Recombinant BACE1 Research Articles

Macrocyclic BACE1 inhibitors with hydrophobic cross-linked structures: Optimization of ring size and ring structure

Based on the X-ray crystallography of recombinant BACE1 and a hydroxyethylamine-type peptidic inhibitor, we introduced a cross-linked structure between the P1 and P3 side chains of the inhibitor to enhance its inhibitory activity. The P1 and P3 fragments bearing terminal alkenes were synthesized, and a ring-closing metathesis of these alkenes was used to construct the cross-linked structure. Evaluation of ring size using P1 and P3 fragments with various side chain lengths revealed that 13-membered rings were optimal, although their activity was reduced compared to that of the parent compound. Furthermore, the optimal ring structure was found to be a macrocycle with a dimethyl branched substituent at the P3 β-position, which was approximately 100-fold more active than the non-substituted macrocycle. In addition, the introduction of a 4-carboxymethylphenyl group at the P1′ position further improved the activity.

Structure–activity relationship study of hydroxyethylamine isostere and P1′ site structure of peptide mimetic BACE1 inhibitors

An aromatic substituent has been introduced into a known hydroxyethylamine (HEA)-type BACE1 inhibitor containing the superior substrate sequence to enhance inhibitory activity. The HEA-type isosteres bearing different hydroxyl group and methyl group configurations were prepared through a branched synthesis approach using intra- and inter-molecular epoxide opening reactions. The effect of their configuration was evaluated, showing that an R-configuration improved the inhibitory activity, while introduction of a methyl group on the isostere decreased the activity. Based on the non-substituted isostere with an R-configuration, 21 derivatives containing various substituents at the P1′ site were synthesized. Our evaluation of the derivatives showed that the structure of the P1′ site had a clear effect on activity, and highly potent inhibitor 40g, which showed sub-micromolar activity against recombinant BACE1 (rBACE1), was identified. The docking simulation of 40g with rBACE1 suggested that a carboxymethyl group at the para-position of the P1′ benzene ring interacted with Lys285 in the S1′ pocket.

Semi-synthetic isoflavones as BACE-1 inhibitors against Alzheimer’s disease

BACE-1 is considered to be one of the targets for prevention and treatment of Alzheimer’s disease (AD). We here report a novel class of semi-synthetic derivatives of prenylated isoflavones, obtained from the derivatization of natural flavonoids from Maclura pomifera. In vitro anti-AD effect of the synthesized compounds were evaluated via human recombinant BACE-1 inhibition assay. Compound 7, 8 and 13 were found to be the most active candidates which demonstrates good correlation between the computational docking and pharmacokinetic predictions. Moreover, cytotoxic studies demonstrated that the compounds are not toxic against normal and cancer cell lines. Among these three compounds, compound 7 enhance the activity of P-glycoprotein (P-gp) on A549 cancer cells and increases the activity of P-gp ATPase with a possible role on the efflux of amyloid-β across the blood- brain barrier. In conclusion, the present findings may pave the way for the discovery of a novel class of compounds to prevent and/or treat AD.

Inhibition of β-Secretase Activity by Monoterpenes, Sesquiterpenes, and C13 Norisoprenoids.

Inhibition of β-secretase (BACE1) is currently regarded as the leading treatment strategy for Alzheimer's disease. In the present study, we aimed to screen the in vitro inhibitory activity of 80 types of aroma compounds (monoterpenes, sesquiterpenes, and C13 norisoprenoids), including plant-based types, at a 200-μM concentration against a recombinant human BACE1. The results showed that the most potent inhibitor of BACE1 was geranyl acetone followed by (+)-camphor, (-)-fenchone, (+)-fenchone, and (-)-camphor with the half-maximal inhibitory concentration (IC50) values of 51.9 ± 3.9, 95.9 ± 11.0, 106.3 ± 14.9, 117.0 ± 18.6, and 134.1 ± 16.4 μM, respectively. Furthermore, the mechanism of inhibition of BACE1 by geranyl acetone was analyzed using Dixon kinetics plus Cornish-Bowden plots, which revealed mixed-type mode. Therefore aroma compounds may be used as potential lead molecules for designing anti-BACE1 agents.

