Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.     

Purification and characterization of human salivary-gland prokallikrein from recombinant baculovirus-infected insect cells.

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.     

Characterization of human recombinant transglutaminase 1 purified from baculovirus-infected insect cells

Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both mu- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.     

Secretion of an active recombinant dog gastric lipase from baculovirus-infected insect cells

An active dog gastric lipase (DGL) belonging to the acid-stable mammalian lipase family, was produced and secreted from baculovirus-infected insect cells using the honeybee melittin and the DGL signal sequences. Both sequences drove the secretion of an active 47 kDa recombinant DGL (rDGL). rDGL was secreted at about 35 mg/L of culture medium. A one step purification using a cation exchange chromatography was used to recover an active electrophoretically pure rDGL from 2 days post-infection supernatant. There were not significant differences between the enzymatic properties of native and recombinant proteins. © Rapid Science Ltd. 1998     

Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells

Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.     

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni²⁺-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M_r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (M_r 77,000) and β (M_r 70,000) sGC subunits. It showed an A₄₃₀/A₂₈₀ = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k_cat/K_m = 1.7 × 10⁻³ s⁻¹ μM⁻¹ that increased to 5.8 × 10⁻¹ s⁻¹ μM⁻¹ upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.     

Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells.

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.     

Expression, purification and characterization of recombinant mouse PC6B from baculovirus-infected insect cells

We constructed a mouse PC6B truncated mutant and introduced a tag of 6 consecutive histidines at its carboxyl terminus for simple purification. Using the baculovirus expression system and standard enzymatic assay, we obtained recombinant mouse PC6B protein and with enzymatic activity.     

Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.     

Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells

Aims: To develop an efficient and facile expression system supply of high purity and stable activity of rFip-fve for oral administration, medicinal study and applications. Methods and Results: A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence, was constructed. The rFip-fve was purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified by Western blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip-fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip-fve was evaluated by measuring the amount of interleukin-2 released from murine splenocytes. Conclusions: A reliable scheme to express and purify active rFip-fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip-fve protein. Significance and Impact of the Study: The rFip-fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip-fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli.     

Secretion of biologically active leech hirudin from baculovirus-infected insect cells.

The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.     

Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells.

The turkey beta-adrenergic receptor (beta-AR), the m1 and m2 forms of the human muscarinic cholingeric receptor (MAChR) and several other mutant and wild-type G protein-coupled receptors were produced in insect Sf9 cells by infection with recombinant baculoviruses. Maximal expression for most receptors was 5-30 pmol receptor/mg protein (2-15 nmol/liter culture). The receptors displayed typical ligand binding characteristics. The beta-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The beta-AR stimulated endogenous adenylyl cyclase in response to beta-adrenergic agonists. The beta-AR and both MAChRs were purified and coreconstituted with various purified G proteins in phospholipid vesicles. The recombinant beta-AR catalyzed the agonist-dependent activation of Gs by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with the same efficiency as did the natural beta-AR. The m2 MAChR efficiently catalyzed GTP gamma S binding to Go and to the recently identified G protein Gz (Gx). The m2 MAChR also catalyzed the activation of Gj,1 and Gj,3 weakly. Activation of these same G proteins by the ml MAChR was much less efficient, consistent with its known selectivity for pertussis toxin-insensitive G proteins ("Gp") that have not yet been isolated. The beta-AR and m2 MAChR were characteristically stimulated by reduction of disulfides. These results demonstrate the general utility of the baculovirus system for production of large quantities of native G protein-coupled receptors.     

Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells

An experimental study was undertaken to quantify the effects of infection cell density, medium condition, and surface aeration on recombinant protein yields in insect cells. In the absence of surface aeration and fresh medium, insect cells generated higher product yields (on a per cell basis) when infected with recombinant baculovirus at low cell densities, LCD (3 x 10(5)-4 x 10(5) cells/mL), than at high cell densities, HCD (>0.9 x 10(6) cells/mL), for two distinct baculovirus types. Surface aeration of a HCD culture infected in spent medium improved beta-glactosidase yields 5-fold over the nonaerated case. Surface aeration and medium replenishment improved beta-galactosidase yields of a HCD culture by 20-fold (compared to a 1.6-fold improvement for a LCD culture), resulting in cultures with productivties that were independent of the cell density at infection.     

Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells

The unprocessed Gag precursor from HIV-1, when expressed in recombinant baculovirus-infected insect cells, is targeted to the plasma membrane and assembles in 100-120 nm particles budding from the cell surface. This process mimics HIV immature particle formation and is dependent on myristoylation of the N-terminal glycine, as deletion of the latter results in particle accumulation in the cytoplasm and, interestingly, in the nucleus, pointing to a potential role of this non-fatty-acid-acylated species in the viral life cycle. Inclusion of the pol gene in the construct results in efficient processing of Pr55gag and a pronounced decrease in particle formation. Deletion of the C terminus (p16) of the Gag precursor, including the finger domains, abolishes particle assembly, but membrane targeting and evagination still occur. Heterologous expression in insect cells may prove very useful for the study of the molecular events leading to retroviral particle morphogenesis.     

Functional characterization and purification of the secretin receptor expressed in baculovirus-infected insect cells

Structural insights into Class II G protein-coupled receptors have been limited by the absence of a plentiful and highly enrichable source such as rhodopsin in the Class I family. With structural differences predicted to exist between these families, and with the key importance of an intact, disulfide-bonded amino-terminal domain for the Class II receptors, an overproduction and purification scheme is critically important. In this work, we have established and characterized a baculoviral expression and purification system for the secretin receptor. Hemagglutinin epitope-tagged wild-type rat secretin receptor construct was expressed using the recombinant baculovirus/Sf9 insect cell-based system, achieving a level of expression substantially higher than that previously achieved in Chinese hamster ovary (CHO-SecR) cells. Receptor expressed in Sf9 cells had similar affinity for secretin (Ki=1.4+/-0.2 nM) and similar potency to stimulate intracellular cAMP in response to this hormone (EC50=194+/-45 pM) as did wild-type receptor expressed in CHO cells. Receptors from Sf9 cells were also affinity labeled saturably and specifically by a photolabile secretin analogue. The receptors were purified to homogeneity by solubilization with sodium deoxycholate, selective ammonium sulfate precipitation, gel filtration and immunoaffinity purification. This expression system should facilitate the structural characterization of this receptor and its important amino-terminal domain.     

Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

Three proteins, namely, the core protein C and envelope glycoproteins E1 and E2, are main structural proteins forming a hepatitis C virus (HCV) virion. The virus structure and assembly and the role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Highly efficient heterologous expression systems make it possible to obtain self-assembled, nonreplicating, genome-lacking particles that are morphologically similar to intact virions. Using recombinant baculoviruses expressing the HCV structural protein genes in insect cells, the individual HCV structural proteins were expressed to 25–35% of the total cell protein, and the CE1 and E1E2 heterodimers and HCV-like particles were obtained. It was demonstrated that the recombinant C, E1, and E2 proteins underwent posttranslational modification, the glycoproteins formed a noncovalent heterodimer, and HCV- like particles were located in endoplasmic reticulum membranes of infected cells. The formation of E1E2 dimers and HCV-like particles was used to study the effect of E1 glycosylation on the expression and processing of the coat proteins.     

Immunocytochemical study of recombinant terminal deoxynucleotidyl transferase (TdT) synthesized by baculovirus-infected insect cells.

The ability to overproduce terminal transferase through recombinant DNA technology should provide alternate means for generating sufficient quantities for structural and mechanistic study of this creative DNA polymerase. In this work we have investigated, at electron microscope level, the morphological modification and ultrastructural localization of synthesized human terminal transferase occurring in Sf-9 cells during recombinant baculovirus infection time. The results obtained showed that TdT is localized and stored only at the cytoplasmic level; the nucleus did not show any specific site able to link the neosynthesized TdT. The amount of the enzyme, estimate by immunostaining analysis, increased with the viral infection time. Morphological changes occurring during viral infection consist mainly of variations of cellular surface, different size and shape of cytoplasmic organelles and modification of nuclear components.     

Purification and characterization of a recombinant rat prohaptoglobin expressed in baculovirus-infected Sf9 insect cells

To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin alphabeta subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 microg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence. The protein was successfully purified by a three step procedure including nickel-linked agarose and DEAE-Sepharose chromatography steps. Hemoglobin was not detected in the purified preparations. Purified recombinant rat prohaptoglobin protein was also found to be glycosylated, and to be capable of forming a complex with rat hemoglobin in vitro.     

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellularviral components, and the heterologous product titers. The model describes the whole processes of viral infection and theeffect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave goodagreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infectionin batch and fed-batch culture were studied using the model.