Production Of Chimeric Protein Research Articles

PROTEIN ENGINEERING OF BST POLYMERASE FOR ISOTHERMAL AMPLIFIСATION PURPOSES

The review is devoted to protein engineering of Bst polymerase using various methods. To modify the enzyme, approaches such as the production of chimeric proteins, directed evolution, directed and random mutagenesis have been used. Examples of successful changes in enzyme properties such as catalytic activity, processivity, thermal stability, and resistance to inhibitors are described.

Recombinant Proteins Combining the Inter-domain Region of Pneumococcal Surface Antigen "A" and Adaptive W-type Polypeptide of Thermotoga Bacteria as a Potential Component Base for the Development of New Diagnostics and Genetically Engineered Subunit Vaccines against Pneumococcal Infection

The problems related to the development of pneumococcal vaccines require combination of traditional solutions and alternative systems optimizing this procedure. Recombinant subunit vaccines have undeniable advantages over inactivated and live-attenuated vaccines: they induce cell-mediated and humoral immunologic responses effectively and with high specificity, but without the risks associated with authentic pathogen processing. However, subunit vaccines require specific adjuvants to enhance the immune response or special fusion partners to improve solubility, expression and optimize subsequent fine purification of the protein of interest. In the framework of this work, a structurally conserved region of the most immunogenic region of the vaccine-valuable surface antigen PspA of Streptococcus pneumoniae was chosen as a model protein, and the adaptive polypeptide CheW from the hyperthermophilic microorganism Thermotoga petrophila was used as a promising fusion protein. Appropriate expression plasmid vectors were designed in silico and constructed in vitro. Efficient E. coli producer strains were obtained and appropriate conditions for heterologous production of chimeric proteins were selected. The fusion partner from T. petrophila positively influenced the properties of the resulting constructs such as thermostability, solubility, and homogeneity. During this work, the optimal pH and temperature ranges of the created proteins were determined, and the principles of low-stage purification were elaborated. We obtained and characterized new proteins, which were not previously found in nature in a similar bioconfiguration. The results indicate that the biotechnologically valuable characteristics of the fusion protein were more expressed when the adaptive CheW protein was combined with the N-terminus of the PspA antigen.

Construction of Solid Binding Peptide (SBP)-Fused Non-Native Chimeric Enzyme to Promote Bienzymatic Cascade Induced Bioelectrocatalysis

In nature, most enzymes form a multienzyme complex called “metabolon” in the cellular system. The natural metabolon has been evolved to increase the efficiency of multi-step biocatalysis involved in such conditions. Such principle of enzymatic cascade reaction could be applied to the system which mimics pertinent reaction in vitro. Especially, its pertaining to bioelectrocatalytic system is quite promising to enhance its performance in terms of electrical generation or biochemical production, depending on systematic applications. In the previous studies , it was abundantly reported that multi-enzymatic reaction has been utilized to generate higher electrical current via sequential enzymatic oxidation of substrates (e.g., methanol, glucose, etc.) in bioelectrochemical systems, and to operate biochemical production system utilizing multi-enzymic conversion or reduction reactions. However, it is still challenging to implement multi-step enzymatic reaction in the electrode platforms due to unmanageable regulations of inter-enzyme distance or electrical connection between enzymatic cofactor and electrode, during multi-enzyme co-immobilization on electrode surface.Herein, we intentionally design and construct variants of chimeric enzyme in which two enzymes, invertase (INV) and glucose dehydrogenase (GDH), are conjugated with peptide linkers. We expected that INV hydrolyzes sucrose into fructose and glucose, then glucose is oxidized at the catalytic subunit of GDH, releasing electrons toward the electrode. It also employed solid binding peptides (SBPs) fused to GDH in chimeric enzyme for the electrical connection of GDH-SBP and electrode. In order to accomplish the desired reaction with chimeric enzyme, we have conducted the following tests; 1) The recombinant plasmid harboring the genetic sequence of two enzymes with an in-between linker was constructed. 2) By production of chimeric proteins, the chain reaction efficiency of fusion constructs was compared depending on the length of the linker. 3) The gold binding peptide (GBP) was fused to sites (N- and C-terminus, or other sites) of the catalytic subunit of GDH. And finally, 4) the electron transfer rates were monitored and compared depending on the GBP tethering sites. Throughout the whole study, we found the factors affecting the occurrence of multienzyme cascade-based bioelectrochemical reactions and how to control these factors to improve the reaction efficiency Figure 1

Regulatory networks and 5' partner usage of miRNA host gene fusions in breast cancer.

Genomic rearrangements in cancer cells can create gene fusions where the juxtaposition of two different genes leads to the production of chimeric proteins or altered gene expression through promoter‐swapping. We have previously shown that fusion transcripts involving microRNA (miRNA) host genes contribute to deregulation of miRNA expression regardless of the protein‐coding potential of these transcripts. Many different genes can also be used as 5′ partners by a miRNA host gene in what we named recurrent miRNA‐convergent fusions. Here, we have explored the properties of 5′ partners in fusion transcripts that involve miRNA hosts in breast tumours from The Cancer Genome Atlas (TCGA). We hypothesised that firstly, 5′ partner genes should belong to pathways and transcriptional programmes that reflect the tumour phenotype and secondly, there should be a selection for fusion events that shape miRNA expression to benefit the tumour cell through the known hallmarks of cancer. We found that the set of 5′ partners in miRNA host fusions is non‐random, with overrepresentation of highly expressed genes in pathways active in cancer including epithelial‐to‐mesenchymal transition, translational regulation and oestrogen signalling. Furthermore, many miRNAs were upregulated in samples with host gene fusions, including established oncogenic miRNAs such as mir‐21 and the mir‐106b~mir‐93~mir‐25 cluster. To the list of mechanisms for deregulation of miRNA expression, we have added fusion transcripts that change the promoter region. We propose that this adds material for genetic selection and tumour evolution in cancer cells and that miRNA host fusions can act as tumour ‘drivers’.

Neuregulin 1 (NRG1) fusion-positive high-grade spindle cell sarcoma: A distinct group of soft tissue tumors with metastatic potential.

Neuregulin 1 (NRG1) is an epidermal growth factor (EGF)-like ligand that activates receptor tyrosine kinases of the ErbB family of receptors. NRG1 gene fusions, which are rare (<1%) but recurrent events in solid tumors, are an emerging oncogenic driver that is potentially actionable using ErbB-targeted tyrosine kinase inhibitors. Largely characterized only in carcinomas, we describe three cases of NRG1-rearranged sarcomas. The patients were all female, aged 32-47 years old. Two cases were deep-seated tumors in the lower extremities (right thigh and calf); one case presented as a uterine mass. The tumors measured 9-11.5 cm in the greatest dimensions. Histologically, all three tumors were high-grade spindle cell sarcomas composed of monomorphic spindle cells arranged in interlacing fascicles. The tumor cells were set in the loose collagenous stroma with branching, curvilinear thin-walled vasculature in the background. Cytologically, the neoplastic cells displayed ovoid to fusiform nuclei with finely stippled chromatin, inconspicuous nucleoli, scant to moderate clear to eosinophilic cytoplasm, occasional cytoplasmic vacuoles, and elongated cytoplasmic processes. Mitotic activity was elevated (> 20/10 high power fields) and tumor necrosis was present. None of the tumors expressed lineage-specific immunophenotypical markers. Targeted RNA-sequencing uncovered gene fusions involving NRG1 and the 5' untranslated regions of PPHLN1, HMBOX1, or MTUS1. In all cases, the C-terminal EGF-like domain of NRG1 was preserved in the predicted chimeric protein product. All three patients developed metastatic disease within 2 years from initial presentation and were alive with disease at last follow-up (mean follow-up period=19 months). In conclusion, we present the first case series of NRG1-rearranged sarcomas characterized by high-grade fascicular spindle cell morphology, non-specific immunoprofile, and aggressive clinical behavior. Further studies are needed to determine whether this distinct subgroup of spindle cell sarcomas are amenable to targeted therapies.

