Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed. Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions. Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation. These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients.     

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.     

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 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) 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.     

Characterization of recombinant human cathepsin B expressed at high levels in baculovirus.

The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper-glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus-expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.     

Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector

The catalytic subunit of protein phosphatase 1 (PP1), a key enzyme in the regulation of many cellular functions, has been expressed in insect cells using a baculovirus vector containing PP1 alpha cDNA. The expressed protein had the same apparent molecular mass as PP1 from rabbit skeletal muscle and comprised up to 25% of the total cellular protein. About 5% of expressed PP1 alpha was present as a soluble active species, representing a 15-fold increase over the endogenous activity. Insoluble protein, comprising about 95% of the expressed PP1 was dissolved in 6 M guanidinium chloride and could be fully reactivated by extensive and rapid dilution with buffers containing Mn2+. By a number of criteria (specific activity towards phosphorylase, interaction with inhibitor-1, inhibitor-2 and okadaic acid), this reactivated species was indistinguishable from authentic PP1, and could be concentrated and purified to homogeneity by a single chromatography on DEAE-Sepharose. This procedure yielded about 10 mg active PP1/1 culture, which will facilitate future structural analyses of native and mutant protein phosphatases.     

Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines.

Recombinant Autographa california baculoviruses expressing genes for pseudorabies virus glycoprotein (gp50T), human plasminogen (HPg), and beta-galactosidase (beta-gal) were used to infect 23 cell lines or strains. The objectives were to compare amounts of recombinant proteins expressed in the cell lines, compare yields from clones and parent lines, investigate the effects of long-term culture in serum-free medium on production, and determine if some lines yield gp50T with different glycosylation patterns. For HPg, IZD-MB0503 had the highest yield and four other lines (IPLB-TN-R2, IPLB-SF-1254, IPLB-LdEIta, and CM-1) had levels above that of SF-9 cells. For gp50T, four lines (IPLB-HvT1, IPLB-SF21AE, IPLB-SF21AE-15, and IPLB-SF-1254) had higher amounts than SF-9 cells. Some lines yielded gp50T with molecular mass about 1000 daltons larger than that from SF-9 cells, which suggests increased oligosaccharide processing. Equally high levels of beta-gal were expressed in three lines (SF-9, IZD-MB0503, and BCIRL-PX2-HNV3). The major conclusion is that no single cell line produced highest yields for all three recombinant proteins. Four lines were cultured in serum-free medium for 31-34 passages and then infected with the three recombinant viruses. For most cell line-recombinant combinations, the yields in serum-free medium were equal to or better than those in serum-supplemented medium. Medium composition had a much stronger effect on foreign gene expression than on susceptibility of cells to wild-type virus.     

Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.     

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.     

Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production.     

Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems.

Rab6 protein (rab6p) belongs to a family of ras-like GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. We have constructed a recombinant baculovirus in order to express rab6p in insect cells. We report here the characterization of four forms of this protein which are found in cytosolic and membrane fractions of infected Sf9 cells. The two major forms are a cytosolic 24 kD protein which represents the unprocessed precursor form of rab6p and a membrane-bound isoprenylated 23 kD protein which represents the processed form. Two other minor forms were also detected: a cytosolic isoprenylated 23 kD protein which may represent a pool in equilibrium with the 23 kD membrane-bound form and a 24 kD non-isoprenylated membrane-bound form which may represent an intermediate in the processing of rab6p.     

Purification and characterization of recombinant human pro-urokinase produced in silkworm using a baculovirus vector

Summary A recombinant baculovirus (BmNPV-pk2) was constructed by inserting the human pro-urokinase(pro-UK) cDNA into the genome of baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) adjacent to the strong polyhedrin promoter. The recombinant virus replicated in silkworm larvae, which synthesized 30g pro-UK/ml in the haemolymph within 4 days post-infection. Purification to near homogeneity was accomplished by fractional precipitation with ammonium sulphate and immunoaffinity chromatography with an overall yield of 23% and a specific activity of 100,000IU/mg in fibrin plate assay. This purified product was comprised of a single chain protein with approximately Mr. 50kDa as determined by SDS-PAGE gel. The N-Terminal amino acids sequence revealed that the secretion signal of pro-UK was correctly processed.     

Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. We expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [3H]-azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents (e.g., daunomycin greater than verapamil greater than vinblastine approximately vincristine) compete with the [3H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.     

Characterization of HCV-like particles produced in a human hepatoma cell line by a recombinant baculovirus

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor

 Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, prosystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively. Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity. Received: 30 August 1999 / Accepted: 6 December 1999     

Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.     

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.