Stable Fusion Protein Research Articles

Halo-RPD: searching for RNA-binding protein targets in plants.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding RNA biology. Currently, various methods are available to investigate these interactions with, RNA immunoprecipitation (RIP) being the most common. The search for RNA targets has largely been conducted using antibodies to an endogenous protein or to GFP-tag directly. Having to be dependent on the expression level of the target protein and having to spend time selecting highly specific antibodies make immunoprecipitation complicated. Expression of the GFP-fused protein can lead to cytotoxicity and, consequently, to improper recognition or degradation of the chimeric protein. Over the past few years, multifunctional tags have been developed. SNAP-tag and HaloTag allow the target protein to be studied from different perspectives. Labeling of the fusion protein with custom-made fluorescent dyes makes it possible to study protein expression and to localize it in the cell or the whole organism. A high-affinity substrate has been created to allow covalent binding by chimeric proteins, minimizing protein loss during protein isolation. In this paper, a HaloTag-based method, which we called Halo-RPD (HaloTag RNA PullDown), is presented. The proposed protocol uses plants with stable fusion protein expression and Magne® HaloTag® magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic Arabidopsis thaliana plants. The key stages described in the paper are as follows: (1) preparation of the magnetic beads; (2) tissue homogenization and collection of control samples; (3) precipitation and wash of RNA-protein complexes; (4) evaluation of protein binding efficiency; (5) RNA isolation; (6) analysis of the RNA obtained. Recommendations for better NGS assay designs are provided.

Designing and computational analyzing of chimeric long-lasting GLP-1 receptor agonists for type 2 diabetes

Glucagon-like peptide-1 (GLP-1) is an intestinally derived incretin that plays a vital role in engineering the biological circuit involved in treating type 2 diabetes. Exceedingly short half-life (1–2 min) of GLP-1 limits its therapeutic applicability, and the implication of its new variants is under question. Since albumin-binding DARPin as a mimetic molecule has been reported to increase the serum half-life of therapeutic compounds, the interaction of new variants of GLP-1 in fusion with DARPin needs to be examined against the GLP-1 receptor. This study was aimed to design stable and functional fusion proteins consisting of new protease-resistant GLP-1 mutants (mGLP1) genetically fused to DARPin as a critical step toward developing long-acting GLP-1 receptor agonists. The stability and solubility of the engineered fusion proteins were analyzed, and their secondary and tertiary structures were predicted and satisfactorily validated. Molecular dynamics simulation studies revealed that the predicted structures of engineered fusion proteins remained stable throughout the simulation. The relative binding affinity of the engineered fusion proteins' complex with human serum albumin and the GLP-1 receptor individually was assessed using molecular docking analyses. It revealed a higher affinity compared to the interaction of the individual GLP-1 and HSA-binding DARPin with the GLP-1 receptor and human serum albumin, respectively. The present study suggests that engineered fusion proteins can be used as a potential molecule in the treatment of type 2 diabetes, and this study provides insight into further experimental use of mimetic complexes as alternative molecules to be evaluated as new bio-breaks in the engineering of biological circuits in the treatment of type 2 diabetes.

A comparative study of the arazyme-based fusion proteins with various ligands for more effective targeting cancer therapy: an in-silico analysis.

Recently, the use of immunotoxins for targeted cancer therapy has been proposed, to find new anticancer drugs with high efficacy on tumor cells with minimal side effects on normal cells. we designed and compared several arazyme (AraA)-based fusion proteins with different ligands to choose the best-targeted therapy for interleukin 13 receptor alpha 2 (IL13Rα2)-overexpressed cancer cells. For this purpose, IL13Rα2 was selected as a receptor and IL13 and IL13.E13K were evaluated as native and mutant ligands, respectively. In addition, Pep-1 and A2b11 were chosen as the peptide ligands for targeted cancer therapy. Several bioinformatics servers were used for designing constructs and optimization. The structures of the chimeric proteins were predicted and verified by I-TASSER, Q-Mean, ProSA, Ramachandran plot, and Verify3D program. Physicochemical properties, toxicity, and antigenicity were predicted by ProtParam, ToxinPred, and VaxiJen. HawkDock, LigPlot+, and GROMACS software were used for docking and molecular dynamics simulation of the ligand-receptor interaction. The in silico results showed AraA-A2b11 has higher values of confidence score and Q-mean score was obtained for high-resolution crystal structures. All chimeric proteins were stable, non-toxic, and non-antigenic. AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 retained its natural structure and based on ligand-receptor docking and molecular dynamic analysis, the binding ability of AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 to IL13Rα2 was sufficiently strong. Based on the bioinformatics result AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 was a stable fusion protein with two separate domains and high affinity with the IL13Rα2 receptor. Therefore, AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 fusion protein could be a new potent candidate for target cancer therapy.

Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches

Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.

Generation of a soluble and stable apoptin-EGF fusion protein, a targeted viral protein applicable for tumor therapy

A promising candidate for tumor targeted toxins is the chicken anemia-derived protein apoptin that induces tumor-specific apoptosis. It was aimed to design a novel apoptin-based targeted toxin by genetic fusion of apoptin with the tumor-directed ligand epidermal growth factor (EGF) using Escherichia coli as expression host. However, apoptin is highly hydrophobic and tends to form insoluble aggregates. Therefore, three different apoptin-EGF variants were generated. The fusion protein hexa-histidine (His)-apoptin-EGF (HAE) was expressed in E. coli and purified under denaturing conditions due to inclusion bodies. The protein solubility was improved by maltose-binding protein (MBP) or glutathione S-transferase. The protein MBP-apoptin-EGFHis (MAEH) was found favorable as a targeted toxin regarding final yield (4–6 mg/L) and stability. MBP was enzymatically removed using clotting factor Xa, which resulted in low yield and poor separation. MAEH was tested on target and non-target cell lines. The targeted tumor cell line A431 showed significant toxicity with an IC50 of 69.55 nM upon incubation with MAEH while fibroblasts and target receptor-free cells remained unaffected. Here we designed a novel EGF receptor targeting drug with high yield, purity and stability.

In silico Evaluation of PLAC1-fliC As a Chimeric Vaccine against Breast Cancer

Background:Breast cancer is one of the most prevalent cancers among women. Common cancer treatment methods are not effective enough, and there is a need for a more efficient treatment procedure. Cancer vaccine is a novel immunotherapy method that stimulates humoral and/or cellular immunity against cancer. PLAC1 is a cancer/testis antigen, prevalent in breast cancer and rarely found in normal tissues. FliC, as a bacterial adjuvant, when fused to PLAC1 can elicit humoral and cellular responses. Therefore, PLAC1-fliC is a chimeric protein, which can be considered a suitable candidate against breast cancer. Methods:ProtParam was used to evaluate the physicochemical properties of PLAC1-fliC. Second structures were determined using the GOR V server. PLAC1-fliC 3D structure was modeled by Phyre2, and it was refined using GalaxyWEB. The refined model was submitted to RAMPAGE, PROCHECK, and ProSA-web for validation. Antigenicity and allergenicity of the construct were predicted by ANTIGENpro, VaxiJen, AllergenFP, and SDAP databases. Then MHC-I- and MHC-II-binding epitopes of PLAC1-fliC were forecasted by NetMHC 4.0 and NetMHCII 2.3 Servers. Finally, Ellipro and CTLpred were employed to predict B-cell and CTL epitopes. Results:The construct was evaluated as a stable fusion protein, which could be antigenic and could stimulate B and T cells against breast cancer. Conclusion:PLAC1-fliC, as a cancer vaccine candidate, might be suitable and specific for breast cancer, which could evoke humoral and cellular immunity against this type of tumor.

