Production of native creatine kinase B in insect cells using a baculovirus expression vector

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.     

A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.     

Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.     

Characterization and purification of human fos protein generated in insect cells with a baculoviral expression vector.

We generated recombinant baculoviruses that contained the human fos gene and that, upon infection of insect cells, synthesized fos protein. The quantity of fos protein produced was at least 10 to 20 times higher than that observed in any mammalian cells reported so far. The fos protein made in insect cells manifested most of the characteristics of mammalian fos protein, which include (i) 55-kilodalton size, (ii) nuclear localization, (iii) phosphoesterification at serine residues, (iv) identical 35S tryptic peptide maps, (v) ability to make heterodimers with the nuclear jun oncoprotein, and (vi) cooperation with the jun protein to bind to a 12-O-tetradecanoyl-phorbol-13-acetate-responsive element. A 100- to 150-fold purification of the fos protein from infected insect cells was achieved in a single step by immunoaffinity chromatography. Availability of authentic fos protein made by baculoviral vectors in insect cells should allow a more rigorous analysis of its biochemical and biological properties.     

Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×10⁷ cells.ml⁻¹, and a β-gal concentration of up to 104,000 unit.ml⁻¹ were achieved.     

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.     

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 adenovirus type 2 DNA polymerase in insect cells infected with a recombinant baculovirus.

Sequences encoding adenovirus type 2 DNA polymerase were placed under control of the polyhedrin promoter and inserted into the baculovirus Autographa californica nuclear polyhedrosis virus by homologous recombination. Insect cells infected with the recombinant virus produced substantial amounts of the adenovirus type 2 DNA polymerase protein which was functional in both DNA polymerase and replication initiation reactions. Thus, the baculovirus expression system can provide active adenovirus type 2 DNA polymerase that is produced in quantities suitable for biochemical and structural analysis.     

Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.     

Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity.     

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.     

Display of a Maize cDNA library on baculovirus infected insect cells

Background  

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings.     

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.     

A baculovirus expression system for insect cells

The review considers the biology of baculoviruses, construction of transfer vectors for the baculovirus expression system, selection of recombinant baculoviruses, approaches to expression of multimeric proteins, and the potentialities and prospects of the system.     

Production of functional hepatocyte growth factor (HGF) in insect cells infected with an HGF-recombinant baculovirus in a serum-free medium

Three insect cell lines, SL-7B cells derived from Spodoptera litura, Sf9, and High Five (Hi-5) cells, were used for the production of pro-hepatocyte growth factor (pro-HGF). Cells were cultured and then infected with a recombinant HGF-containing baculovirus in a serum-free medium. In SL-7B cells, pro-HGF is synthesized and excreted from the cells and late in infection is converted to a heterodimeric form of HGF even when the cells are grown in serum free medium. Conversion of a single-chain form of HGF (pro-HGF) into an HGF heterodimer was unexpected, as pro-HGF is normally cleaved by a serum protease called HGF activator. The proliferation activity of heparin-affinity-purified HGF from serum-free culture supernatant of SL-7B cells is comparable to that obtained from HGF converted by serum proteases, suggesting that SL-7B cells produce a functionally analogous protease to correctly process pro-HGF. This work reports, for the first time, on the feasibility of properly processing pro-HGF to form functional HGF by proteases from invertebrate cells in serum-free media. Avoiding the supplementation of sera provides the advantages of a low production cost, zero contamination of infectious agents from sera, and simple downstream product purification. Experimental results further demonstrate that the conversion of pro-HGF by insect cells is cell-line-dependent, because proteases in Hi-5 or Sf9 cells could not process pro-HGF as efficiently and properly as those in SL-7B cells.