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At least 775 records · Page 43Linked to original sources
PubMed1994

Cloning and production of functional active recombinant hepatitis B virus surface antigen binding protein.

Endonexin II present on the surface of human hepatocytes has recently been identified as a hepatitis B virus surface antigen (HBsAg) binding protein. A full-length cDNA clone encoding human endonexin II was isolated from a human liver cDNA library and was placed under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Infection of Spodoptera frugiperda cells with recombinant virus resulted in the production of high amounts of recombinant protein. This protein has the same molecular weight and iso-electric point as native human endonexin II. It can be easily purified by methods analogous to those described for the native protein. Moreover, the recombinant product binds very efficiently to hepatitis B surface proteins (HBsAg) in a similar fashion as native human endonexin II.

Animals↗
PubMed1995

Signal transduction in SF9 insect cells: endocytosis of recombinant CD4 after phorbol ester treatment.

In Spodoptera frugiperda (Sf9) insect cells infected with a recombinant baculovirus carrying a gene construct encoding human CD4 endocytosis of CD4 is induced after stimulation with phorbol-12-myristate-13-acetate (PMA). Stimulation of endocytosis with PMA reduced the amount of full-length CD4 on the plasma membrane of Sf9 cells by 50% after 2 hours. Endocytosis of CD4 is blocked after intracellular delivery by cationic liposomes of a monoclonal antibody directed against a cytoplasmic sequence of CD4. Endocytosis is also blocked by the calmodulin inhibitor W7. The PKC inhibitor H7 does not inhibit PMA-induced endocytosis. A truncated CD4, in which the last 32 C-terminal amino acids were deleted did, not respond to PMA. Our results show that PMA can stimulate the calmodulin-dependent signal transduction for endocytosis of full-length CD4 in Sf9 cells. Phosphorylation of CD4 in Sf9 cells was not detectable after PMA treatment and PKC is not required for endocytosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

High level production and localization of bovine rod cGMP-gated cation channel subunit 1 in baculovirus-infected insect cells and Saccharomyces cerevisiae.

The cGMP-gated cation channel is responsible for the last step in the vertebrate phototransduction cascade which couples light activation of rhodopsin to a change in membrane permeability. Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Saccharomyces cerevisiae, were used for the overproduction of the cGMP-gated cation channel subunit 1 of bovine rod cells. Presence of recombinant channel protein was monitored by SDS-PAGE and Western-blot analysis. Through immunogold labeling, the heterologously expressed cation channel was found to be localized predominantly in the inner compartments of the infected insect cells and in the vacuole of recombinant yeast cells.

Animals↗
PubMed1996

Functional expression of the Aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system.

A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein.

Animals↗
PubMed1996

Isoform-specific intracellular vesicle formation by recombinant annexin XI-A in Sf9 cells.

Annexins are a group of structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and have the ability to self-aggregate and to promote vesicle aggregation and membrane fusion. Two isoforms of annexin XI, termed XI-A and XI-B, were previously identified by screening a bovine chondrocyte cDNA library. But little is known about differences in their biological function. In the present study, we therefore examined the results of expression of the two proteins in Spodoptera frugiperda (Sf9) insect cells, and in mammalian COS-7 cells. Annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. Recombinant annexin XI-A but not XI-B caused formation of spherical "annexin XI-associated vesicles, " in the cytoplasm of Sf9 cells. Furthermore, indirect immnocytofluorescence studies showed similar phenomenon, that of local aggregation, with transfected annexin XI-A in COS-7 cells, whereas annexin XI-B remained diffusely distributed throughout the cytoplasm. Since annexin XI isoforms differ in amino acid sequence only in the alternative splicing region of the N-terminal domain, these findings suggest that this domain has distinct biological significance in terms of aggregation and vesicle formation.

Amino Acid Sequence↗
PubMed1996

Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells.

Trichoplusia ni (High Five) and Spodoptera frugiperda (Sf21) cells were engineered for expression of epitope (Flag)-tagged signal peptide-green fluorescent protein (GFP) fusions to examine the suitability of GFP as a secretory marker. The recombinant baculovirus-infected cells became fluorescent, and the High Five cells but not Sf21 cells secreted GFP in the culture medium as detected by the presence in the culture supernatant of a Flag-immunoreactive 30-kDa species and the characteristic 510-nm GFP fluorescence peak. Signal peptides derived from ecdysteroid UDP-glucosyltransferase of Autographa californica nuclear polyhedrosis virus and from rat brain glutamate receptor were both able to promote secretion of GFP. GFP may thus be used as a research tool in the study of the secretory process in insect cells both in cell biology and in biotechnological applications.

Amino Acid Sequence↗
PubMed1997

The production and purification of functional decorin in a baculovirus system.

Human decorin was expressed in Spodoptera frugiperda 21 (Sf21) insect cells. A full-length cDNA encoding preprodecorin of 359 amino acids from a human fibroblast library was cloned into baculovirus transfer vector pVL1392, and transfected into Sf21 insect cells. The infected cells secreted the mature decorin into the culture medium. The secreted decorin lacked glycosaminoglycan but was N-glycosylated, whereas the unmodified decorin was present in the cell lysates, suggesting that N-glycosylation is required for decorin secretion from Sf21 cells. The recombinant decorin was then efficiently purified from the conditioned medium by two chromatographic procedures, hydroxyapatite Sepharose and Con A-Agarose, under nondenaturing conditions. The purified decorin was more potent, as evaluated by the inhibition of collagen fibrillogenesis, than that obtained from bovine tissues under denaturing conditions. The final yield of recombinant decorin was 1.5 mg in 200 ml culture medium of 3 x 10(8) cells. The biologically active decorin produced in Sf21 cells is a potentially useful probe for investigating the molecular interactions of this protein with other extracellular matrix proteins and may also have therapeutic applications.

Animals↗
PubMed1997

Expression of a fungal cellobiohydrolase in insect cells.

The gene for Trichoderma reesei cellobiohydrolase I (CBHI) was expressed with a recombinant baculovirus and high levels of secreted protein were produced in Spodoptera frugiperda and Trichoplusia ni insect cells. Electrophoretic analysis indicated that the recombinant CBHI (rCBHI) was similar in apparent molecular weight to the native form and immunoblotting with anti-CBHI monoclonal antibodies confirmed its identity. The rCBHI was easily purified by affinity and hydrophobic interaction chromatography and demonstrated enzymatic activity on soluble substrate.

Amino Acid Sequence↗
PubMed1998

Suppression of cell growth by heavy chain ferritin.

While producing recombinant rat liver H and L chain ferritin homopolymers using the baculovirus expression system, we noticed that rat liver H chain ferritin, but not L chain ferritin, had a suppressive effect on the growth of Spodoptera frugiperda (Sf-21) cells. Suppression was observed immediately after infection with recombinant H chain ferritin baculovirus prepared from lysed infected cells. Immediate suppression was observed when purified with either recombinant H chain apoferritin or various holoferritins (loaded with 1,970 +/- 50 or 2,520 +/- 90 atoms of iron/ferritin) indicating that suppression was not due to sequestration of iron required for cell growth. Suppression by H chain ferritin was also observed upon attempting to express the protein in Escherichia coli. Strategies for expression of recombinant rat liver H and L ferritin homopolymers in both prokaryotic and eukaryotic expression systems were developed.

Animals↗
PubMed1999

Expression, purification, and characterization of the cytoplasmic domain of the human IGF-1 receptor using a baculovirus expression system.

The cytoplasmatic domain of the beta-subunit of the human IGF-1 receptor (residues 929-1337) has been overexpressed in insect cells using the baculovirus expression system. Synthesis of the soluble protein (IGFK, M(r) 46 kDa) in Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation was achieved 40-48 h postinfection. Rapid purification to near homogeneity (>/=95% pure protein) was accomplished by sequential chromatography on Resource-Q and phenyl-Sepharose with a specific activity of 142 nmol/min/mg using poly[Glu:Tyr] as substrate. The purified IGFK showed a preference for Mn(2+) ions and a linear incorporation of (32)P from [gamma-(32)P]ATP over a 20-fold dilution of the protein and was stimulated 20-fold by the polycation poly-L-lysine. Interestingly, the kinase autophosphorylated on tyrosine and serine residues. In contrast, a kinase-negative mutant, IGFK-K1003A, did not undergo phosphorylation on tyrosine or serine residues, respectively, suggesting that IGF-1 receptor kinase is a dual specific kinase.

Adenosine Triphosphate↗
PubMed2001

Recombinant expression and purification of human androgen receptor in a baculovirus system.

A full-length human androgen receptor (hAR) cDNA was used to produce recombinant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this virus expressed protein with an N-terminal hexahistidine tag (His(6)-hAR) in soluble and insoluble forms. The soluble cytosolic His(6)-hAR demonstrated similar association and dissociation half-times for mibolerone, similar binding affinity for mibolerone, and similar steroid specificity as bona fide AR. Under native conditions, the soluble cytosolic His(6)-hAR was purified to apparent homogeneity in the presence of dihydrotestosterone, using metal ion affinity chromatography. The insoluble pellet fraction was solubilized with strong denaturant 6 M guanidine HCl, and His(6)-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His(6)-hAR could be renatured to bind mibolerone. The baculovirus system will therefore provide an efficient means for producing hAR for ligand-binding assays, as well as purifying hAR for detailed molecular analyses.

Animals↗
PubMed1995

The P1A alloantigen system is a sensitive indicator of the structural integrity of the amino-terminal domain of the human integrin beta 3 subunit.

Within the native integrin alpha pi b beta 3, the conformation of the amino-terminal domain of the beta 3 subunit has a significant influence on the availability of the Leu33/Pro33 polymorphism that defines the A1 and A2 alleles, respectively, of the P1A alloantigen system. The majority of anti-P1A1 IgG antibodies, affinity-purified by adsorption to either (A1/A1)-platelets or purified alpha pi b(A1) beta 3, fail to bind to the Leu33 polymorphic loop within Cys26-Cys38 in native beta 3 unless this sequence is maintained in a proper orientation by two noncontiguous human beta 3 sequences. By comparing IgG binding to recombinant beta 3 molecules composed of segments of human, Xenopus and avian sequences expressed by Spodoptera frugiperda cell lines, we have found that neither region alone is sufficient for full expression of the epitope. One sequence, that most proximal to Leu33/Pro33, lies within beta 3[54-133], and a second distal sequence is located within beta 3[435-490]. Proximity of the distal sequence to the Cys26-Cys38 loop in native beta 3 is confirmed by the fact that both anti-P1A1 IgG and the murine monoclonal antibody SZ21, specific for beta 3[28-35], completely inhibit the binding of rabbit polyclonal IgG specific for the sequence beta 3[479-485]. Anti-P1A1 IgG antibodies from all of the 7 donors selectively bind to the epitope within the native conformation of beta 3. However, in one of the seven donors, roughly 60% of the anti-P1A1IgG binds exclusively to denatured (A1) beta 3, and not to either denatured (A2) beta 3 or nondenatured (native) (A1) beta 3. This IgG subpopulation cannot be purified by adsorption to intact (A1/A1)-platelets or to native alpha pi b(A1) beta 3 integrin. The physiologic relevance of this exceptional type of anti-P1A1 antibody is debatable, but the presence of IgG with this specificity only in an immunized subject suggests that the epitope may well be presented on a "denatured" form of beta 3 in vivo. Regardless, our results indicate that the amino-terminal domain of the native beta 3 molecule associates with noncontiguous beta 3 sequences, and the epitope recognized by a majority of anti-P1A1 antibodies is a sensitive indicator of correct tertiary structure within this region of beta 3.

