Sf9 Cell Lysates Research Articles

Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography.

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈1014 vp mL-1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%).

Baculovirus display of single chain antibody (scFv) using a novel signal peptide

BackgroundCells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.ResultsTwo versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.ConclusionExpression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.

Stress response of a p53 homologue in the radioresistant Sf9 insect cells

Purpose: To investigate homology and stress response of p53 (a 53 kDa tumor suppressor protein) orthologue in Sf9 Lepidopteran insect cell line that exhibits very high radioresistance.Materials and methods: Western immunoblotting, immunoprecipitation, degenerate RT-PCR (reverse transcription-polymerase chain reaction), electrophoretic gel mobility shift assay, flow cytometry and immuno-fluorescence microscopy were used for characterizing structural and functional features of Sfp53 (Spodoptera frugiperda p53) in γ-irradiated or etoposide-treated Sf9 insect and BMG-1 (brain malignant glioma) human cells. Cells were pre-treated with caffeine for inhibiting ATM/ATR (ataxia-telangiectasia mutated protein/ATM and Rad-3-related protein) activation, wherever required.Results: A 47–49 kDa protein band was observed with antibodies against three different epitopes, demonstrating conservation of respective domains in Sfp53. Immunoprecipitation also yielded similar-sized protein. Degenerate RT-PCR resulted in product of same size in both cell lines. Similar gel mobility shift of p53-binding oligonucleotide with BMG-1 and Sf9 cell lysates indicated analogous transcriptional activity of Sfp53. Constitutive Sfp53 level was higher than hp53 (human p53) and showed primarily cytoplasmic localization. Radiation-induced accumulation was considerably less in Sf9 even as an analogous ATM/ATR-dependent nuclear translocation was observed following γ-irradiation and etoposide.Conclusions: A smaller-sized Sfp53 orthologue shows highly conserved native structure with DNA-binding, N-terminus and C-terminus domains, and has analogous p53 transcriptional activity. While its nuclear translocation and ATM/ATR dependence were similar to hp53, the cytoplasmic localization and subdued accumulation following γ-irradiation indicate functional differences from human cells.

Inhibition of UDP‐glucuronosyltransferase (UGT) enzymes by protein kinase C inhibitors

UGTs are involved in the clearance of ∼10% of the top drugs on the market. Evidence for a novel mechanism of UGT inhibition involving protein kinase C (PKC) is accumulating. Therefore, we screened a panel of ten PKC inhibitors, including PKC‐α, β, δ, and η specific inhibitors, to determine their effect on acetaminophen (APAP) glucuronidation in human LS180 colon cells. Of these, only calphostin‐C (selective PKC inhibitor), curcumin (non‐specific PKC), hypericin (non‐specific PKC), and rottlerin (PKC‐δ selective) decreased glucuronidation by more than 50%. To ensure that this inhibition was not due to direct effects, these compounds were counterscreened in human liver microsomes (HLM), where these inhibitors were 5 to 15 times less potent. Curcumin and calphostin‐C were also investigated for their effects on serotonin glucuronidation in human UGT1A6‐infected Sf9 insect cells and cell lysates. Calphostin‐C was at least 3 times more active in the whole cells (IC50 = 0.7 μM) compared to cell lysates (IC50 &gt; 2 μM), while curcumin was a potent direct inhibitor of UGT1A6 and so effects via PKC could not be differentiated. These data further support the role of PKC in phosphorylating and activating UGTs and indicates that PKC‐δ, but perhaps not α, β, or η, is involved in phosphorylating the UGTs required for APAP glucuronidation. This work was made possible by grants F31AT003973, R01GM061834, and R21GM074369 from the NIH.

Reconstitution and subunit geometry of human condensin complexes

Vertebrate cells possess two different condensin complexes, known as condensin I and condensin II, that play a fundamental role in chromosome assembly and segregation during mitosis. Each complex contains a pair of structural maintenance of chromosomes (SMC) ATPases, a kleisin subunit and two HEAT-repeat subunits. Here we use recombinant human condensin subunits to determine their geometry within each complex. We show that both condensin I and condensin II have a pseudo-symmetrical structure, in which the N-terminal half of kleisin links the first HEAT subunit to SMC2, whereas its C-terminal half links the second HEAT subunit to SMC4. No direct interactions are detectable between the SMC dimer and the HEAT subunits, indicating that the kleisin subunit acts as the linchpin in holocomplex assembly. ATP has little, if any, effects on the assembly and integrity of condensin. Cleavage pattern of SMC2 by limited proteolysis is changed upon its binding to ATP or DNA. Our results shed new light on the architecture and dynamics of this highly elaborate machinery designed for chromosome assembly.