Camelid heavy chain only antibody fragment domain against β-site of amyloid precursor protein cleaving enzyme 1 inhibits β-secretase activity in vitro and in vivo.

Accumulation and aggregation of the amyloid-β (Aβ) peptide is associated with Alzheimer's disease (AD). Aβ is generated from the amyloid precursor protein by the successive action of two membrane-associated processing enzymes: β-secretase or β-site of amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase. Inhibition of one or both of these enzymes prevents Aβ generation and the accompanying Aβ accumulation. Antigen binding fragments from camelid heavy chain only antibodies (VHHs) were found to exert excellent enzyme inhibition activity. In the present study, we generated VHHs against BACE1 by active immunization of Lama glama with the recombinant BACE1 protein. Two classes of VHHs were selected from a VHH-phage display library by competitive elution with a peptide encoding the Swedish mutation variant of the BACE1 processing site. One VHH was found to inhibit the enzyme activity of BACE1 in vitro and in cell culture, whereas two other VHHs were found to stimulate BACE1 activity under the same conditions in vitro. Furthermore, an in vivo study with a transgenic AD mouse model, using intracisternal injection of the inhibitory VHH, led to acute reduction of the Aβ load in the blood and brain. This inhibitory VHH may be considered as a candidate molecule for a therapy directed towards reduction of Aβ load and prevention of AD progression. Both the inhibitory and stimulatory VHH may be useful for improving our understanding of the structure-function relationship of BACE1, as well as its role in AD progression. The GenBank sequence accession numbers are KR363186 for VHH B1a; KR363187 for VHH B3a; and KR363188 for VHH B5a.

Evaluation of transition-state mimics in a superior BACE1 cleavage sequence as peptide-mimetic BACE1 inhibitors

A superior substrate sequence for BACE1 containing transition-state mimics at the scissile site was evaluated as a protease inhibitor. Hydroxymethylcarbonyl (HMC) and hydroxyethylamine (HEA) isosteres were selected as the transition state mimics, and incorporated into the scissile site of the superior sequence covering the P4 to P1’ sites (Glu-Ile-Thi-Thi*Nva; *denotes the cleavage site). Isosteres having different absolute configurations of the hydroxyl group were synthesized separately, and the effect of the configuration was evaluated. Configuration of the hydroxyl group of each isostere showed a marked effect on the inhibitory activity; anti-configuration to the scissile site substituent had potent inhibitory activity in an HMC-type inhibitor, whereas anti-configuration of HEA-type inhibitors showed no inhibitory activity. Structural evaluations based on X-ray crystallographic analyses of recombinant BACE1 in complex with each inhibitor provided insights into the protein–ligand interactions, especially at the prime sites.

Platelet-Derived Secreted Amyloid-Precursor Protein-β as a Marker for Diagnosing Alzheimer’s Disease

A marker of Alzheimer's disease (AD) with a high sensitivity and specificity would facilitate a diagnosis at early stages. Blood platelets may be of particular interest in search of biomarkers, because they express amyloid-precursor protein (APP), and display a dysfunctional processing in AD. The aim of the present study is to establish and validate an assay for secreted amyloid-precursor protein (sAPP)-α and -β in platelets of AD and mild cognitively impaired (MCI) subjects, compared to healthy young and old controls. Freshly isolated platelet extracts (25 µg) were incubated with or without recombinant BACE1 (beta-site APP-Cleaving Enzyme; β-secretase, 8U) at 37°C and low pH and the levels of sAPP-α and sAPP-b were measured by specific ELISAs. Our data show that sAPP-α levels were not different between AD, MCI and control subjects. However, sAPP-β levels in MCI and AD were significantly elevated relative to controls. When recombinant BACE1 was added, no changes were seen in sAPP-α levels, but the processed sAPP-β levels were again markedly increased. The sAPP-β processing was specific and selective after 2.5 hours at 37°C, and was possibly mediated by exogenous BACE1, because it was blocked by a BACE1 inhibitor and BACE1 enzyme levels were enhanced in AD patients. Our data reveal that quantitive analysis of platelet sAPP-β assay by ELISA may be a novel diagnostic biomarker for MCI and AD.

