Expression of functional recombinant human tissue transglutaminase (TG2) using the bac-to-bac baculovirus expression system

Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bacto- Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. © 2016 The Authors

Expression of an innate immune element (mouse hepcidin-1) in baculovirus expression system and the comparison of its function with synthetic human hepcidin-25

Hepcidin is an innate immune element which decreases the iron absorption from diet and iron releasing from macrophage cell. In contrast to the chemical iron chelators, there has been limited effort applied to the specific use of hepcidin as a new drug for decreasing the iron overload. Hepcidin is produced in different biological systems. For instance, E-coli is used for human hepcidin expression, however, post-translational modification is impaired. We have used a simple baculovirus expression system (BES) to improve the hepcidin folding and activity. Hepcidin Messenger Ribonucleic acid (mRNA) was isolated from mouse liver cells and its complementary Deoxyribonucleic acid (cDNA) was produced and amplified. PFastBac HTB vector was used for recombinant bacmid production. Recombinant baculovirus was produced using SF-9 cell line. The mouse hepcidin-1 protein was expressed in a large quantity and functional tests were performed for this recombinant peptide. The yield of hepcidin in BES was 20 μg/mL and anti-histidine (anti-His) tag antibody was used for the confirmation of hepcidin on western blot nitrocellulose paper. Functional tests showed that mouse hepcidin accumulates iron in the macrophage cell line J774A.1 up to 63%. In addition, our data showed that the mouse hepcidin-1 has less toxicity compared to the synthetic human hepcidin-25 (p = 0.000). © 2011 by School of Pharmacy

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective conditio

Helicoverpa armigera nucleopolyhedrovirus occlusion-derived virus-associated protein, HA100, affects oral infectivity in vivo but not virus replication in vitro

ORF100 (ha100) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been reported as one of the unique genes of group II alphabaculoviruses encoding a protein located in the occlusion-derived virus (ODV) envelope and nucleocapsid. The protein consists of 510 aa with a predicted mass of 58.1 kDa and is a homologue of poly(ADP–ribose) glycohydrolase in eukaryotes. Western blot analysis detected a 60 kDa band in HearNPV-infected HzAM1 cells starting at 18 h post-infection. Transient expression of GFP-fused HA100 in HzAM1 cells resulted in cytoplasmic localization of the protein, but after superinfection with HearNPV, GFPfused HA100 was localized in the nucleus. To study the function of HA100 further, an ha100-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the ha100-null virus had similar budded virus production kinetics to that of the parental virus. Electron microscopy revealed that deletion of HA100 did not alter the morphology of ODVs or occlusion bodies (OBs). However, bioassays in larvae showed that the 50% lethal concentration (LC50) value of HA100-null OBs was significantly higher than that of parental OBs; the median lethal time (LT50) of ha100-null OBs was about 24 h later than control virus. These results indicate that HA100 is not essential for virus replication in vitro. However, it significantly affects the oral infectivity of OBs in host insects, suggesting that the association HA100 with the ODV contributes to the infectivity of OBs in vivo

A full UL13 open reading frame in Marek’s disease virus (MDV) is dispensable for tumor formation and feather follicle tropism and cannot restore horizontal virus transmission of rRB-1B in vivo

peer reviewedMarek’s disease virus (MDV) is an oncogenic alphaherpesvirus that is highly contagious in poultry. Recombinant RB-1B (rRB-1B) reconstituted from an infectious genome cloned as a bacterial artificial chromosome (BAC) is unable to spread horizontally, quite in contrast to parental RB-1B. This finding suggests the presence of one or several mutations in cloned relative to parental viral DNA. Sequence analyses of the pRB-1B bacmid identified a one-nucleotide insertion in the UL13 orthologous gene that causes a frame-shift mutation and thereby results in a theoretical truncated UL13 protein (176 aa vs. 513 aa in parental RB-1B). UL13 genes are conserved among alphaherpesviruses and encode protein kinases. Using two-step “en passant” mutagenesis, we restored the UL13 ORF in pRB-1B. After transfection of UL13-positive pRB-1B DNA (pRB-1B*UL13), the resulting, repaired virus did not exhibit a difference in cell-to cell spread (measured by plaque sizes) and in UL13 transcripts in culture to parental rRB-1B virus. Although 89% of the chickens inoculated with rRB-1B*UL13 virus developed tumors in visceral organs, none of the contact birds did. MDV antigens were clearly expressed in the feather tips of rRB-1B infected chickens, suggesting that the UL13 gene mutation did not alter virus tropism of the feather follicle. The results indicate that the correction in UL13 gene alone is not sufficient to restore in vivo spreading capabilities of the rRB-1B virus, and that other region(s) of pRB-1B might be involved in the loss-of-function phenotype. This finding also shows for the first time that a full UL13 ORF is dispensable for MDV tumor formation and feather follicle tropism

Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% ß-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infectio

Construction of Fluorescently-Tagged and Adenosine Nucleotide-Binding Mutations of the Human MutS Homolog Heterodimer MSH2-MSH3

