High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.

Two forms of herpes simplex virus glycoprotein gD were recombined into Autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected Spodoptera frugiperda (Sf9) cells. Each protein was truncated at residue 306 of mature gD. One form, gD-1(306t), contains the coding sequence of Patton strain herpes simplex virus type 1 gD; the other, gD-1(QAAt), contains three mutations which eliminate all signals for addition of N-linked oligosaccharides. Prior to recombination, each gene was cloned into the baculovirus transfer vector pVT-Bac, which permits insertion of the gene minus its natural signal peptide in frame with the signal peptide of honeybee melittin. As in the case with many other baculovirus transfer vectors, pVT-Bac also contains the promoter for the baculovirus polyhedrin gene and flanking sequences to permit recombination into the polyhedrin site of baculovirus. Each gD gene was engineered to contain codons for five additional histidine residues following histidine at residue 306, to facilitate purification of the secreted protein on nickel-containing resins. Both forms of gD-1 were abundantly expressed and secreted from infected Sf9 cells, reaching a maximum at 96 h postinfection for gD-1(306t) and 72 h postinfection for gD-1(QAAt). Secretion of the latter protein was less efficient than gD-1(306t), possibly because of the absence of N-linked oligosaccharides from gD-1(QAAt). Purification of the two proteins by a combination of immunoaffinity chromatography, nickel-agarose chromatography, and gel filtration yielded products that were > 99% pure, with excellent recovery. We are able to obtain 20 mg of purified gD-1(306t) and 1 to 5 mg of purified gD-1(QAAt) per liter of infected insect cells grown in suspension. Both proteins reacted with monoclonal antibodies to discontinuous epitopes, indicating that they retain native structure. Use of this system for gD expression makes crystallization trials feasible.     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera.

The terminal proteins TP1 and TP2 are putative products of Epstein-Barr virus (EBV) genes expressed during the latent cycle of the virus. They are predicted to code for 53- and 40-kilodalton integral membrane proteins. We used the baculovirus Autographa californica nuclear polyhedrosis virus as an expression vector to produce TP1 in large amounts in insect cells. The DNA sequences used to express TP1 originated from a TP1 cDNA derived from an M-ABA/CBL1 cDNA library. Rabbit antisera raised against procaryotic TP1 fusion proteins recognized a monomer and a dimer of the recombinant TP1 protein in the infected insect cells. Immunofluorescence studies of living insect cells showed that the recombinant protein is located in the plasma membrane. The insect cells infected with the recombinant baculovirus producing TP1 provided a test system to screen human antisera for TP1 antibodies. A total of 168 human EBV-positive and EBV-negative antisera were studied. TP1 antibodies were detected only in sera from nasopharyngeal carcinoma patients (16 out of 42). Rabbit antiserum raised against the recombinant TP1 protein expressed in the baculovirus system specifically recognized a protein of about 54 kilodaltons in the lymphoblastoid cell lines M-ABA and M-ABA/CBL1 and in the Burkitt's lymphoma cell lines BL18 and BL72. This protein could be located in the total membrane fraction of M-ABA cells and is upregulated by treating the cells with 12-O-tetradecanoylphorbol-13-acetate.     

核衣壳蛋白VP39融合TAT转导肽对杆状病毒转导哺乳动物细胞的影响

为了提高昆虫杆状病毒在哺乳动物细胞中转导基因的效率,构建了重组杆状病毒AcRed-tat和AcRed。两者都能在哺乳动物细胞内表达红色荧光蛋白作为报告基因。同时,AcRed-tat带有HIV-1Tat转导肽、病毒主要衣壳蛋白基因vp39及增强型绿色荧光蛋白(egfp)三者的融合基因,并由杆状病毒多角体启动子表达,能够在昆虫细胞中表达该Tat融合蛋白,并掺入子代病毒粒子。而AcRed作为相应的对照病毒,带有多角体启动子表达vp39和egfp的融合基因。2株病毒分别转导哺乳动物细胞后,利用流式细胞仪检测报告基因的表达水平,发现在CHO和Vero细胞中AcRed-Tat介导的报告基因表达水平明显高于AcRed,而在HEK293细胞中2株病毒介导的报告基因表达水平差异不显著。结果表明Tat转导肽可以提高杆状病毒对一部分哺乳动物细胞的转导效率,为改进杆状病毒-哺乳动物细胞转导载体提供了新的思路。     

Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.     

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector

Summary The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein.     

Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors.

A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots (immunoblots).     

Baculovirus lef-12 is not required for viral replication

The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.     

Molecular biology of terminal transferase

Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes. The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure. Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000. The two peptide structure found earlier was caused by proteolysis. Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography. In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments. Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein. The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein. Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.     

Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus.

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4.     

Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector

A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes.     

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.     

A baculovirus superinfection system: efficient vehicle for gene transfer into Drosophila S2 cells

The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood Drosophila S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a Drosophila heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized Drosophila S2 cells.     