Development and Characterization of a Novel Membrane Assay for Full-Length BACE-1 at pH 6.0

β-Site amyloid precursor protein cleaving enzyme–1 (BACE-1) is a transmembrane aspartic protease that mediates the initial cleavage of the amyloid precursor protein (APP), leading to the generation of amyloid-β (Aβ) peptides that are thought to be causative of Alzheimer’s disease (AD). Consequently, inhibition of BACE-1 is an attractive therapeutic approach for the treatment of AD. In general, in vitro biochemical assays to monitor BACE-1 activity have used the extracellular domain of the protein that contains the catalytic active site. This form of BACE-1 is catalytically active at acidic pH and cleaves APP-based peptide substrates at the β-site. However, this form of BACE-1 does not mimic the natural physiology of BACE-1 and shows minimal activity at pH 6.0, which is more representative of the pH within the intracellular compartments where BACE-1 resides. Moreover, high-throughput screens with recombinant BACE-1 at pH 4.5 have failed to identify tractable leads for drug discovery, and hence, BACE-1 inhibitor development has adopted a rational drug design approach. Here we describe the development and validation of a novel membrane assay comprising full-length BACE-1 with measurable activity at pH 6.0, which could be used for the identification of novel inhibitors of BACE-1.

Human recombinant beta-secretase immobilized enzyme reactor for fast hits’ selection and characterization from a virtual screening library

In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the β-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC50 and Ki determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30μM for the fast hits’ selection and for mode of action determination.

Expression analysis of human β-secretase in transgenic tomato fruits

An emerging strategy in biomanufacturing involves using transgenic plants to express recombinant pharmaceutical and industrial proteins in large quantities. β-Site APP cleaving enzyme 1 (β-secretase 1, BACE1) is an enzyme involved in the abnormal production of Aβ42, the major component of senile plaques in Alzheimer’s disease (AD). Thus, BACE1 represents a key target protein in the development of new potential drugs to treat Alzheimer’s disease. We aimed to develop a tomato-derived recombinant BACE1 (rBACE1) protein to serve as a vaccine antigen that would promote an immune response. We utilized a plant expression cassette, pE8BACE, to optimize BACE1 expression in tomato fruits. Polyemerase chain reaction and Southern blot analyses verified integration of the BACE1 gene into the plant genome. Northern and Western blot analyses demonstrated successful mRNA and protein expression of rBACE1, respectively; the Sensizyme assay kit estimated the expression level of rBACE1 protein at 136±7ngmg−1 total soluble protein. The tomato-derived rBACE1 retains its activity for a long storage period at cool or room temperature, and is highly resistant to degradation in conditions such as low acidity. Tomato-derived rBACE1 was severely degraded by heat or boiling. The proteolytic activity of tomato-derived rBACE1, confirmed by fluorescence resonance transfer assay, was similar to that of a commercial sample of Escherichia coli-derived BACE1.

Flavonoids lower Alzheimer's Aβ production via an NFκB dependent mechanism.