DNA mismatch repair (MMR) is a highly conserved system for correcting mispaired nucleotides arising from misincorporation errors during DNA replication, genetic recombination, and chemical or physical damage. The MutS homologues (MSH) and MutL homologues (MLH/PMS) are the fundamental components of MMR and are conserved from bacteria to humans. The MSH proteins initiate MMR via mismatch legion recognition. One human MSH complex in particular, hMSH2-hMSH3, recognizes small insertion deletion loops (IDL) and repetitive DNA sequences. Inherited mutations in many MMR genes including hMSH2 lead to a predisposition for colorectal cancer (hereditary non-polyposis colorectal cancer, HNPCC). Also, the hMSH2-hMSH3 complex has been implicated in the expansion of tri-nucleotide repeats in disorders such as Huntington’s disease and myotonic dystrophy. The role hMSH2-hMSH3 plays in this expansion remains enigmatic. Two mutations made to the Walker A nucleotide binding domain of hMSH2 and hMSH3 will allow for a detailed study of the mechanics of this complex in recognizing and binding DNA lesions, as well as the signaling of downstream MMR components. A detailed study of the conformational changes the protein undergoes in lesion recognition will also be possible via fluorescently tagged MSH2 and MSH3 subunits for use in fluorescence resonance energy transfer.Dean's Undergraduate Research Fund AwardNo embarg

Untersuchungen zu einem tumorrelevanten, FGF-bindenden Protein (FGF-BP)

Die nahezu ubiquitär vorkommenden Wachstumsfaktoren FGF-1 und FGF-2 spielen neben ihren physiologischen Funktionen auch eine wichtige Rolle beim Wachstum vieler neoplastischer Erkrankungen. Allerdings werden FGF-1 und FGF-2 nach ihrer Sekretion aus der Zelle in der extrazellulären Matrix immobilisiert und dadurch daran gehindert, ihre Wirkung durch das Binden an spezifische extrazelluläre Rezeptoren zu entfalten. Ein sekretiertes Protein, FGF-Bindeprotein (FGF-BP), bindet reversibel und nicht-kovalent an FGF-1 und FGF-2, löst sie aus der extrazellulären Matrix und ermöglicht ihnen so, mit ihren Rezeptoren zu interagieren. Gerade bei neoplastischen Erkrankungen kommt dieser FGF-BP-vermittelten Aktivität von FGF-1 und FGF-2 eine Schalterfunktion bei der Wachstumsförderung von Tumoren, auch bedingt durch die Induktion der Angiogenese, zu. Diese zentrale regulierende Rolle von FGF-BP in malignen Erkrankungen macht es zu einem eventuell vielversprechenden therapeutischen bzw. diagnostischen Ansatzpunkt in der Krebsbehandlung. Im ersten Teil dieser Arbeit sollte die biologische Aktivität von exogen zugesetztem rekombinantem FGF-BP auf Tumor- bzw. Endothel-Zelllinien näher definiert werden. Hierzu wurde rekombinantes, gereinigtes FGF-BP mittels eines Baculovirus-Expressions-System in Insektenzellen gewonnen (Bv-(FGF-BP)), die Suspensionskultur für dieses System etabliert und gezeigt, dass diese der Adhäsionskultur in Bezug auf Effizienz und Aufwand überlegen ist. Die parakrine, FGF-2-abhängige Bioaktivität des rekombinanten Bv-(FGF-BP) wurde mittels eines Softagar-Assays mit SW-13-Nebennieren-Karzinom- und DU-145-Prostata-Karzinom-Zellen nachgewiesen. Durch den Zusatz von Bv-(FGF-BP) konnte eine deutliche Steigerung der Wachstumsaktivität dieser Tumorzellen erreicht werden, welche sich durch den Zusatz von FGF-2-Antikörpern komplett blockieren ließ. Auch das Wachstum von HUVECs, humanen venösen Nabelschnur-Endothelzellen, konnte in Proliferations-Assays durch Bv-(FGF-BP) beschleunigt werden. Diese Ergebnisse zeigen, dass FGF-BP das Wachstum von Tumoren auf zwei verschiedene Arten anregt: direkt durch Stimulation der Tumorzellen und indirekt durch die Förderung der Blutversorgung des Tumors. Im zweiten Teil dieser Arbeit sollten die abnorme Molmasse des vollständigen, rekombinanten Bv-(FGF-BP) und das Auftreten höhermolekularer kovalenter FGF-BP-Komplexe, die auch schon in anderen Arbeiten beschrieben worden waren, untersucht werden. Nachdem eine mögliche Glykosilierung als Ursache für die zu hohe Molmasse ausgeschlossen werden konnte, wurden FGF-BP und FGF-2 in Insektenzellen koexprimiert, um Hinweise auf ein eventuelle intrazelluläre Komplexbildung zu erhalten. Trotz erfolgreicher Koexpression wurde unter den gewählten Bedingungen keine Komplexbildung beobachtet. Im dritten Teil gelang es erstmals, endogen exprimiertes FGF-BP mittels Immunhistochemie auf Prostata-Karzinom-Schnitten nachzuweisen und zu quantifizieren. Hierbei wurde bei allen 129 Schnitten von 88 Patienten eine immunhistochemische Reaktion beobachtet. Die Ergebnisse dieser Arbeit geben weiteren Aufschluss über die biologische Wirkung von FGF-BP, zeigen seine FGF-2-vermittelte, duale wachstumssteigernde Wirkung auf Tumoren und beweisen dessen Expression im Prostatakarzinom. Diese Erkenntnisse unterstreichen die mögliche Bedeutung dieses Proteins als therapeutisches Zielmolekül

Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy.

Soluble guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) in mammalian nitric oxide signaling. We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy. NO and the sGC-specific stimulator YC-1 induce a 71° rotation of the heme-binding β H-NOX and PAS domains. Repositioning of the β H-NOX domain leads to a straightening of the coiled-coil domains, which, in turn, use the motion to move the catalytic domains into an active conformation. YC-1 binds directly between the β H-NOX domain and the two CC domains. The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering (SAXS). These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO

MultiBac: expanding the research toolbox for multiprotein complexes

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms