Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors

Luckow, Verne A.; Summers, Max D.

doi:10.1016/0042-6822(88)90054-2

Cited by 197 publications

(115 citation statements)

References 29 publications

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“…Although three potential N-glycosylation sites are located at the N-terminal region (Asnl4, Asn27) and in the first extracellular loop (Asn197) of the receptor, it seems that the V,, receptor expressed in Sf9 cells is poorly glycosylated if not at all. In addition, it has been reported that N-glycosylation of membrane proteins expressed in this insect cell line is an incomplete process (Luckow and Summers, 1988). Since very little material aggregated at the top of the gel and only one broad band was detected, we determined the covalent binding yield of the probe for this receptor.…”

Section: Residuementioning

confidence: 99%

Efficient Photoaffinity Labeling of the Rat V_1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Carnazzi

Aumelas

Phalipou

et al. 1997

European Journal of Biochemistry

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We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-~-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-ArgTyr(31)-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V,, receptor [dissociation constant (Kd) = 54 2 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-18491 and for both the rat vasopressin V,, receptor expressed in Sixx.iopteru frugiperdu 9 cells (Sf9 cells, Kd = 688 i 35 pM) and in COS-7 cells (Kd = 320 2 20 pM). This probe labels specifically the V,, receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Znz+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V,, receptor and for mapping its antagonist-binding site.Keywords: photoaffinity ; vasopressin; antagonist; receptor; proteolysis.Vasopressin and oxytocin are two neurohypophyseal hormones that exert a wide range of physiological effects upon binding to different guanine-nucleotide-binding-regulatory(G)-protein-coupled receptor subtypes (Jard, 1983). More than a dozen of these oxytocin and vasopressin receptor subtypes have been cloned in several species, among which the vasopressin V,, subtype cloned in the rat (Morel et al., 1992;Innamorati et al., 1995), the sheep (Hutchins et al., 1995) and the human (Thibonnier et al., 1994). One of the most recently cloned members of this family is the mesotocin receptor, described by Akhundova et al. (1996). These receptors are able to specifically bind the natural hormones or synthetic peptide analogues by distinguishing subtle structural differences in their sequence (Akhundova et al., 1996). In addition, numerous agonist and antagonist ligands have been described over 40 years for this receptor family (Manning and Sawyer, 1993). Thus, these membrane proteins represent a good model for the study of the molecular mechanisms involved in hormone-receptor interactions.The analysis of the hydrophobicity profile of these receptor sequences confirms that they belong to the seven-transmembrane-domain receptor family. The comparison of the amino acid composition of all the known members of this family has revealed the presence of conserved residues and led to some structure/activity presumptions. A...

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Section: Residuementioning

confidence: 99%

Efficient Photoaffinity Labeling of the Rat V_1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Carnazzi

Aumelas

Phalipou

et al. 1997

European Journal of Biochemistry

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“…T he baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) 3 has long been used as a biopesticide and as a tool for efficient recombinant protein production in insect cells (1,2). Its host specificity was originally thought to be restricted to cells derived from arthropods.…”

Section: Baculovirus Induces An Innate Immune Response and Confers Prmentioning

confidence: 99%

Baculovirus Induces an Innate Immune Response and Confers Protection from Lethal Influenza Virus Infection in Mice

Abe¹,

Takahashi

Hamazaki³

et al. 2003

The Journal of Immunology

221

173

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A recombinant baculovirus expressing the hemagglutinin gene of the influenza virus, A/PR/8/34 (H1N1), under the control of the chicken β-actin promoter, was constructed. To determine the induction of protective immunity in vivo, mice were inoculated with the recombinant baculovirus by intramuscular, intradermal, i.p., and intranasal routes and then were challenged with a lethal dose of the influenza virus. Intramuscular or i.p. immunization with the recombinant baculovirus elicited higher titers of antihemagglutinin Ab than intradermal or intranasal administration. However, protection from a lethal challenge of the influenza virus was only achieved by intranasal immunization of the recombinant baculovirus. Surprisingly, sufficient protection from the lethal influenza challenge was also observed in mice inoculated intranasally with a wild-type baculovirus, as evaluated by reductions in the virus titer, inflammatory cytokine production, and pulmonary consolidations. These results indicate that intranasal inoculation with a wild-type baculovirus induces a strong innate immune response, which protects mice from a lethal challenge of influenza virus.

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“…The system includes Spodeptera frugiperda (Sf 21) as the insect cell line, pBlueBac 4.5 (Luckow and Summers, 1988) as a transfer vector, and engineered baculovirus Autographa californica multiple polyhedrosis virus (Bac-N-Blue DNA) as an expression vector. In brief, the cDNA of the AtLACS6 was inserted into the pBlueBac 4.5 transfer vector and cotransfected together with linearized baculoviral Bac-N-Blue DNA in insect cells.…”

Section: Expression Of Recombinant Atlacs6 From Insect Cellsmentioning

confidence: 99%

Molecular Characterization of an Arabidopsis Acyl-Coenzyme A Synthetase Localized on Glyoxysomal Membranes

et al. 2002

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In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the β-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designatedAtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). TheAtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that theAtLACS6 is localized on glyoxysomal membranes.AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and β-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid β-oxidation activating the fatty acids.

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Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors

Cited by 197 publications

References 29 publications

Efficient Photoaffinity Labeling of the Rat V_1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Efficient Photoaffinity Labeling of the Rat V_1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Baculovirus Induces an Innate Immune Response and Confers Protection from Lethal Influenza Virus Infection in Mice

Molecular Characterization of an Arabidopsis Acyl-Coenzyme A Synthetase Localized on Glyoxysomal Membranes

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Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors

Cited by 197 publications

References 29 publications

Efficient Photoaffinity Labeling of the Rat V1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Efficient Photoaffinity Labeling of the Rat V1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Baculovirus Induces an Innate Immune Response and Confers Protection from Lethal Influenza Virus Infection in Mice

Molecular Characterization of an Arabidopsis Acyl-Coenzyme A Synthetase Localized on Glyoxysomal Membranes

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Efficient Photoaffinity Labeling of the Rat V_1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

Efficient Photoaffinity Labeling of the Rat V_1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist