online.

Synonymous Modification Enables High Fidelity Expression of Biosensors and Probes with Repetitive Protein and Nucleotide Sequences

Repetitive nucleotide or amino acid sequences are often engineered into biosensors and probes to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously difficult to construct and maintain for stable expression. They are easily deleted or truncated, which occurs randomly and is not a priori predictable, resulting in aberrant expression and impacting the ability to correctly detect and to interpret biological functions. Here, we introduce a simple and generally applicable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them non-repetitive but still functional (“synonymous”). We first demonstrate the procedure by designing a cassette of synonymous MS2 RNA aptamers, and tandem coat proteins for RNA imaging and show a dramatic improvement in signal and reproducibility in single RNA detection in live cells. The same approach is extended to enhance the stability of engineered fluorescent biosensors containing FRET-pair of fluorescent proteins, upon which a great majority of systems thus far in the field are based. Using the synonymous modified FRET biosensors, we achieve correct expression of full-length sensors, eliminating the aberrant truncation products that were often assumed to be due to non-specific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus the method we describe here should be routinely employed in generation of probes and sensors containing multiple, repetitive motifs to achieve correct expression these state-of-the-art tools.

Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitiveproteins.

Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinantexpression.

The New Era of Virus-Like Particles Based Vaccines

Virus-like particles or what is called VLPs for short are considered a useful tool in the development of vaccines. These VLPs-based vaccines have attracted the attention of many biotech and pharmaceutical companies as effective treatment for many types of non-infectious chronic diseases. VLPs simply resemble viruses; however they are noninfectious due to the lack of viral genome. The expression of envelop or capsid proteins result in the self-assembly of the particle, this assembly can easily be carried out in bacteria, yeast, plant cells or insect cell lines. The presence of repetitive viral surface protein epitopes is successful in eliciting strong B cell responses. VLPs have also high safety profile due to the lack of viral genome and lack of the ability to replicate. This area of research is not really new, the discovery of hepatitis B virus derived VLPs goes back to 1976.

Use of Human Pluripotent Stem Cells to Model the Hematopoietic Defect and Repair of Human Telomerase Deficiency

Telomeres are repetitive DNA protein structures that cap the ends of chromosomes, protect chromosome ends from degradation and fusion, and are essential for maintenance of genomic integrity. Telomere length has been shown to gradually shorten over time as cells divide. When telomeres become critically short, the cells enter a state of senescence. As such, telomere shortening has been implicated in accelerated aging. Defects in telomerase function have been associated with the development of bone marrow failure. Patients with inherited mutations in telomerase components have significantly shortened telomere lengths and reductions in telomere length have been associated with a worse prognosis in myelodysplastic syndrome (MDS) and Aplastic anemia (AA). We have isolated fibroblasts from patients with a novel mutation in the telomerase RNA component (TERC), a 6 nucleotide in frame duplication at position +334, which results in bone marrow failure. We have derived several lines of human induced pluripotent stem cells (iPSCs) from these patients. We have demonstrated that these telomerase-deficient iPSCs appropriately express markers of pluripotency: Oct4, Sox2, SSEA4, and Nanog. Using quantitative real time PCR, we were able to measure the average telomere length as a T/S ratio of kilobases of telomere length per genome. We have determined that reprogramming results in significant increase in telomere length of control fibroblasts. However despite the typical induction of endogenous TERT expression during reprograming in our telomerase-deficient iPSCs, these TERC-mutant iPSCs did not demonstrate significant telomere elongation. We found telomerase-deficient fibroblasts have telomere lengths of 130-150kb/diploid genome compared to normal human fibroblasts with telomeres of ~250kb/diploid genome. After reprogramming, the iPSCs generated from wild type fibroblasts can have markedly increased telomere lengths to as high as 2000kb/diploid genome. The telomere lengths in TERC-mutant iPSCs from two different patients are less than 300kb/diploid genome (replicates=3). Expression of telomerase components TERT and TERC, DKC was compared to the mRNA level by qRT-PCR in the TERC-mutant iPSCs. We found significant variation in mRNA expression levels of telomerase components the different lines of telomerase deficient iPSCs, and even variation among different clones of the same telomerase deficient line. We have also found that mutations in TERC results in defective hematopoietic differentiation from these iPSCs in in vitro assays. In the TERC-mutant iPSCs, the proportion of CD34+CD45+ hematopoietic cells was reduced compared to wild type controls. Wild type iPSCs produce 30% CD34+ cells compared to 15-20% in TERC-mutant iPSCs (n=3). Additionally, wild type iPSC controls produce 9-10% CD34+CD45+ hematopoietic progenitor cells compared to 1-2% of TERC mutant cells. Interestingly, approximately equal proportions (8-9%) of wild type and TERC-mutant cells differentiate into CD34+CD31+ endothelial cells, suggesting this pathway is less affected by the TERC mutation. The TERC-mutant iPSCs demonstrate reduced development of hematopoietic progenitor cells in standard hematopoietic colony forming cell assays: 100 CFCs per 50,000 differentiated wild type iPSC-derived cells compared to 65 CFCs per 50,000 differentiated TERC mutant iPSC-derived cells. In order to rescue the hematopoietic defect in telomerase deficient TERC-mutant iPSCs we have used the Sleeping Beauty transposon system to over express these telomerase components TERC and TERT in the TERC mutant iPSCs. We are currently characterizing the effect of overexpression of these telomerase components on hematopoietic differentiation to determine if this provides a strategy to enable use of gene-corrected iPSCs to provide a future therapy for patients with bone marrow failure due to defined telomerase deficiencies. DisclosuresNo relevant conflicts of interest to declare.

Characterization of the Secondary Structure of CP30, a Highly Repetitive Ampholytic Protein in Beetle Elytral Cuticle

SummaryCP30 is a major component protein in the rigid cuticle of the beetle elytron which we recently identified. It has a highly unusual amino acid sequence comprised largely of alternating blocks of 3–5 anionic and cationic amino acid residues (three charged residues, glutamic acid, arginine and histidine, make up 76% of the sequence) which suggest an unusual stuctural role. The secondary structure of CP30 was evaluated at a variety of pH, ionic strength and temperature conditions. Circular dichroism (CD), fluorescence spectroscopy and static and dynamic light scattering showed that the protein had an extended α‐helical structure at pH 3, 5 and 7 which unfolded around 60°C and reformed upon cooling back to 10°C. The protein aggregated or adhered to the dialysis membrane at pH 5, low salt, which is close to the protein's isoelectric point (5.82), preventing analysis at that condition. Further studies aimed at solubilizing the protein near that condition may uncover evidence of assembly. While the structural function of CP30 is currently unknown, the sequence of CP30 may offer new design motifs for the development of novel polymer or gel biomaterials.