Abstract 3479: Functional significance of ESR1 fusions with diverse gene partners in endocrine therapy resistant breast cancer

Abstract Emerging evidence suggests that ESR1 fusion transcripts that produce functional fusion proteins in estrogen receptor positive (ER+) breast cancer play a role in acquired endocrine therapy resistance. We recently reported two in-frame ESR1 fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, identified in patients with endocrine therapy resistant breast cancer. Both ESR1 fusions generated stable in-frame, fusion proteins that were transcriptionally active, driving endocrine therapy resistant proliferation and also promoting an epithelial-to-mesenchymal transition (EMT) transcriptional program leading to metastasis in experimental models. Here, we extend our studies by examining properties of additional ESR1 fusions with diverse partner genes, ESR1-DAB2, ESR1-GYG1, ESR1-SOX9, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B, all identified in endocrine-refractory breast tumors, to better understand the role of ESR1fusion gene formation in endocrine therapy resistance. Structurally, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B fusions retain the first 6 exons of ESR1, therefore lacking ESR1 exons encoding the ligand-binding domain (LBD) that endocrine therapies recognize, but are instead fused in-frame to C-terminal sequences from partner genes, which follows the same fusion pattern of previously identified ESR1-YAP1, ESR1-PCDH11X, ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 fusions. ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B constructs, along with ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 constructs were stably expressed in an ER+ breast cancer cell line, T47D, and all produced stable fusion proteins. ESR1-ARNT2 and ESR1-SOX9 promoted hormone-independent and fulvestrant-resistant cell proliferation that was sensitive to cyclin-dependent kinase (CDK) 4/6 inhibitors, palbociclib and abemaciclib. In addition, ESR1-ARNT2 induced hormone-independent activation of estrogen responsive genes (TFF1, GREB1, and PGR), and EMT genes (SNAI1 and VCAN) along with upregulation of Snail protein. These results indicate that ESR1-ARNT2 and ESR1-SOX9 have similar functional and pharmacological properties to our previously described ESR1-YAP1 and ESR1-PCDH11X fusions. These data further support a role for ESR1 fusions in driving not only endocrine therapy resistance but also activating the metastatic process. Ongoing studies are examining the ability of ESR1 fusions to induce EMT phenotypes and to determine structure function relationships of ESR1 fusions. Although the formation of ESR1 fusions likely confers resistance to all endocrine therapies that target the LBD, ESR1 fusion driven growth remained sensitive to CDK4/6 inhibitor treatment therefore providing rationale for testing ESR1 fusion detection in guiding treatment with CDK4/6 monotherapy. Citation Format: Jonathan T. Lei, Xuxu Gou, Sinem Seker, Vaishnaivi Devorakonda, Kimberly R. Holloway, Adrian V. Lee, Dan R. Robinson, Matthew J. Ellis. Functional significance of ESR1 fusions with diverse gene partners in endocrine therapy resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3479.

An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

Bacterial Outer Membrane Vesicles (OMVs)-based Dual Vaccine for Influenza A H1N1 Virus and MERS-CoV.

Vaccination is the most functional medical intervention to prophylactically control severe diseases caused by human-to-human or animal-to-human transmissible viral pathogens. Annually, seasonal influenza epidemics attack human populations leading to 290–650 thousand deaths/year worldwide. Recently, a novel Middle East Respiratory Syndrome Coronavirus emerged. Together, those two viruses present a significant public health burden in areas where they circulate. Herein, we generated a bacterial outer membrane vesicles (OMVs)-based vaccine presenting the antigenic stable chimeric fusion protein of the H1-type haemagglutinin (HA) of the pandemic influenza A virus (H1N1) strain from 2009 (H1N1pdm09) and the receptor binding domain (RBD) of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (OMVs-H1/RBD). Our results showed that the chimeric antigen could induce specific neutralizing antibodies against both strains leading to protection of immunized mice against H1N1pdm09 and efficient neutralization of MERS-CoV. This study demonstrate that OMVs-based vaccines presenting viral antigens provide a safe and reliable approach to protect against two different viral infections.