Functional expression of the human growth factor activatable Na+/H+ antiporter (NHE-1) in baculovirus-infected cells.

We constructed a recombinant baculovirus, based on Autographa californica nuclear polyhedrosis virus, containing the human Na+/H+ antiporter cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the Sf9 cell line, derived from Spodoptera frugiperda, expresses a fully functional Na+/H+ antiporter as measured by the generation of an amiloride-sensitive Na+ influx in response to an acid load. The Na+/H(+)-exchange activity, not detectable in Sf9 cells, emerges 18 h after infection and continues to increase over the next two days to reach a maximal value about 20-fold higher than in cultured mammalian fibroblasts. Parallel to this activity, infected cells express a single immunoreactive polypeptide of 85 kDa that represents a non-glycosylated form of the 110-kDa mature human antiporter. We estimated that only 10% of the expressed protein is in a functional state. Not only is the antiporter expressed in insect cells phosphorylated, but also, like in mammalian cells, phosphorylation is increased in response to phorbol esters and okadaic acid. Moreover, tumor promoters apparently modify the same antiporter site in both insect and mammalian cells. We conclude that, with this high level of functional expression and apparently conserved signaling machinery, the present system opens the way to the biochemistry of the transporter including identification of the growth factor stimulated phosphorylation sites.     

Expression of the cardiac Na(+)-Ca2+ exchanger in insect cells using a baculovirus vector.

We constructed a recombinant baculovirus containing cardiac Na(+)-Ca2+ exchanger cDNA under control of the polyhedrin promoter. When either Sf9 or Sf21 insect cells are infected with the recombinant baculovirus, both Na(+)-Ca2+ exchanger protein and Na(+)-Ca2+ exchange activity are expressed at high level. The exchanger protein can be detected either by immunoblot or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysate. At maximal expression, the exchanger protein comprises about 3-5% of total cell protein. The Na(+)-Ca2+ exchanger can be purified by alkaline extraction of infected cells followed by elution from a Bio-Rad Prep Cell. The expressed exchanger, in contrast to the native sarcolemmal exchanger, is not glycosylated. Sf9 cells expressing the exchanger are intensely stained by anti-exchanger antibodies as observed by immunofluorescence. The expressed exchanger is predominantly in the cell plasma membrane since it is susceptible to extracellular trypsin. In 45Ca2+ flux experiments, the expressed Na(+)-Ca2+ exchange activity is about 4-fold higher than that in cultured neonatal rat heart cells. The expressed exchanger was also analyzed electrophysiologically using whole cell patch clamp techniques. The characteristics of inward exchange currents in infected Sf21 cells are very similar to those of ventricular myocytes, although of a larger magnitude.     

Use of flow cytometry to monitor infection and recombinant human alpha-1,3/4 fucosyltransferase production in baculovirus infected Sf9 cell cultures

This paper describes the setup and the use of a flow cytometric method for monitoring Sf9 insect cell infection by a recombinant baculovirus expressing the human alpha1,3/4 fucosyltransferase Fuc-TIII. Using side scattered light coupled to green fluorescence detection after immunolabeling of the recombinant protein, this method made it possible to monitor baculovirus infection of Sf9 cells grown in batch cultures and infected at different cell densities and multiplicities of infection. The method was able to precisely assess the extent of infection of the insect cells from 60 h postinfection. In asynchronously infected Sf9 cell cultures, the two-step infection process (primary and secondary infection) was well-characterized using this technique. Finally, a reduced sensitivity to baculovirus infection was observed for cells infected at the end of the growth phase compared to the cells infected during exponential growth phase.     

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.     

杆状病毒AcMNPV p48基因在昆虫细胞中的表达

【目的】p48(ac103)基因在昆虫杆状病毒中高度保守,暗示其具有重要的生物学功能。为了研究该基因的功能,我们首先对该基因的表达特征进行描述。【方法】以杆状病毒代表种——苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)的p48基因为研究对象,利用Bac-to-Bac杆状病毒表达载体系统分别构建了在P48蛋白N-端和C-端融合HA-标签,并且携带绿色荧光蛋白基因和多角体蛋白基因的重组Bacmid。将重组Bacmid转染Sf9细胞,收集含病毒的上清去感染Sf9细胞,在感染后不同时间点收集细胞进行SDS-PAGE电泳,利用商业化的HA抗体进行Western blot分析以检测融合蛋白在昆虫细胞中的表达情况。【结果】用C-端融合HA-标签的重组病毒感染细胞后12h即可检测到一条43kDa左右、能与HA抗体发生特异性结合的蛋白条带,该特异性蛋白的表达一直持续到病毒感染后96h。从感染后48h起一直到96h,均能检测到另外一条约26kDa的蛋白条带也能与HA抗体发生特异性结合。在N-端融合HA-标签的重组病毒感染的细胞中没有检测到与HA抗体特异结合的蛋白。【结论】结果表明,p48基因是个晚期基因,在病毒感染的晚期表达,并且该蛋白在昆虫细胞中表达时N-端可能被剪切。     

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.