Molecular Mechanisms of Import of Peroxisome-targeting Signal Type 2 (PTS2) Proteins by PTS2 Receptor Pex7p and PTS1 Receptor Pex5pL

In the present study, we investigated molecular mechanisms underlying the import of peroxisome-targeting signal type 2 (PTS2) proteins into peroxisomes. Purified Chinese hamster Pex7p that had been expressed in an Sf9/baculovirus system was biologically active in several assays such as those for PTS2 binding and assessing the restoration of the impaired PTS2 protein import in Chinese hamster ovary (CHO) pex7 mutant ZPG207. Pex7p was eluted as a monomer in gel filtration chromatography. Moreover, the mutation of the highly conserved cysteine residue suggested to be involved in the dimer formation did not affect the complementing activity in ZPG207 cells. Together, Pex7p more likely functions as a monomer. Together with PTS1 protein, the Pex7p-PTS2 protein complex was bound to Pex5pL, the longer form of Pex5p, which was prerequisite for the translocation of Pex7p-PTS2 protein complexes. Pex5pL-(Pex7p-PTS2 protein) complexes were detectable in wild-type CHO-K1 cells and were apparently more stable in pex14 CHO cells deficient in the entry site of the matrix proteins, whereas only the Pex7p-PTS2 protein complex was discernible in a Pex5pL-defective pex5 CHO mutant. Pex7p-PTS2 proteins bound to Pex14p via Pex5pL. In contrast, PTS2 protein-bound Pex7p as well as Pex7p directly and equally interacted with Pex13p, implying that the PTS2 cargo may be released at Pex13p. Furthermore, we detected the Pex13p complexes likewise formed with Pex5pL-bound Pex7p-PTS2 proteins. Thus, the Pex7p-mediated PTS2 protein import shares most of the steps with the Pex5p-dependent PTS1 import machinery but is likely distinct at the cargo-releasing stage.

Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1.

We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.

Alsin is a Rab5 and Rac1 guanine nucleotide exchange factor.

ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.

Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1.

We recently characterized the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1, whereas the amino-terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP-1 and overexpression of PARP-1. Our results unequivocally show that the presence of PARP-1 regulates the basal expression of wt p53 in unstressed cells.

Central and carboxy-terminal regions of human p53 protein are essential for interaction and complex formation with PARP-1.

It has been previously described by different groups that poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 form tight complexes. We investigated which domains of human PARP-1 and of human wild-type p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length protein or distinct functional domains of both proteins. Baculovirally expressed wild-type p53 was posttranslationally modified. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1. The amino-terminal part harboring the transactivation functional domain of p53 was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. Finally, we explored the functional significance of the interaction between both proteins. Inactivation of PARP-1 resulted in the reduction of p53 steady-state levels. Inhibition of nuclear export by leptomycin B prevented accelerated degradation of p53 in PARP-1 KO cells and led to accumulation of p53 protein. Considering the fact that the accelerated p53 nuclear export in the absence of PARP-1 contributes to enhanced p53 degradation, we conclude that PARP-1 may mask the NES of p53 through complex formation with its carboxy-terminal part, thereby preventing the export.

Regulation of Drosophila TRPL Channels by Immunophilin FKBP59

Transient receptor potential and transient receptor potential-like (TRPL) are Ca(2+)-permeable cation channels found in Drosophila photoreceptor cells associated with large multimeric signaling complexes held together by the scaffolding protein, INAD. To identify novel proteins involved in channel regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein, FKBP52 (previously known as FKBP59). To determine the function of dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca(2+) influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunoprecipitation resulted in a temperature-dependent dissociation of dFKBP59 and TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lysates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in vivo. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lysates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutagenesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709) eliminated interaction of the TRPL with dFKBP59. These results provide strong support for the hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an important role in modulation of channel activity via interaction with conserved leucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel.

