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1.
J Virol ; 74(24): 11873-80, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11090187

ABSTRACT

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.


Subject(s)
Baculoviridae , Drosophila/virology , Genetic Vectors , Superinfection , Transfection , Animals , Cells, Cultured , Gene Expression Regulation, Viral , Recombination, Genetic
2.
Article in English | MEDLINE | ID: mdl-10099898

ABSTRACT

The successful use of live attenuated viral and bacterial vaccines depends not only on the proper choice and delivery of the microorganisms, but also on maintaining the sufficient potency required for an immune response. The inherent lability of live organisms presents a particular formulation challenge in terms of stabilizing and preserving vaccine viability during manufacturing, storage, and administration. This review examines pharmaceutical approaches to the stabilization, formulation, and lyophilization of biological macromolecules in general, as well as the specific applicability of these principles to live attenuated viral and bacterial vaccines. Several formulation development case studies with live vaccines are presented. In addition, comparative stability data are summarized for many other live viral and bacterial preparations. Various pharmaceutical issues with conventional and novel delivery systems for administration of parenteral and oral live vaccines are also discussed.


Subject(s)
Bacterial Vaccines/administration & dosage , Bacterial Vaccines/chemical synthesis , Viral Vaccines/administration & dosage , Viral Vaccines/chemical synthesis , Chemistry, Pharmaceutical , Drug Administration Routes , Drug Carriers , Drug Stability , Humans , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/chemical synthesis
3.
Neuroscience ; 83(1): 7-14, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9466395

ABSTRACT

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with diverse biological functions. Signal transduction of GDNF is mediated by binding to a glycosyl-phosphatidylinositol (GPI)-linked receptor GDNFR-alpha and activation of c-RET tyrosine kinase. The recent discovery of a new GDNF homolog neurturin raises the possibility that multiple receptors exist for the members in the GDNF family. Here we report isolation of the gene encoding a new receptor called GDNFR-beta. Sequence analysis indicated that GDNFR-beta is also a GPI-linked protein, with 47% identity to GDNFR-alpha. The GDNFR-beta transcript was preferentially expressed in the brain, spleen and lung, but moderate levels of GDNFR-beta mRNA were also found in kidney and the entire gastrointestinal track. In situ hybridization revealed high expression levels in the entorhinal cortex and olfactory bulb, followed by cortex, septum, inferior and superior colliculus, and zona inserta. A laminar pattern of expression was detected in layer III of the cortex. Treatment with GDNF of PC12 cells transfected with the GDNFR-beta gene activated mitogen-activated protein kinase (MAPK) and elicited neurite outgrowth. GDNFR-alpha and GDNFR-beta together form a new family of GPI-linked receptors for GDNF-like molecules.


Subject(s)
Drosophila Proteins , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Amino Acid Sequence , Animals , Brain/anatomy & histology , Brain Chemistry , Cloning, Molecular , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , Molecular Sequence Data , Proto-Oncogene Proteins c-ret , Rats , Tissue Distribution
4.
J Biol Chem ; 272(36): 22556-63, 1997 Sep 05.
Article in English | MEDLINE | ID: mdl-9278409

ABSTRACT

Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenbaugh, M. J. (1996) Protein Expression Purif. 7, 281-288). PDI is known to include catalytic active sites in two separate thioredoxin-like domains, one near the amino terminus and another near the carboxyl terminus. To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with cysteine to serine mutations at the first cysteine of one or both of the CGHC active site sequences. Trichoplusia ni insect cells were coinfected with a baculovirus vector coding for IgG in concert with either the wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl-terminal mutant (PDI-C), or mutant with both active sites altered (PDI-NC). Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells. To evaluate the effect of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with 35S-amino-acids for different periods, and the soluble immunoglobulins were immunoprecipitated from clarified cell lysates and culture medium using anti-IgG antibodies. Only coinfections with the wild-type PDI and PDI-N mutant led to increased immunoglobulin solubility and higher IgG secretion. In contrast, infection with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus. Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI protein were detected in soluble form. The capacity for PDI-N to increase immunoglobulin solubility whereas the PDI-C mutant lowered solubility indicates that the amino- and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the active site. Furthermore, mutations at the active site of the carboxyl-terminal thioredoxin domain result in PDI variants that can act as anti-chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).