Study of neurotrophic factor receptor tyrosine kinase gene fusion in the precise treatment of wild-type gastrointestinal stromal tumor

The neurotrophin receptor kinase (NTRK) gene encodes neurotrophic factor receptor tyrosine kinase (NTRK), which plays an important role in the development and function of the nervous system. NTRK gene fusion mutation results in the production of chimeric NTRK proteins, which have carcinogenic potential through constitutive activation or overexpression. NTRK gene fusion mutation can lead to a special type of wild type gastrointestinal stromal tumor (GIST), whose clinical manifestations and treatment are completely different from other types of GIST. This fusion mutation can be detected clinically by a variety of methods, including tumor DNA and RNA sequencing and immunohistochemical staining. In patients with NTRK fusion positive tumors, NTRK inhibitors such as larotrectinib and entrectinib have shown good antitumor efficacy, with clinical response rates as high as 75%. Therefore, there is a need to improve the recognition and detection of fuch patients and to improve their prognosis by individualized and precise treatment with TRK inhibitors.

Immunization with oral and parenteral subunit chimeric vaccine candidate confers protection against Necrotic Enteritis in chickens

Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM.The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.

Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays.

NTRK fusions involving three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, and NTRK3 and a variety of fusion partners were identified as oncogenic drivers across many cancer types. Drugs that target the chimeric protein product require the identification of the underlying gene fusion. This advocates the diagnostic use of molecular assays ranging from fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR)/Sanger approaches to targeted next-generation sequencing (NGS). Immunohistochemistry may be used as a screening tool and adjunct diagnostic assay in this context. Although FISH and RT-PCR/Sanger approaches are widely adopted in routine diagnostics, current experience with targeted RNA-based NGS is limited. Here, we report on the analysis of major assays (TruSight TST170 and TruSight RNA Fusion [Illumina]; Archer FusionPlex Solid Tumor, Archer FusionPlex Lung, and Archer FusionPlex Oncology [Archer]; Oncomine Comprehensive Assay v3 RNA and Oncomine Focus RNA [Thermo Fisher Scientific]) that are commercially available. The data set includes performance results of a multicentric comparative wet-lab study as well as an in silico analysis on the ability to detect the broad range of NTRK fusions reported until now. A test algorithm that reflects assay methodology is provided. This data will support implementation of targeted RNA sequencing in routine diagnostics and inform screening and testing strategies that have been brought forward.

Designing of suitable linkers for the chimeric proteins to achieve the desired flexibility and extended conformation

The designing and production of therapeutic chimeric proteins is the central focus area of many industrial R & D and research institutes. The efficient productions of chimeric proteins not only require suitable domains or partner proteins and intact receptor binding sites, but the selection of linkers is equally important. The linkers are essential to provide space between two domains, prevent from steric hindrance and most importantly to make the chimeric proteins flexible. In this in silico study, we have systematically designed various linkers, tested their feasibility and evaluated their essentiality. Here, we fused cytokine of interest (i.e., IL-2) with IgG1 Fc via various linkers. We designed linkers of various lengths and amino acid composition and tested their ability to provide the extended conformation and desired flexibility and also to minimise the conformational changes. Additionally, we have evaluated the role of the linker in dimerisation of chimeric proteins. We next tested the influence of linkers on stability and functionality of chimeric proteins.

Designing of suitable linkers for the chimeric proteins to achieve the desired flexibility and extended conformation

The designing and production of therapeutic chimeric proteins is the central focus area of many industrial R & D and research institutes. The efficient productions of chimeric proteins not only require suitable domains or partner proteins and intact receptor binding sites, but the selection of linkers is equally important. The linkers are essential to provide space between two domains, prevent from steric hindrance and most importantly to make the chimeric proteins flexible. In this in silico study, we have systematically designed various linkers, tested their feasibility and evaluated their essentiality. Here, we fused cytokine of interest (i.e., IL-2) with IgG1 Fc via various linkers. We designed linkers of various lengths and amino acid composition and tested their ability to provide the extended conformation and desired flexibility and also to minimise the conformational changes. Additionally, we have evaluated the role of the linker in dimerisation of chimeric proteins. We next tested the influence of linkers on stability and functionality of chimeric proteins.

Recombinant approach to develop ScFv antibodies and vaccines against viral diseases as Ebola, flu (AH1N1) and cancer (polyomavirus)

Currently, production of vaccines and diagnostic systems for infectious diseases has failed to provide a systematic vision that merges state-of-the-art technologies with industry to provide an effective commercial solution. Infectious and rapidly transmitted diseases, such as cancer, Ebola and influenza, should be a focus of interest for these prospects. While technological advances of recent years have been revolutionizing the life sciences industry, specifically in the biopharma field, these advances have been disproportional in terms of their applications towards infectious diseases. Working on the development, recombinant technology is needed for the production of chimeric proteins using mammalian, yeast, and bacterial cells modified for those purposes. Proteins developed through a process of molecular engineering, which begins with in silico bioinformatic processes, using validations and algorithms, subsequently through synthetic biology, molecular biology, genetic engineering, and Bioprocess development.

Alpha repeat proteins (αRep) as expression and crystallization helpers.

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

Comparative functional analysis of the molecular network of 7 selected MLL fusion proteins

The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. MLL-rearrangements are primarily associated with pediatric leukemias with poor prognosis. Balanced translocations result in the fusion of the MLL gene to over 60 fusion partner genes, leading to the production of novel chimeric proteins. Even though critical effectors of distinct MLL fusion proteins have been identified, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist.

Towards A Superior Streptokinase for Fibrinolytic Therapy of Vascular Thrombosis

Medical intervention with fibrinolytic drugs such as tissue plasminogen activator (tPA) and streptokinase (SK) is the principal treatment for life-treating thromboembolic disorders. Contrary to tPA, SK is a heterogenic and non-human (bacterial) protein produced by streptococci and its medical application may elicit sever immune and anaphylactic responses that restrict its utilization. Besides, human plasminogen (HPG) activation by SK is not blood-clot specific and associated with a risk of hemorrhage. Despite these limitations, comparative clinical trials on various thrombolytic agents suggested that SK is the most cost-effective fibrinolytic drug and almost as safe as its other counterparts such as tPA. Therefore, a number of studies were conducted to provide structurally modified SK with reduced immunogenicity, higher blood-clot specificity and half-lives. Although there are extensive overlaps in SK structural domains responsible for functionality, immunogenicity and stability that may limit its modifications, various strategies such as genetic manipulations (amino acid substitution /addition /deletion or domain fusions through production of chimeric SK proteins linked to HPG or hirudin) and chemical modification such as (homogenous/site-specific) PEGylation have been employed to develop a superior SK. In addition, data of the latest studies on SK screened from different streptococcal sources indicated the possibility of retrieving naturally occurring SKs with higher activities, less antigenicity and/or more fibrinspecificity. In the present review, after a survey on structure function relationships of SK domains and different strategies for SK improvement, recent advances and potential application of computer and matrix-based analyses for design and introduction of superior SKs will be presented.

Abstract C213: Protein interaction analysis of mll-aff4 and mll-fel oncogenic fusion proteins using data mining approach.

Abstract Mixed-lineage leukemia (MLL) is a transcriptional co-activator that plays an essential role in early development and hematopoiesis. MLL H3K4 methyltransferase activity mediates chromatin remodeling associated with epigenetic regulation of transcription activation. Multiple chromosomal translocations of MLL gene lead to its fusion to numerous partner genes and production of chimeric proteins, which are enrolled in progression of similar phenotypes of leukemia [5]. However, protein interaction models of different MLL fusions are still debated. To assess the pattern of protein interactions of MLL chimeras, we have performed protein-protein interaction (PPI) network analysis for two MLL fusion proteins, MLL-FEL - t(4;11)(q21;q23) and MLL-AFF4 - ins(5;11)(q31;q13q23), which are involved in acute leukemia progression [2]. Protein interaction data was extracted from data mining software GPS-Prot v. 2.0 [4]. PPI network visualization and network parameters evaluation were performed using Cytoscape v. 2.8 [7]. Cluster analysis was made by MCODE plug-in. The statistical significance of cluster extraction was assessed as described [3]. Comparative experimental interaction data taken from previously published papers were manually discarded from our analysis. Our results show that each of the components of studied fusion proteins in native form compose highly interconnected distinct regions. MLL PPI network analysis reveals a clique cluster with cluster density equal to 1, indicating that MLL interacters are united in protein macromolecular complex, which is in a good accordance with previously published experimental data [6]. Worth of mention, that FEL and AFF4 proteins form the same clique clusters, which also is in a good congruence with experimentally obtained data [1]. Therefore, we assume that the MLL-AFF4 and MLL-FEL fusion proteins generate the identical PPI network, which underlie the manifestation of similar malignancy. Further corroboration of this result will confirm the assumption that different fusions can bring to the same disease network, which could possibly has its impact in leukemia therapeutical approaches. To obtain more detailed pattern of protein interactions of MLL-caused leukemias further network analysis is required. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C213. Citation Format: Grant Henry Hovhannisyan, Ashot Vardan Marqaryan. Protein interaction analysis of mll-aff4 and mll-fel oncogenic fusion proteins using data mining approach. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C213.

Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry

Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.

Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

Membrane FasL is the natural trigger of Fas-mediated apoptosis. A soluble homotrimeric counterpart (sFasL) also exists which is very weakly active, and needs oligomerization beyond its trimeric state to induce apoptosis. We recently generated a soluble FasL chimera by fusing the immunoglobulin-like domain of the leukemia inhibitory factor receptor gp190 to the extracellular region of human FasL, which enabled spontaneous dodecameric homotypic polymerization of FasL. This polymeric soluble human FasL (pFasL) displayed anti-tumoral activity in vitro and in vivo without systemic cytotoxicity in mouse. In the present work, we focused on the improvement of pFasL, with two complementary objectives. First, we developed more complex pFasL-based chimeras that contained a cell-targeting module. Secondly, we attempted to improve the production and/or the specific activity of pFasL and of the cell-targeting chimeras. We designed two chimeras by fusing to pFasL the extracellular portions of the HLA-A2 molecule or of a human gamma-delta TCR, and analyzed the consequences of co-expressing these molecules or pFasL together with sFasL on their heterotopic cell production. This strategy significantly enhanced the production of pFasL and of the two chimeras, as well as the cytotoxic activity of the two chimeras but not of pFasL. These results provide the proof of concept for an optimization of FasL-based chimeric proteins for a therapeutic use.

Protein chimerism: Novel source of protein diversity in humans adds complexity to bottom‐up proteomics

Three main molecular mechanisms are considered to contribute expanding the repertoire and diversity of proteins present in living organisms: first, at DNA level (gene polymorphisms and single nucleotide polymorphisms); second, at messenger RNA (pre-mRNA and mRNA) level including alternative splicing (also termed differential splicing or cis-splicing); finally, at the protein level mainly driven through PTM and specific proteolytic cleavages. Chimeric mRNAs constitute an alternative source of protein diversity, which can be generated either by chromosomal translocations or by trans-splicing events. The occurrence of chimeric mRNAs and proteins is a frequent event in cells from the immune system and cancer cells, mainly as a consequence of gene rearrangements. Recent reports support that chimeric proteins may also be expressed at low levels under normal physiological circumstances, thus, representing a novel source of protein diversity. Notably, recent publications demonstrate that chimeric protein products can be successfully identified through bottom-up proteomic analyses. Several questions remain unsolved, such as the physiological role and impact of such chimeric proteins or the potential occurrence of chimeric proteins in higher eukaryotic organisms different from humans. The occurrence of chimeric proteins certainly seems to be another unforeseen source of complexity for the proteome. It may be a process to take in mind not only when performing bottom-up proteomic analyses in cancer studies but also in general bottom-up proteomics experiments.

Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.