Amino Acid Sequence↗
PubMed1995

Characterization of two human cAMP-specific phosphodiesterase subtypes expressed in baculovirus-infected insect cells.

Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Production of biologically active human RelA (p65) in baculovirus-infected insect cells.

A recombinant baculovirus was constructed to express a cDNA encoding RelA (p65), a member of the NF-kappa B/Rel family of proteins. Infection of Spodoptera frugiderda insect cells with the recombinant baculovirus resulted in the production of the biologically active protein as measured by immunoblotting using RelA-specific antisera and by electrophoretic mobility shift assays. The recombinant protein bound specifically to an oligonucleotide containing the NF-kappa B consensus motif but not to that containing the unrelated Oct-1 consensus motif. Thus insect cell-derived RelA possess properties similar to the native protein and may be used in physical, biochemical, and pharmacological studies.

Animals↗
PubMed1999

Biological functions of recombinant bovine interleukin 6 expressed in a baculovirus system.

The cDNA encoding bovine interleukin 6 (IL-6) was obtained from messenger RNA extracted from lipopolysaccharide-stimulated bovine Kupffer cells by the reverse transcription polymerase chain reaction (RT/PCR), and cloned into the baculovirus vector pVL 1392. Insect cells (Sf21AE derived from Spodoptera frugiperda) infected with the recombinant baculovirus secreted a large amount of 23.7 kD protein into the culture medium. This protein was capable of causing increased haptoglobin production and decreased albumin production in primary cultured bovine hepatocytes. The swine and human IL-6s were also able to decrease albumin production in bovine hepatocytes. This recombinant IL-6 did not stimulate the proliferation of 7TD1 cells (a murine IL-6-dependent cell line), whereas the recombinant swine IL-6 which was expressed in the same baculovirus system, and recombinant human IL-6 derived from Escherichia coli were each capable of stimulating proliferation of 7TD1 cells, respectively. This suggests a species restriction between bovine IL-6 and murine IL-6 dependent cell lines.

Acute-Phase Proteins↗
PubMed2001

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.

Animals↗
PubMed2002

Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line.

Animals↗
PubMed1997

Paraquat inhibits the processing of human manganese-dependent superoxide dismutase by SF-9 insect cell mitochondria.

Precursor human manganese-dependent superoxide dismutase (hMn-SOD) was expressed using the baculovirus system in Spodoptera fungiperda (Sf-9) insect cells. Following infection of Sf-9 cells with hMn-SOD-expressing baculovirus, mature hMn-SOD was expressed at 15-25% of total cellular protein, with the recombinant protein localized in the mitochondrial matrix. Partial amino acid sequencing and SOD activity assays indicated that the hMn-SOD was correctly processed and assembled by insect mitochondria. This expression system was used to study the effects of paraquat and menadione, two intracellular superoxide generators, on processing of precursor hMn-SOD by insect mitochondria. Paraquat was found to potently inhibit mitochondrial processing of hMn-SOD, leading to the accumulation of precursor hMn-SOD and a decrease in measurable Mn-SOD activity. In contrast, menadione treatment was not found to affect the ratio of precursor to mature Mn-SOD. Paraquat did not lead to lower total production of hMn-SOD or cellular toxicity at the concentrations which were found to block processing of precursor hMn-SOD. These results indicate that mitochondrial processing and import of the precursor protein hMn-SOD are early events susceptible to dysfunction induced by the redox-cycling agent paraquat. These results also emphasize that mitochondrial processing of precursor proteins represents a parameter of cellular function which may be compromised, preceding the appearance of more generalized deterioration of cellular function, under certain toxic or pathological conditions.

Animals↗