Subject(s)
Isomerases/metabolism , Molecular Chaperones/antagonists & inhibitors , Thioredoxins/metabolism , Animals , Baculoviridae/genetics , Cell Line , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Isomerases/genetics , Mutagenesis, Site-Directed , Precipitin Tests , Protein Disulfide-Isomerases , Solubility , Spodoptera
5.
J Biol Chem ; 272(14): 9062-70, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9083032

ABSTRACT

Structures of the N-linked oligosaccharide attached to the heavy chain of a heterologous murine IgG2a produced from Trichoplusia ni (TN-5B1-4, High Five) insect cells were characterized. Coexpression of the chaperone immunoglobulin heavy chain-binding protein (BiP) in the baculovirus-infected insect cells increased the soluble intracellular and secreted IgG level. This facilitated the detailed analysis of N-glycans from both intracellular and secreted IgG. Following purification of the immunoglobulins using Protein A-Sepharose, glycopeptides, prepared by trypsin-chymotrypsin digestion, were further digested with glycoamidase from sweet almond emulsin to obtain the oligosaccharide moieties. The resulting oligosaccharides were then reductively aminated with 2-aminopyridine and the structures identified by two-dimensional high performance liquid chromatography mapping (Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171, 73-90). The N-glycans obtained from the secreted IgG contain 35% complex type, some with terminal galactose residues at either alpha1, 3-Man or alpha1,6-Man branches of the Man3GlcNAc2 core. The remaining oligosaccharides detected in the secreted IgG were principally hybrid (30%) and paucimannosidic (35%) type N-glycans. Most (84%) of these secreted glycoforms contained fucose alpha1, 6-linked to the innermost GlcNAc residue and the presence of a potentially allergenic fucose alpha1,3-linked to the innermost GlcNAc residue was also detected. In contrast, the intracellular immunoglobulins included 50% high mannose-type N-glycans with lower levels of complex, hybrid, and paucimannosidic-type structures. Reverse phase one-dimensional high performance liquid chromatography analysis of the IgG N-glycans in the absence of heterologous BiP exhibited a similar distribution of intracellular and secreted glycoforms. These studies indicate that Trichoplusia ni TN-5B1-4 cells are capable of terminal galactosylation. However, the processing pathways in these cell lines appear to diverge from mammalian cells in the formation of paucimannosidic structures, in the presence of alpha1,3-fucose linkages, and in the absence of sialylation.


Subject(s)
Immunoglobulin G/chemistry , Polysaccharides/chemistry , Animals , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Culture Media , Lepidoptera , Mass Spectrometry , Molecular Sequence Data , Oligosaccharides/chemistry
6.
Biotechnol Prog ; 13(1): 96-104, 1997.
Article in English | MEDLINE | ID: mdl-9041711

ABSTRACT

Infection of Trichoplusia ni (BTI-TN5B1-4) insect cells with a baculovirus coding for immunoglobulin G resulted in significant intracellular insolubility of the immunoglobulin chains. In order to increase the immunoglobulin solubility, the chaperone BiP was coexpressed in the insect cells using a separate baculovirus vector. This heterologous BiP was observed to associate with immunoglobulin chains in vivo and enhance the level of soluble intracellular and secreted IgG obtained from T.ni. Pulse chase studies indicated that the heterologous BiP increased the level of soluble nascent immunoglobulin chains and assembly intermediates to suggest that BiP is acting as a true molecular chaperone. The effect of heterologous BiP became more significant with time post-infection as secreted IgG levels increased by 90% after 3.4 days of baculovirus infection. Even following the treatment of cells with tunicamycin, BiP coexpression still enhanced immunoglobulin solubility and secretion to indicate that BiP does not function specifically to retain unglycosylated proteins in the endoplasmic reticulum. Thus, coexpression of a molecular chaperone may be used to enhance cellular productivity and protein secretion provided that the chaperone is involved with post-translational processing and significant protein aggregation is observed.


Subject(s)
Carrier Proteins/genetics , Heat-Shock Proteins , Immunoglobulin G/genetics , Insecta/genetics , Molecular Chaperones/genetics , Animals , Baculoviridae/genetics , Carrier Proteins/metabolism , Cloning, Molecular , Electrophoresis, Gel, Pulsed-Field , Endoplasmic Reticulum Chaperone BiP , Genetic Vectors , Immunoglobulin G/metabolism , Mice , Molecular Chaperones/metabolism , Solubility , Tunicamycin/pharmacology
7.
Biotechnol Bioeng ; 56(1): 106-16, 1997 Oct 05.
Article in English | MEDLINE | ID: mdl-18636615

ABSTRACT

A model for immunoglobulin G (IgG) production in the baculovirus-insect cell system was developed that incorporates polypeptide synthesis, oligomer assembly, protein aggregation, and protein secretion. In addition, the capacity of a chaperone to protect heavy and light chain polypeptides from protein aggregation was considered by including in vitro chaperone-peptide binding and dissociation kinetic constants from the literature. Model predictions were then compared to experiments in which the chaperone immunoglobulin heavy chain binding protein, BiP, was coexpressed by coinfecting insect cells with BiP-containing baculovirus. The model predicted a nearly twofold increase in intracellular and secreted IgG that was similar to the behavior observed experimentally after approximately 3 days of coexpressing heterologous IgG and BiP. However, immunoglobulin aggregation was still significant in both the model simulation and experiments, so the model was then used to predict the effect of strategies for improving IgG production even further. Increasing expression of the chaperone BiP by 10-fold over current experimental levels provided a 2.5-fold increase in secreted IgG production over IgG assembly without BiP. Alternatively, the expression of BiP earlier in the baculovirus infection cycle achieved a twofold increase in protein secretion without requiring excessive BiP production. The potential effect of cochaperones on BiP activity was considered by varying the BiP binding and release constants. The utilization of lower binding and release kinetic constants led to a severalfold increase in IgG secretion because the polypeptides were protected from aggregation for greater periods. An optimized strategy for chaperone action would include the rapid peptide binding of a BiP-ATP conformation along with the slow peptide release of a BiP-ligand conformation. However, even with an optimized chaperoning system, limitations in the secretion kinetics can result in the accumulation of intracellular IgG. Thus, the entire secretory pathway must be considered when enhanced secretion of heterologous proteins is desired.

8.
Protein Expr Purif ; 7(3): 281-8, 1996 May.
Article in English | MEDLINE | ID: mdl-8860653

ABSTRACT

A substantial fraction of immunoglobulin heavy and light chain polypeptides were insoluble when expressed in the baculovirus-insect cell expression system. In the presence of coexpressed heterologous protein disulfide isomerase (PDI), however, the solubility of the immunoglobulins was enhanced and IgG was secreted at higher levels from baculovirus-infected Trichoplusia ni insect cells. Pulse-chase experiments indicated that some immunoglobulin polypeptides were initially insoluble in the presence of PDI but subsequently were rescued in a soluble form competent for IgG assembly and secretion. Recovery of the insoluble immunoglobulins was not observed in the absence of coexpressed PDI. Even after treatment of insect cells with tunicamycin to inhibit N-glycosylation of immunoglobulin heavy chains, coexpressed PDI was able to salvage insoluble immunoglobulins and secrete these modified glycoforms. The capacity for PDI to rescue immunoglobulins was also demonstrated in vitro where immunoglobulin heavy chains and light chain dimers were salvaged from aggregates of denatured IgG. PDI-mediated rescue of proteins, perhaps assisted by chaperones and other foldases, may be important in vivo where insolubility is a common occurrence for newly synthesized polypeptides.


Subject(s)
Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Light Chains/biosynthesis , Isomerases/metabolism , Nucleopolyhedroviruses/metabolism , Protein Processing, Post-Translational , Animals , Cells, Cultured , Genetic Vectors , Goats , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Insecta/metabolism , Insecta/virology , Isomerases/genetics , Nucleopolyhedroviruses/genetics , Protein Disulfide-Isomerases , Protein Folding , Protein Processing, Post-Translational/drug effects , Tunicamycin/pharmacology
9.
Cytotechnology ; 20(1-3): 149-59, 1996 Jan.
Article in English | MEDLINE | ID: mdl-22358480

ABSTRACT

CONCLUSIONS: The BEVS has become widely utilized for production of recombinant proteins. However, protein aggregation and inefficient processing often limit yields, especially for secreted and membrane proteins. Since many proteins of pharmaceutical interest require similar posttranslational processing steps, engineering the folding, assembly, and secretion pathway may enhance the production of a wide variety of valuable complex proteins. Efforts should be undertaken to coexpress the relevant chaperones or foldases at low levels in concert with the final product to ensure the ideal folding and assembly environment. In the future, expression of oligosaccharide modifying enzymes and secretion factors may further improve secretion rates of assembled proteins and provide heterologous proteins with altered glycoforms. Also significant is the use of BEVS as an in vivo eucaryotic laboratory to study the fundamental roles of differnt chaperones, foldases, and secretion factors. The coexpression of chaperones and foldases will complement other approaches such as the development of alternative insect cell lines, promoters, and signal peptides to optimize the baculovirus-insect cell expression system for generating high yields of valuable proteins.

10.
Protein Expr Purif ; 5(6): 595-603, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7858430

ABSTRACT

The assembly pathway of the insect cell Spodoptera frugiperda (Sf-9) was engineered to include expression of the murine chaperone immunoglobulin heavy chain binding protein (BiP) using the baculovirus vector. The impact of BiP coexpression on the production and secretion of functional and soluble recombinant immunoglobulin IgG levels was evaluated. Recombinant BiP was found to associate specifically with immunoglobulins in immunoprecipitation studies. Coinfection of insect cells with a BiP-containing baculovirus and baculoviruses coding for two different murine IgG proteins increased intracellular functional antibody activity levels substantially above the levels observed in the absence of BiP. Soluble intracellular immunoglobulin levels were found to increase as well. However, secreted functional antibody levels did not increase significantly. Also, degradation of heavy chain immunoglobulin in insect cells was indicated by the accumulation of lower molecular weight immunoglobulins at 4 days postinfection. Coexpression of light chains reduced the level of these lower molecular weight immunoglobulins while BiP coexpression led to enhanced levels. These findings suggest that coexpressed BiP can increase intracellular soluble and functional antibody yields but that secretion in the baculovirus-insect cell system must be limited at some post-translational step.


