Human tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells

A cDNA fragment encoding the human tissue-type plasminogen activator was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. The induction kinetics of t-PA was followed, after infection of Spodoptera frugiperda cells, at both mRNA and protein levels. Fibrinolytically active plasminogen activator accumulated in the culture medium and reached 2.5 micrograms/ml after 120 h. The protein was compared with recombinant plasminogen activator produced in mouse cells and was found to be slightly smaller. This difference in size was found to be caused by N-linked oligosaccharides which are shorter in the recombinant activator obtained from insect cells. The molecules produced in such cells contain at least two different types of N-linked glycans, since only one out of three oligosaccharides is sensitive to endoglycosidase H. However, all glycan structures bind strongly to concanavalin A-Sepharose.     

High-Level Synthesis and Fate of Acetylcholine in Baculovirus-Infected Cells: Characterization and Purification of Recombinant Rat Choline Acetyltransferase

Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.     

Trichoplusia ni attacin A,a differentially displayed insect gene coding for an antibacterial protein

The mRNA differential display method was used to isolate antibacterial defense genes from Trichoplusia ni. The mRNA population in last-instar T. ni larvae injected with bacteria was compared to that of untreated larvae. Using a PCR amplified probe corresponding to an induced mRNA, we were able to clone an attacin homolog from a λ cDNA library from vaccinated larvae. The corresponding protein showed 63% identity to Hyalophora cecropia acidic attacin. The induction kinetics of T. ni attacin A gave optimal mRNA levels at 20 h post-infection. Genomic analysis showed this to be a single-copy gene with two introns.     

Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme

Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5.     

Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis.

We have constructed a recombinant baculovirus containing a 4.0-kilobase dengue virus cDNA sequence that codes for the three virus structural proteins, capsid (C) protein, premembrane (PreM) protein, and envelope glycoprotein (E), and nonstructural proteins NS1 and NS2a. Infection of cultured Spodoptera frugiperda cells with this recombinant virus resulted in the production of E and NS1 proteins that were similar in size to the corresponding viral proteins expressed in dengue virus-infected simian cells. Other dengue virus-encoded proteins such as PreM and C were also synthesized. Rabbits immunized with the dengue virus protein products of the recombinant virus developed antibodies to PreM, E, and NS1, although the titers were low, especially to PreM and E. Nevertheless, the dengue virus antigens produced by the recombinant virus induced resistance in mice to fatal dengue encephalitis.     

Insect immunity. The primary structure of the antibacterial protein attacin F and its relation to two native attacins from Hyalophora cecropia

The attacins are antibacterial proteins present in the hemolymph of the pupae of the silk moth Hyalophora cecropia after bacterial infection. We present the primary structure of one attacin, the F form. We show that this protein is derived by proteolysis from the native protein, attacin E. Using a method for rapid purification from the hemolymph of immunized pupae of the neutral attacin E and a basic attacin, both proteins were found in freshly collected immune hemolymph. We conclude that they are the native products of two attacin genes, the existence of which was inferred from the isolation of two cDNA clones as described in the accompanying paper. The two proteins, which differed in their pIs (7 and 9), were found to have similar mol. wts. (20 000) and closely related primary structures, displaying a total of 40 amino acid substitutions, 12 of which were of a non-conservative nature.     

Expression of the gene encoding firefly luciferase in insect cells using a baculovirus vector

A cDNA encoding the firefly luciferase [Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was cloned downstream from the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda clone-9 cells. Synthesis of luciferase (Luc) was accurately measured in insect cells growing in a 96-well plate, by a simple, rapid, nonradioactive, inexpensive and sensitive method based on fogging of x-ray film. Luc was produced in a coordinate fashion during virus infection. The Luc synthesized in insect cells was not secreted into the medium but was contained within the cell. Our findings suggest that Luc can be used as a superior reporter enzyme for molecular genetic analyses of baculovirus regulatory signals involved in high level expression of foreign genes, protein processing, targeting and stability in insect cells.     

Plasmid DNA encoding replicating foot-and-mouth disease virus genomes induces antiviral immune responses in swine.

DNA vaccine candidates for foot-and-mouth disease (FMD) were engineered to produce FMD virus (FMDV) particles that were noninfectious in cell culture or animals. The prototype plasmid, pWRM, contains a cytomegalovirus immediate-early promoter-driven genome-length type A12 cDNA followed by the bovine growth hormone polyadenylation site. BHK cells transfected with this plasmid produced virus, but the specific infectivity of pWRM was much lower than that achieved with in vitro-generated RNA genomes. To improve the infectivity of the plasmid, a cDNA encoding the hepatitis delta virus ribozyme was added to the 3' end of the FMDV cDNA. The resulting plasmid, pWRMH, exhibited slightly increased infectivity in cell culture and produced virus when inoculated into suckling mice. A third plasmid, pWRMHX, was created by removal of the sequences encoding the cell binding site found in capsid protein VP1 of pWRMH. Although cells transfected with pWRMHX produced viral capsids, this plasmid was not lethal in suckling mice, indicating that particles lacking the cell binding site were not able to initiate secondary infectious cycles. Swine inoculated with pWRMHX did not show any signs of disease and produced neutralizing antibodies to FMDV, and 20% of the vaccinated animals were protected from challenge. A derivative of pWRMHX, pWRMHX-pol-, harboring a mutation designed to inactivate the viral polymerase was much less immunogenic, indicating that immunogenicity of pWRMHX resulted, in part, from amplification of the viral genome in the animal.     

Insect immunity. Isolation and sequence of two cDNA clones corresponding to acidic and basic attacins from Hyalophora cecropia

The Cecropia moth has three known classes of antibacterial immune proteins, attacins, lysozyme and cecropins (earlier referred to as P5, P7 and P9, respectively). Six attacins with different isoelectric points have been purified. The N-terminal sequences for five of these forms imply that only two different genes exist. We have now isolated and sequenced two cDNA clones, one for the basic attacin and one for the acidic form. The two mature proteins show 76% homology at the nucleotide level, while the regions beyond the stop codons are 36% homologous. The differences in the content of aspartic acid accounts for the difference in net charge between the acidic and basic attacin. Further differences in charge can be obtained by post-translational removal of a lysine-containing tetrapeptide at the C-terminal end of the two proteins. Evidence for a prepro form of the basic attacin is presented.     

Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.     

Baculovirus-Mediated Large-Scale Expression and Purification of a Polyhistidine-Tagged Rubella Virus Capsid Protein

The capsid protein of rubella virus was produced in baculovirus-infectedSpodoptera frugiperdainsect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal–ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen–antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies.     

On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process

On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.     

Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.

A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active.     

Anti-attacin polyclonal antibody from an in vitro derived antigen used for immunoblot to quantify attacin expressed in transgenic apple

A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l^–1) by Ni²⁺-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.