scholarly journals Large-Scale, pH-Dependent, Quaternary Structure Changes in an RNA Virus Capsid Are Reversible in the Absence of Subunit Autoproteolysis

2002 ◽  
Vol 76 (19) ◽  
pp. 9972-9980 ◽  
Author(s):  
Derek J. Taylor ◽  
Neel K. Krishna ◽  
Mary A. Canady ◽  
Anette Schneemann ◽  
John E. Johnson
Keyword(s):  
Coat Protein ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Quaternary Structure ◽  
Rna Virus ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Ph Dependent

ABSTRACT The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus, Nudaurelia capensis ω virus (NωV), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 Å in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-Å-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, NωV provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in NωV.

scholarly journals Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system

1999 ◽  
Vol 342 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Corné H. W. KLAASSEN ◽  
Petra H. M. BOVEE-GEURTS ◽  
Godelieve L. J. DECALUWÉ ◽  
Willem J. DEGRIP
Keyword(s):  
Membrane Proteins ◽  
Large Scale ◽  
Structural Changes ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Scale Production ◽  
Protein Activation ◽  
Bovine Rhodopsin ◽  
Lipid Environment

Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni2+-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Biosynthesis of recombinant human pro-α1(III) chains in a baculovirus expression system: production of disulphide-bonded and non-disulphide-bonded species containing full-length triple helices

1995 ◽  
Vol 312 (3) ◽  
pp. 847-853 ◽  
Author(s):  
M Tomita ◽  
N Ohkura ◽  
M Ito ◽  
T Kato ◽  
P M Royce ◽  
...  
Keyword(s):  
Insect Cells ◽  
Incubation Temperature ◽  
Expression System ◽  
Significant Proportion ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Dermal Fibroblasts ◽  
Full Length ◽  
Type Iii Collagen ◽  
Procollagen Iii

We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the pro-alpha1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displayed a melting temperature (Tm) of approx. 38 degrees C as assessed by its resistance to digestion by a mixture of trypsin and chymotrypsin, slightly lower than that of 39.5 degrees C for procollagen III synthesized by cultured human dermal fibroblasts, and reflected a slight degree of under-hydroxylation of prolyl residues. This is possibly a consequence of the lower incubation temperature of insect cells, or of an insufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of similar thermal stability containing an apparently full-length triple-helical region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the system promises to be useful in the study of procollagen chain association and subsequent folding.

Production of recombinant androgen receptor in a heterologous expression system

1993 ◽  
Vol 39 (2) ◽  
pp. 346-352 ◽  
Author(s):  
O A Jänne ◽  
J J Palvimo ◽  
P Kallio ◽  
M Mehto ◽  
Y B Xie ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Binding Activity ◽  
Full Length ◽  
Biologically Active ◽  
Receptor Protein ◽  
Cell Protein ◽  
Domain Specific

Abstract To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.

Structural implications for the transformation of the Bacillus thuringiensis δ-endotoxins from water-soluble to membrane-inserted forms

2001 ◽  
Vol 29 (4) ◽  
pp. 571-577 ◽  
Author(s):  
J. Li ◽  
D. J. Derbyshire ◽  
B. Promdonkoy ◽  
D. J. Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Insect Cells ◽  
Water Soluble ◽  
Single Amino Acid ◽  
The Core ◽  
High Affinity Site ◽  
Temperature Factors

Crystal structures combined with biochemical data show that the δ-endotoxins from Bacillus thuringiensis are structurally poised towards large-scale, irreversible conformational changes that transform them from the soluble protein bound at the cell surface into a membrane-embedded form causing lysis of susceptible insect cells. Cry δ-endotoxins are made of a helix bundle, a β-prism and a β-sandwich. The conformational change involves an umbrella-like opening between the helix-4,5-hairpin and the remaining helices, and between the helical domain and the two sheet domains. Comparison of Cry1Ac structures with and without the bound receptor ligand GalNAc associates occupation of the high-affinity site on the β-sandwich with an increase of temperature factors in the helical, pore-forming domain, which may indicate how receptor binding could trigger the required major conformational change. The structure of Cyt δ-endotoxins indicates that the surface helix hairpins must peel away to expose the β-strands for membrane attack. Single amino acid substitutions in hinge residues or the core can restore activity following an inhibitory mutation.

scholarly journals A Molecular View of Lipid-mediated Activation Process of Apolipoprotein E

2020 ◽  
Author(s):  
Dube Dheeraj Prakashchand ◽  
Jagannath Mondal
Keyword(s):  
Apolipoprotein E ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Dynamics Simulation ◽  
Lipid Transport ◽  
Activation Process ◽  
Rapid Separation ◽  
Terminal Domain ◽  
Complexation Process

AbstractApolipoprotein E (ApoE) is a major determinant protein of lipid-metabolism and actively participates in lipid transport in plasma and central nervous system. As a part of its lipid-transport activity, low-density-lipid receptor (LDLR) needs to recognise apoE as a ligand. But, all prior evidences point to the fact that the recognition of apoE by LDLR only takes place in presence of lipid molecules which are assumed to play an important role in conformationally activating apoE upon binding. However, the molecular mechanism underlying the complexation process of apoE with lipid molecules and associated lipid-induced conformational change of apoE are currently elusive. Here we capture the spontaneous complexation process of monomeric apoE3 and phospholipid molecules by employing molecular dynamics simulation at multiple resolution. In particular, our multi scale simulations demonstrate a large-scale conformational change of the full-length apoE3, triggered by two-stage apoE-lipid complexation process. At first stage, lipid molecules assemble close to C-terminal domain of the protein and induce a rapid separation of C-terminal domain of monomeric apoE3 from rest of its tertiary fold. In the second and final stage, long-time scale simulation captures a slow on-the-fly lipid-induced inter-helix separation process in N-terminal domain of the protein. The resultant equilibrated complex, as obtained in the current work resembles an ‘open conformation’ of lipid-stabilised apoE, previously hypothesised based on small-angle X-ray scattering experiments. Taken together, the simulations provide a molecular view of kinetic interplay of apoE and lipid complexation multi-stage process leading to conformational changes in protein, potentially making it conducive for recognising LDLR.

Expression of H5N1Avian influenza virushaemagglutinin in a baculovirus expression system

2007 ◽  
Vol 4 (3) ◽  
pp. 233-237 ◽  
Author(s):  
Dun Jifeng ◽  
Pu Juan ◽  
Zhou Yingchun ◽  
Liu Jinhua
Keyword(s):  
Shuttle Vector ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Sf9 Cells ◽  
Ha Gene ◽  
Sds Page ◽  
Reaction Specificity ◽  
H5 Subtype ◽  
Ha Protein

AbstractThe haemagglutinin (HA) gene of H5N1Avian influenza virus(AIV) was amplified from the plasmid pGEM-HA and cloned into the baculovirus transfer plasmid pFastBacHT to construct the recombinant transfer vector pFastBacHT-HA. The pFastBacHT-HA was transformed into DH10Bac competent cells, transposed with a shuttle vector (Bacmid) and the transposition rBacmid-HA was constructed. The recombinant baculovirus was harvested from sf9 cells transfected with rBacmid-HA. The expressed HA protein was identified and analysed by SDS–PAGE, Western blot and haemadsorption assay. The 66 kDa protein could only react with chicken serum positive to H5 subtype AIV. The haemadsorption assay showed that the sf9 cells infected with rBacmid-HA baculovirus could absorb chicken red blood cells. These results indicated that the HA protein was successfully expressed in sf9 cells, with reaction specificity to H5 subtype antiserum.

scholarly journals Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

2010 ◽  
Vol 43 (3) ◽  
pp. 229-233 ◽  
Author(s):  
Alex Martins Machado ◽  
Glauciane Garcia Figueiredo ◽  
Gelse Maria Campos ◽  
Mario Enrique Lozano ◽  
Aline Rafaela da Silva Rodrigues Machado ◽  
...  
Keyword(s):  
Expression System ◽  
Recombinant Baculovirus ◽  
Hemorrhagic Fever ◽  
Baculovirus Expression System ◽  
Elisa Test ◽  
Serum Samples ◽  
Serological Screening ◽  
Mato Grosso ◽  
Junin Virus ◽  
Junín Virus

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

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