Subject(s)
Baculoviridae/metabolism , Immunoglobulins/metabolism , Molecular Chaperones/metabolism , Animals , Cell Line , Enzyme-Linked Immunosorbent Assay , Immunoassay , Protein Binding , Protein Folding , Recombination, Genetic , Spodoptera
11.
Virology ; 204(2): 854-9, 1994 Nov 01.
Article in English | MEDLINE | ID: mdl-7524242

ABSTRACT

To facilitate studies of the individual viral proteins, two Borna disease virus proteins, p24 and p38/40, were synthesized in vitro by means of a baculovirus expression system and examined for antigenic identity to viral proteins from BDV-infected cells. Recombinant proteins p24 and p38/40 were nearly identical in size to the viral proteins from BDV-infected cells. Immunoblot and immunocytochemistry analysis of BDV proteins from infected tissue culture cells and rat brain showed binding of antisera directed against the recombinant proteins. Specific recognition of the recombinant proteins by Borna disease virus-specific convalescent antisera and monoclonal antibodies further demonstrated that the antigenic characters of the p24 and p38/40 had been conserved. Polyclonal antibody directed against either of the recombinant proteins recognized only the protein used as immunogen, without cross reactivity with the other recombinant protein, indicating no common epitopes. Moreover, these data confirmed the proposed gene coding assignments of ORF I and II of BDV p38/40 and p24, respectively. Both of the recombinant proteins were secreted into the media of insect cells in tissue culture, but secretion of recombinant p24 was evident only as a dimeric form and not with the monomeric form. Immunoprecipitation studies performed with monoclonal antibodies and BDV proteins from infected rat brain suggested that a heterodimer forms via binding of p40 to the p24.


Subject(s)
Borna disease virus/immunology , Viral Proteins/immunology , Viral Structural Proteins/immunology , Animals , Baculoviridae/genetics , Cells, Cultured , Epitopes , Immunoblotting , Rabbits , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Spodoptera , Viral Proteins/biosynthesis , Viral Structural Proteins/biosynthesis
12.
Ann N Y Acad Sci ; 721: 208-17, 1994 May 02.
Article in English | MEDLINE | ID: mdl-8010671

ABSTRACT

The synthesis of complex biological structures such as antibodies using recombinant DNA technology is a major biotechnological advance. Active murine antibody (IgG) oligomers, composed of two heavy (H) and two light (L) polypeptide chains, have been expressed and secreted by the baculovirus-insect cell expression system. Unfortunately, expression of the functional antibodies is accompanied by the formation of abnormal protein complexes and aggregates in which the polypeptide chains are bound together into incorrect associations. The formation of these abnormal complexes or protein aggregates in insect cells may be caused by insufficient intracellular levels of two catalytic proteins, immunoglobulin binding protein (BiP or GRP78), and protein disulfide isomerase (PDI). Consequently, we obtained the genes coding for murine BiP and PDI and cloned the genes into the baculovirus vector (Autographa californica nuclear polyhedrosis virus) to obtain AcBB-BiP and AcBB-PDI. Infection of Spodoptera frugiperda (Sf-9) insect cells with these two baculoviruses yielded recombinant proteins of the correct size that were recognized by antibodies to these proteins. Cloning these genes into the baculovirus vector is one approach to engineering the assembly pathway in order to lower aggregation and increase production of functionally active proteins and oligomers.


Subject(s)
Genetic Engineering , Heat-Shock Proteins , Molecular Chaperones , Nucleopolyhedroviruses/genetics , Animals , Antibody Formation/genetics , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cells, Cultured , Cloning, Molecular , DNA, Recombinant/genetics , Endoplasmic Reticulum Chaperone BiP , Gene Expression , Genetic Vectors , Isomerases/biosynthesis , Isomerases/genetics , Moths , Protein Disulfide-Isomerases , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
13.
Biotechnol Bioeng ; 21(12): 2235-46, 1979 Dec.
Article in English | MEDLINE | ID: mdl-117848

ABSTRACT

A convenient method for enzyme kinetic studies is introduced. The method includes identification of reaction mechanism and estimation of the associated kinetic constants with a minimum number of experiments. The application of the method is illustrated by using literature data. Factors limiting the application of this method are also discussed.


Subject(s)
Enzymes/metabolism , Models, Chemical , Binding, Competitive , Cellobiose/metabolism , Enzyme Inhibitors/pharmacology , Glucose/biosynthesis , Hydrolysis , Kinetics , beta-Glucosidase/metabolism
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