scholarly journals Tolerance of the Ralstonia eutropha Class I Polyhydroxyalkanoate Synthase for Translational Fusions to Its C Terminus Reveals a New Mode of Functional Display

2009 ◽  
Vol 75 (17) ◽  
pp. 5461-5466 ◽  
Author(s):  
Anika C. Jahns ◽  
Bernd H. A. Rehm
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Class I ◽  
Functional Protein ◽  
Reporter Protein ◽  
Positive Interaction ◽  
C Terminus ◽  
N Terminus ◽  
Polyhydroxyalkanoate Synthase

ABSTRACT Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.

scholarly journals Whole-Genome Analysis ofBacillus thuringiensisRevealing Partial Genes as a Source of Novel Cry Toxins

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Muhammad Sajid ◽  
Ce Geng ◽  
Miaomiao Li ◽  
Yueying Wang ◽  
Hualin Liu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Evolutionary Strategy ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Cry Toxins ◽  
Content Type ◽  
Crystal Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACTDespite the successful application of crystal proteins (Cry) fromBacillus thuringiensisas biological control agents against insects, there is an increasing demand to identify new Cry toxins having higher toxicity and broad-spectrum activity against insects and plant-parasitic nematodes. To find novel Cry toxins, we screened 100 whole-genome sequences ofB. thuringiensis. Surprisingly, in addition to full Cry toxins, we found partial sequences, such as typical N-terminal or C-terminal regions with conserved domains, widely distributed among 20 strains ofB. thuringiensis. In order to further elucidate the functions of partial genes, here, we selected a partial sequence from strain C15, having 28% similarity with the N terminus of Cry5Ba and lacking a typical C terminus, and denoted it Cry5B-like N terminus. This fragment when coexpressed as a fusion protein with the C terminus of Cry5Ba (N-C fusion protein) produces pyramidal crystals. A recombinant N-C fusion protein having a 50% lethal concentration (LC50) of 23.7 μg/ml severely affected the life span, growth, and survival rate of nematodes. Light microscopy showed damage to the intestine of nematodes, confirming the pathogenicity of the N-C fusion protein. Last, the green fluorescent protein (GFP)-labeled mutantCaenorhabditis elegansFT63 showed significant damage to the intestine upon feeding N-C fusion toxin compared to the control. These results imply that partial genes can be a source of new Cry toxins, and further understanding about functions of partialcrygenes can help in the study of the evolutionary strategy ofB. thuringiensisto produce the multidomain toxins.IMPORTANCEGenomic analysis revealed that coding sequences for N termini and C termini of crystal proteins are widely distributed inB. thuringiensis. We found Cry5B-like N terminus, lacking typical C terminus, was unable to be expressed in wild-type strain C15. However, its fusion with the C terminus of Cry5Ba not only was successfully expressed but also exhibited activity against the nematodes. This study provides insight into a potential source for novel Cry toxins.

A fusion protein with improved thrombolytic effect and low bleeding risk

2009 ◽  
Vol 102 (12) ◽  
pp. 1194-1203 ◽  
Author(s):  
Lisheng Wang ◽  
Qinglin Zhang ◽  
Yide Qin ◽  
Chutse Wu ◽  
Xiudong Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Anticoagulant Activity ◽  
Bleeding Risk ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Recognition Sequence ◽  
Linker Peptide ◽  
C Terminus ◽  
N Terminus

SummaryTo resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH.We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE andWestern blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin’s anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay.Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

scholarly journals Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags

2009 ◽  
Vol 84 (3) ◽  
pp. 1477-1488 ◽  
Author(s):  
Natalya L. Teterina ◽  
Eric A. Levenson ◽  
Ellie Ehrenfeld
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Viral Rna ◽  
Progeny Virus ◽  
C Terminus ◽  
Membrane Reorganization ◽  
Infected Cells ◽  
Viral Polyprotein ◽  
Fluorescent Protein Tags

ABSTRACT The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.

scholarly journals Analysis of the hypoxia-sensing pathway in Drosophila melanogaster

2005 ◽  
Vol 393 (2) ◽  
pp. 471-480 ◽  
Author(s):  
Nathalie Arquier ◽  
Paul Vigne ◽  
Eric Duplan ◽  
Tien Hsu ◽  
Pascal P. Therond ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fusion Protein ◽  
Gene Transcription ◽  
Fluorescent Protein ◽  
Hypoxia Inducible Factor ◽  
Glutathione S Transferase ◽  
Reporter Protein ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Green Fluorescent

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1α (hypoxia-inducible factor-1 α subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1α to the ubiquitin/proteasome pathway. HIF-1α thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1α and Similar, the Drosophila homologue of HIF-1α. The enzyme promotes human and Drosophila [35S]VHL binding to GST (glutathione S-transferase)–ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD–GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD–GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.

scholarly journals The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16

2017 ◽  
Vol 28 (18) ◽  
pp. 2420-2433 ◽  
Author(s):  
Yuqing Hou ◽  
George B. Witman
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Intraflagellar Transport ◽  
Critical Importance ◽  
C Terminus ◽  
N Terminus ◽  
Dynein Arms

Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multisubunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that the IFT46 N-terminus, ODA16, and outer arm dynein interact for IFT of the latter.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3271-3280 ◽  
Author(s):  
Jan Hänisch ◽  
Marc Wältermann ◽  
Horst Robenek ◽  
Alexander Steinbüchel
Keyword(s):  
Fusion Protein ◽  
Mycobacterium Smegmatis ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Rhodococcus Opacus ◽  
Egfp Fusion Protein ◽  
Green Fluorescent ◽  
Cell Fractions

In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli β-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia–Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1–eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1–eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1–LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of β-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.

scholarly journals The N- and C-Terminal Regions of Rotavirus NSP5 Are the Critical Determinants for the Formation of Viroplasm-Like Structures Independent of NSP2

2003 ◽  
Vol 77 (22) ◽  
pp. 12184-12192 ◽  
Author(s):  
K. V. K. Mohan ◽  
J. Muller ◽  
C. D. Atreya
Keyword(s):  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Nonstructural Proteins ◽  
Binding Region ◽  
Native Form ◽  
Functional Studies ◽  
Molecular Events ◽  
C Terminus ◽  
N Terminus ◽  
Green Fluorescent

ABSTRACT Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.

scholarly journals Mucosal Immunization with Recombinant Fusion Protein DnaJ-ΔA146Ply Enhances Cross-Protective Immunity against Streptococcus pneumoniae Infection in Mice via Interleukin 17A

2014 ◽  
Vol 82 (4) ◽  
pp. 1666-1675 ◽  
Author(s):  
Yusi Liu ◽  
Hong Wang ◽  
Shuai Zhang ◽  
Lingbin Zeng ◽  
Xiuyu Xu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Secretory Iga ◽  
Pneumococcal Infections ◽  
Protective Mechanisms ◽  
Mucosal Adjuvant ◽  
Content Type ◽  
Interleukin 17A ◽  
C Terminus ◽  
N Terminus ◽  
Single Deletion

ABSTRACTPneumolysin (Ply) and its variants are protective against pneumococcal infections in animal models, and as a Toll-like receptor 4 agonist, pneumolysin has been reported to be a mucosal adjuvant. DnaJ has been approved as a useful candidate vaccine protein; we therefore designed novel fusion proteins of DnaJ with a form of Ply that has a deletion of A146 (ΔA146Ply-DnaJ [the C terminus of ΔA146Ply connected with the N terminus of DnaJ] and DnaJ-ΔA146Ply [the C terminus of DnaJ connected with the N terminus of ΔA146Ply]) to test whether they are protective against focal and lethal pneumococcal infections and their potential protective mechanisms. The purified proteins were used to intranasally immunize the animals without additional adjuvant. Immunization with DnaJ-ΔA146Ply or DnaJ plus ΔA146Ply (Ply with a single deletion of A146) could significantly reduceS. pneumoniaecolonization in the nasopharynx and lung relative with DnaJ alone. Additionally, we observed the best protection for DnaJ-ΔA146Ply-immunized mice after challenge with lethal doses ofS. pneumoniaestrains, which was comparable to that achieved by PPV23. Mice immunized with DnaJ-ΔA146Ply produced significantly higher levels of anti-DnaJ IgG in serum and secretory IgA (sIgA) in saliva than those immunized with DnaJ alone. The production of IL-17A was also striking in DnaJ-ΔA146Ply-immunized mice. IL-17A knockout (KO) mice did not benefit from DnaJ-ΔA146Ply immunization in colonization experiments, and sIgA production was impaired in IL-17A KO mice. Collectively, our results indicate a mucosal adjuvant potential for ΔA146Ply and that, without additional adjuvant, DnaJ-ΔA146Ply fusion protein exhibits extensive immune stimulation and is effective against pneumococcal challenges, properties which are partially attributed to the IL-17A-mediated immune responses.

scholarly journals Interactions between the N- and C-termini of the mechanosensitive ion channel AtMSL10 are consistent with a three-step mechanism for activation

2020 ◽  
Vol 71 (14) ◽  
pp. 4020-4032 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M Shoots ◽  
Elizabeth S Haswell
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Adaptive Responses ◽  
C Terminus ◽  
N Terminus ◽  
Mechanosensitive Ion Channel ◽  
Green Fluorescent

Abstract Although a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to green fluorescent protein (GFP) triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag, and could also be induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus could be transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.

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