Abstract P5-04-01: Functional and therapeutic significance of ESR1 fusions in metastatic ER+ breast cancer

Abstract Background. Next-generation sequencing methods have identified several ESR1 fusion genes in treatment refractory ER+ breast cancer, however detailed functional studies in experimental models are lacking and how they might be targeted remains poorly understood. We recently reported two transcriptionally active, in-frame ESR1 fusions, ESR1-YAP1 and ESR1-PCDH11X, identified in a small cohort of metastatic ER+ cases, that induce not only pan-endocrine therapy resistance but also metastatic disease progression (Lei et al., Cell Reports, in press). Limited characterization of ESR1-DAB2 and ESR1-GYG1, also identified in metastatic ER+ disease from a recent study, suggests these two ESR1 fusions also drive estrogen-independent gene activation (Hartmaier et al., Annals of Oncology, 2018). Here, we functionally characterize ESR1-DAB2 and ESR1-GYG1 along with additional ESR1 fusions discovered in metastatic ER+ breast tumors to further support a causal role for in-frame ESR1 fusions in driving endocrine therapy resistance and promoting metastasis-associated biology, and explore therapeutic vulnerabilities induced by ESR1 fusion gene formation. Methods. RNA-seq identified ESR1 fusions from treatment refractory, ER+ metastatic breast tumors. In-frame ESR1 fusions constructs were generated and stably expressed in ER+ breast cancer cell lines: T47D, MCF7, and ZR75-1. Estrogen-independent and fulvestrant-resistant growth was monitored in hormone-deprived stable cell lines. mRNA-qPCR was performed to examine expression of estrogen responsive and epithelial-to-mesenchymal transition (EMT) genes. In vitro sensitivity to CDK4/6 inhibition was tested with palbociclib and abemaciclib. Results. In addition to previously described ESR1-YAP1, ESR1-PCDH11X, ESR1-DAB2, and ESR1-GYG1, that follow a pattern retaining the first 6 exons of ESR1 (ESR1-e6) fused in-frame to C-terminal sequences provided by the partner gene, additional in-frame ESR1-e6 fusions, ESR1-PCMT1, ESR1-ARNT2, and ESR1-ARID1B, all identified in metastatic ER+ samples, were found to follow the same fusion pattern. ESR1-DAB2 and ESR1-GYG1 produced stable ESR1 fusion proteins in ER+ breast cancer cell lines. In T47D, these two fusions drove estrogen-independent and fulvestrant-resistant growth. In addition, T47D and ZR75-1 models revealed that ESR1-DAB2 drove estrogen-independent expression of estrogen responsive genes and also EMT genes, including SNAI1, suggesting this fusion, like ESR1-YAP1 and ESR1-PCDH11X, could also drive metastasis. Treatment with CDK4/6 inhibitors suppressed growth induced by ESR1-DAB2 and ESR1-GYG1. Conclusion. The majority of in-frame ESR1 exon 6 fusions found in metastatic ER+ breast are transcriptionally active, drive endocrine therapy resistant proliferation, and induce an EMT-like transcriptional program. The ability to block ESR1 fusion induced growth with a CDK4/6 inhibitor is clinically significant as ESR1 fusion gene formation renders ER insensitive to all endocrine therapies that target the ligand binding domain. Furthermore, clinical diagnosis of an active ESR1 fusion could potentially stratify patients for CDK4/6 inhibitor treatment. This presentation is the most complete description of the role for ESR1 fusions in endocrine therapy resistance and metastasis described to date. Citation Format: Lei JT, Gou X, Seker S, Haricharan S, Lee AV, Robinson DR, Ellis MJ. Functional and therapeutic significance of ESR1 fusions in metastatic ER+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-01.

In Silico Evaluation of Two Targeted Chimeric Proteins Based on Bacterial Toxins for Breast Cancer Therapy

Background: Despite major advances in cancer research, breast cancer still remains the most common cancer in women. Breast cancer is a heterogeneous disease, including at least 5 subtypes. Overexpression of human epidermal growth factor receptor 2 (HER2) in the patients denotes poor prognosis leading to a reduced survival rate compared to other subtypes of breast cancer. Therefore, HER2 can be a potential therapeutic target. To enhance the potency of HER2 blockers, the conjugation of specific cytotoxic agents with these types of anticancer agents may be successful. Application of antibody-based agents are important emerging anticancer therapies. One novel approach to increase the potency of monoclonal antibodies (mAbs) is combining them with toxic molecules. Objectives: Pseudomonas exotoxin A (PE), ricin toxin (RT), and others in very minor quantities can be more potent and biologically active for this purpose. Methods: In this study, we used trastuzumab as a ligand for HER2 receptor along with Pseudomonas exotoxin A (PE38) and A subunit of Shiga toxin 2a (Stx2a). This fusion protein selectively bound to the HER2 receptor. Upon uptake by the target cells, apoptosis and cell eradication was observed. An in silico method was used before the in vitro study to illustrate the properties and construction of the protein. Physicochemical properties, structure, stability, and ligand-receptor interaction of this chimeric protein were predicted by means of computational and bioinformatics tools and servers. Results: The results of this study showed that codon adaptation index of s1 and p2 fusion gene has improved to 0.98 and .99, respectively. The mfold result has revealed that s1 and p2 mRNA were stable sufficient for efficient translation in the new host. Based on Ramachandran plot, s1 and p2 were categorized as constant fusion protein. Conclusions: Finally, based on docking software analysis, the binding ability of Herceptin was robust enough to its receptor, so these constructs could be assigned as a new antitumor candidate in cancer therapy. The results suggested that s1 and p2 were stable fusion proteins with accurate affinity to the overexpressed receptors making them potential candidates for inducing apoptosis in breast cancer cells.

Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells.

Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the periplasm bacterium E. coli. The production of soluble scFvs is more effective in quantity, however, under the reducing conditions of the E. coli bacterial cytoplasm it is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMALc-TNN and pMALc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by Tobacco Etch Virus (TEV) protease in order to obtain untagged scFv.The second protocol is for efficient production of Fab antibody fragments as MBP fusion proteins secreted by transiently transfected mammalian cells. While transient transfection is a fast and effective way of obtaining several mgs of antibody for initial screening and validation of antibodies, some antibody sequences express poorly or not at all. For such antibodies, fusion to MBP provides an effective approach for solving the expression problem.

Functional Annotation of ESR1 Gene Fusions in Estrogen Receptor-Positive Breast Cancer

SUMMARYRNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective.

Abstract 5240: Functional and therapeutic significance of ESR1 gene fusions in breast cancer

Abstract RNA sequencing detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor positive (ER+) breast cancer but their role in disease pathogenesis remains unclear. Herein we examined multiple in-frame and out-of-frame ESR1 fusions and found only two, both identified in advanced endocrine treatment resistant disease, encoded stable and functional in-frame fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, the N-terminal, DNA binding and dimerization motifs encoded by exons 2-6 were fused to C-terminal sequences from the partner gene. Functional properties included estrogen-independent growth, constitutive expression of ER target genes, anti-estrogen resistance, induction of cellular motility in vitro and the development of lung metastasis in vivo. Chromatin immunoprecipitation and RNA sequencing experiments showed both fusions uniquely activated a metastasis-associated transcriptional program. ESR1-e6>YAP1 and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, palbociclib, and a patient-derived xenograft (PDX) expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective. Citation Format: Jonathan T. Lei, Jieya Shao, Jin Zhang, Michael Iglesia, Doug W. Chan, Jin Cao, Meenakshi Anurag, Purba Singh, Xiaping He, Yoshimasa Kosaka, Ryoichi Matsunuma, Robert Crowder, Jeremy Hoog, Chanpheng Phommaly, Rodrigo Goncalves, Susana Romalho, Raquel M. Peres, Nindo Punturi, Cheryl Schmidt, Alex Bartram, Eric Jou, W V. Lai, Oliver Hampton, Anna Rogers, Ethan Tobias, Poojan Parikh, Sherri R. Davies, Shunqiang Li, Cynthia X. Ma, Vera Suman, Kelly K. Hunt, Mark A. Watson, Katherine A. Hoadley, E A. Thompson, Xi Chen, Shyam M. Kavuri, Chad J. Creighton, Christopher A. Maher, Charles M. Perou, Svasti Haricharan, Matthew J. Ellis. Functional and therapeutic significance of ESR1 gene fusions in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5240.

Engineered adenovirus fiber shaft fusion homotrimer of soluble TRAIL with enhanced stability and antitumor activity.

Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, earlier studies with recombinant TRAIL revealed many shortcomings, including a short half-life, off-target toxicity and existence of TRAIL-resistant tumor cells. In this study, we developed a novel engineering strategy for recombinant soluble TRAIL by redesigning its structure with the adenovirus knobless fiber motif to form a stable homotrimer with improved antitumor activity. The result is a highly stable fiber-TRAIL fusion protein that could form homotrimers similar to natural TRAIL. The recombinant fusion TRAIL developed here displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. This construct will serve as a foundation for a new generation of recombinant proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.

Is it possible to optimize the protein production yield by the generation of homomultimeric fusion enzymes?

BackgroundThe supply of industrially relevant biocatalysts demands an easy and efficient protein production in high yield. In a conventional approach, a recombinant protein is produced in a heterologous host enabling the manipulation of multiple parameters including expression plasmids, growth conditions and regulation of protein biosynthesis. In this study, the generation of homomultimeric fusion genes is tested as an additional parameter to increase the production yield of a heat-stable cellulase.FindingsThe LE (LguI/Eco81I)-cloning strategy was used to generate a set of plasmids containing a single copy or two to four repetitions of the endoglucanase-encoding gene cel5A from the thermophilic anaerobe Fervidobacterium gondwanense. Serial up-scaling of shaking flask volumes from 50 to 500 mL were used to determine the production yield of active cellulolytic enzyme Cel5A in recombinant form in Escherichia coli. Monitoring the cellular wet weight and total protein proved that the bacterial growth rate is not depending on the production of fusion enzymes, however activity assays in combination with Western blotting analyses indicated instability effects of large homomultimeric fusion enzymes.ConclusionThe production yield of fusion cellulases is constant with increasing molecular weights, but improved activities were not observed for recombinant Cel5A homomultimers. This strategy may serve as a starting point for further studies to generate more stable fusion proteins with improved catalytic activities and higher protein yield in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1968-0) contains supplementary material, which is available to authorized users.

Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in Escherichia coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

A potent multivalent vaccine for modulation of immune system in atherosclerosis: an in silico approach

PurposeAtherosclerosis is classically defined as an immune-mediated disease characterized by accumulation of low-density lipoprotein cholesterol over intima in medium sized and large arteries. Recent studies have demonstrated that both innate and adaptive immune responses are involved in atherosclerosis. In addition, experimental and human models have recognized many autoantigens in pathophysiology of this disease. Oxidized low-density lipoproteins, β2 glycoprotein I (β-2-GPI), and heat shock protein 60 (HSP60) are the best studied of them which can represent promising approach to design worthwhile vaccines for modulation of atherosclerosis.Materials and MethodsIn silico approaches are the best tools for design and evaluation of the vaccines before initiating the experimental study. In this study, we identified immunogenic epitopes of HSP60, ApoB-100, and β-2-GPI as major antigens to construct a chimeric protein through bioinformatics tools. Additionally, we have evaluated physico-chemical properties, structures, stability, MHC binding properties, humoral and cellular immune responses, and allergenicity of this chimeric protein by means of bioinformatics tools and servers.ResultsValidation results indicated that 89.1% residues locate in favorite or additional allowed region of Ramachandran plot. Also, based on Ramachandran plot analysis this protein could be classified as a stable fusion protein. In addition, the epitopes in the chimeric protein had strong potential to induce both the B-cell and T-cell mediated immune responses.ConclusionOur results supported that this chimeric vaccine could be effectively utilized as a multivalent vaccine for prevention and modulation of atherosclerosis.

Use of the Uteroglobin Platform for the Expression of a Bivalent Antibody against Oncofetal Fibronectin in Escherichia coli

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646–26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50–70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.

Tumor Suppressor RASSF2 Is Downregulated By The RUNX1-ETO Fusion Protein In t(8;21)+ Acute Myeloid Leukemia

IntroductionThe t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/ResultsWe analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. DiscussionBased on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures:No relevant conflicts of interest to declare.