Chondrocyte-derived ezrin-like domain containing protein (CDEP), a rho guanine nucleotide exchange factor, is inducible in chondrocytes by parathyroid hormone and cyclic AMP and has transforming activity in NIH3T3 Cells

Objective The purpose of this study is to investigate stage- and hormone-dependent expression of chondrocyte-derived ezrin-like domain containing protein (CDEP), a putative guanine nucleotide exchange factor (GEF) for Rho in chondrocytes, and demonstrate the guanine nucleotide exchange activity of this protein in vitro, as well as the transforming activity in NIH3T3 cells. Methods The expression of CDEP mRNA in growth plate chondrocytes in vivo and in vitro was examined by RT-PCR Southern analysis. The guanine nucleotide exchange activity was determined using a recombinant CDEP peptide containing the DH and PH domains in Sf9 cell lysates. The transforming activity was examined using NIH3T3 cells transiently transfected with a truncated CDEP cDNA. Results CDEP mRNA was expressed at the highest level in the hypertrophic (terminal) stage of chondrocytes in vivo and in vitro. Parathyroid hormone (PTH) elicited a biphasic increase of CDEP mRNA in chondrocytes. The CDEP mRNA level increased within 1 h, then decreased nearly to the control level at 3 h. Thereafter the mRNA level started to increase at 6 h, reaching a plateau at 24 h. Dibutyryl cyclic AMP had a similar effect on CDEP expression in chondrocytes. The dissociation of [ 3H]GDP from RhoA was stimulated dose-dependently by Sf9 cell lysates containing the CDEP peptide. Furthermore, transfection of a truncated CDEP cDNA induced focus formation in NIH3T3 cultures. Conclusions CDEP is a novel GEF for Rho family GTPases with the transforming activity. CDEP may play a role in mediating or modulating the action of cAMP-elevating hormones on maturing chondrocytes.

Characterizing the Interactions between the Two Subunits of the p101/p110γ Phosphoinositide 3-Kinase and Their Role in the Activation of This Enzyme by Gβγ Subunits

Recently, we have reported the purification and cloning of a novel G protein betagamma subunit-activated phosphoinositide 3-kinase from pig neutrophils. The enzyme comprises a p110gamma catalytic subunit and a p101 regulatory subunit. Now we have cloned the human ortholog of p101 and generated panels of p101 and p110gamma truncations and deletions and used these in in vitro and in vivo assays to determine the protein domains responsible for subunit interaction and activation by betagamma subunits. Our results suggest large areas of p101 including both N- and C-terminal portions interact with the N-terminal half of p110gamma. While modifications of the N terminus of p110gamma could modulate its intrinsic catalytic activity, binding to the N-terminal region of p101 was found to be indispensable for activation of heterodimers with Gbetagamma.

182 Expression of prolebetase from Vipera lebetina by baculovirus system. Plasminogen activating activity of SF-9 cell lysates

182 Expression of prolebetase from Vipera lebetina by baculovirus system. Plasminogen activating activity of SF-9 cell lysates

Cloning and expression of a putative glycoprotein gene of channel catfish virus using baculovirus expression system.

The 1035 base-pair (bp) open reading frame 59 (ORF59) of channel catfish virus (CCV) codes for an abundant hydrophobic membrane glycoprotein considered to be the major envelope glycoprotein of the virus. The ORF59 gene of CCV was cloned into the baculovirus genome by site-specific transposition and expressed in Spodoptera frugiperda cells (Sf9 cells). Expressed protein could be recognized by polyclonal antibody against CCV and appeared to have the same electrophoretic mobility as native protein. Crude lysates of Sf9 cells infected with recombinant baculovirus containing ORF59 gene inhibited plaque formation of CCV on channel catfish ovary cells.

Calmodulin Activates Phosphatidylinositol 3-Kinase

Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.

Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3.

The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with ICP0, we used a domain of exon II of ICP0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed cyclin D3 colocalized with ICP0 in nuclear structures characteristic of ND10 and which earlier have been reported to contain ICP0. (iv) The accumulation of cyclin D3 protein in Vero cells infected with an alpha0 deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha0 sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST-alpha0(20-241) or GST-alpha0(543-768) fusion protein or immunoprecipitated ICP0 proteins. Moreover, the chimeric GST-ICP0(exon II) protein shown to bind cyclin D3 had no effect on the activity of the kinase on GST-pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4. These results indicate that ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.

Role of one N-linked oligosaccharide chain on canine herpesvirus gD in its biological activity.

The YP11mu strain of a plaque-selected canine herpesvirus (CHV) encoded a smaller molecular weight (MW) of gD than those of other strains including YP2 strain (Xuan et al., 1990). When nucleotide sequence of the mutated gD of YP11mu strain (gD(YP11mu)) was compared with that of gDs of other CHV strains, gD(YP11mu) lacked 12 nucleotides encoding 4 amino acids, NKTI, including one predicted potential N-linked glycosylation site and no other change was found in other regions. When the gD(YP11mu) and gD of YP2 strain (gD(YP2)) expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells were compared each other, both gDs reacted with a panel of monoclonal antibodies (MAbs) against CHV gD by indirect immunofluorescence analysis and the gD(YP11mu) possessed an MW of approximately 47-51 and 39-44 kDa in COS-7 and Sf9 cells, respectively, which were smaller than the expressed gD(YP2) (approximately 51-55 and 41-46 kDa, respectively) by immunoblot analysis. After treatment with tunicamycin, the MW of both gDs in Sf9 cells became approximately 37 kDa. When hemagglutination (HA) test using canine red blood cells (RBC) were carried out, lysates of Sf9 cells expressing CHV gDs agglutinated canine RBC. Serum from mice inoculated with lysates of Sf9 cells expressing the gDs possessed a high titer of virus-neutralizing (VN) activities against CHV. These results indicated that the deletion of 4 amino acids possessing approximately 4 kDa of glyco-chain from gD of CHV in mammalian cells does not affect HA activity and VN antibody-inducing activity and that this deletion of gD(YP11mu) might be a good selective marker for development of recombinant viruses as a live vaccine.

Characterization of Canine Herpesvirus Glycoprotein D (Hemagglutinin).

Glycoprotein D (gD) of canine herpesvirus (CHV) YP2 strain was expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells. The gDs expressed in COS-7 and Sf9 cells reacted with a panel of monoclonal antibodies (MAbs) against CHV gD (hemagglutinin) and an MAb 25C9 against feline herpesvirus type 1 (FHV-1) gD by indirect immunofluorescence assay, and possessed a molecular weight (MW) of approximately 51-55 and 41-46 kilodalton (kDa), respectively, when examined by immunoblot analysis. After treatment with tunicamycin, the MW of the gD expressed in Sf9 cells became approximately 37 kDa. By hemadsorption (HAD) tests using canine or feline red blood cells (RBC), COS-7 cells expressing CHV gD adsorbed only canine RBC, but not feline RBC, whereas control COS-7 cells expressing FHV-1 gD adsorbed feline RBC, but not canine RBC. By hemagglutination (HA) tests, lysates of Sf9 cells expressing CHV gD agglutinated canine RBC, but not feline RBC. These HA and HAD activities were inhibited by HA-inhibition MAbs against CHV gD. Control lysates of Sf9 cells expressing FHV-1 gD agglutinated only feline RBC. Serum from mice inoculated with lysates of Sf9 cells expressing CHV gD possessed a high titer of virus-neutralizing activities against CHV infection. These results indicated that CHV gD is structurally similar to FHV-1 gD, but is functionally different from FHV-1 gD.

Expression and purification of the seven nonstructural proteins of the flavivirus Kunjin in the E. coli and the baculovirus expression systems.

All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) ranging from NS1 to NS5 were expressed either alone or as fusion proteins with Glutathione-S-transferase (GST). High level expression of recombinant proteins was achieved in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system in contrast to the low level of expression in E. coli. The order of the level of expression of the recombinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields approximately 4-5 mg) > GST-delta NS3 (approximately 1-2 mg) > GST-4A (approximately 1 mg) > GST-2B (approximately 0.5-1 mg) > GST-2A (approximately 0.5 mg) > GST-4B (approximately 0.1-0.2 mg). NS1 protein was expressed in a native form at the level of approximately 2-4 mg per 4 x 10(7) Sf9 cells. All the GST-fusion proteins were purified by adsorption on Glutathione Sepharose (GS) beads from solubilized lysates of Sf9 cells infected with the recombinant baculoviruses, or of E. coli cultures transformed with the expression plasmid and induced with IPTG. Only delta NS3 protein was recovered intact by removing GST from the fusion protein by digestion with Factor Xa protease. Attempts to cleave off the GST moiety from all the other purified recombinant proteins resulted either in inefficient cleavage or in degradation of the proteins. No GST-NS5 but from 20 to 50% of the purified GST-NS2A, GST-NS2B, GST-delta NS3, GST-NS4A, and GST-NS4B was eluted off the GS beads by adding glutathione. Thus, KUN purified recombinant proteins, either in eluted form or while immobilized on GS beads, could be used to raise monospecific antibodies, to perform functional assays or to participate in protein-protein or RNA-protein binding reactions.