Alzheimer's disease (AD) is characterized by the brain accumulation of Aβ peptides and by the presence of neurofibrillary tangles. Aβ is believed to play an important role in AD and it has been shown that certain flavonoids can affect Aβ production. Recently, it was suggested that the Aβ lowering properties of flavonoids are mediated by a direct inhibition the β-secretase (BACE-1) activity, the rate limiting enzyme responsible for the production of Aβ peptides. Westernblots and ELISAs were employed to monitor the impact of flavonoids on amyloid precursor protein processing and Aβ production. A cell free chemoluminescent assay using human recombinant BACE-1 was used to assess the effect of flavonoids on BACE-1 activity. The effect of flavonoids on NFκB activation was determined by using a stable NFκB luciferase reporter cell line. Molecular docking simulations were performed to predict the binding of flavonoids to the BACE-1 catalytic site. Real time quantitative PCR was used to determine the effect of flavonoids on BACE-1 transcription. We show in a cell free assay that flavonoids are only weak inhibitors of BACE-1 activity. Docking simulation studies with different BACE-1 structures also suggest that flavonoids are poor BACE-1 inhibitors as they appear to adopt various docking poses in the active site pocket and have weak docking scores that differ as a function of the BACE-1 structures studied. Moreover, a weak correlation was observed between the effect of flavonoids on Aβ production in vitro and their ability to lower BACE-1 activity suggesting that the Aβ lowering properties of flavonoids in whole cells are not mediated via direct inhibition of BACE-1 activity. We found however a strong correlation between the inhibition of NFκB activation by flavonoids and their Aβ lowering properties suggesting that flavonoids inhibit Aβ production in whole cells via NFκB related mechanisms. As NFκB has been shown to regulate BACE-1 expression, we show that NFκB lowering flavonoids inhibit BACE-1 transcription in human neuronal SH-SY5Y cells. Altogether, our data suggest that flavonoids inhibit Aβ and sAPPβ production by regulating BACE-1 expression and not by directly inhibiting BACE-1 activity.

Evaluation of superior BACE1 cleavage sequences containing unnatural amino acids

A recombinant form of BACE1 (β-site amyloid precursor protein cleaving enzyme-1) corresponding to positions 46–454 of the extracellular domain of the original membrane enzyme was prepared. The recombinant BACE1 (rBACE1) had the kinetic parameters K m = 5.5 μM and k cat = 1719 s −1. Using several libraries of substrates containing unnatural amino acids, the specificity of rBACE1 was evaluated. LC/MS of digests derived from the libraries clarified that a dodecapeptide containing unnatural amino acids at P 2 to P 1 ′ was a superior cleavage sequence.

Decrease in age-adjusted cerebrospinal fluid β-secretase activity in Alzheimer's subjects

Objectives To develop a novel cerebrospinal fluid (CSF) β-secretase-1 activity assay and evaluate β-secretase-1 (BACE-1) activity as a potential biomarker in human Alzheimer's disease. Methods The assay consisted of an enzymatic reaction of CSF samples with an optimized β-secretase peptide substrate and the cleavage products were detected using a neo-epitope specific antibody. Results The CSF BACE-1 activity assay described exhibits time, temperature, dose, and pH dependence, with sensitivity down to < 1 pM of recombinant BACE-1 enzyme, and is completely blocked by BACE-1 inhibitors. The endogenous BACE-1 enzyme in CSF appears to exist as a c-terminally truncated protein, based on both western blotting and capture-based activity assays. In a small cohort of human subjects, an age-dependent increase in CSF BACE activity was observed (~ 1.0 pM/year, p < 0.05). In Alzheimer's disease subjects, a significant decline in age-adjusted CSF BACE activity was observed compared to controls (56% in the log-transformed scale, p = 0.02). Conclusion We have developed a robust assay to measure CSF BACE-1 activity which could serve as a potential biomarker in human Alzheimer's disease subjects.

Promising anti-Alzheimer’s dimer bis(7)-tacrine reduces β-amyloid generation by directly inhibiting BACE-1 activity

The regulation of α-, β-, (BACE-1), and γ-secretase activities to alter β-amyloid (Aβ) generation is considered to be one of the most promising disease-modifying therapeutics for Alzheimer’s disease. In this study, the effect and mechanisms of bis(7)-tacrine (a promising anti-Alzheimer’s dimer) on Aβ generation were investigated. Bis(7)-tacrine (0.1–3μM) substantially reduced the amounts of both secreted and intracellular Aβ in Neuro2a APPswe cells without altering the expression of APP. sAPPα and CTFα increased, while sAPPβ and CTFβ decreased significantly in Neuro2a APPswe cells following the treatment with bis(7)-tacrine, indicating that bis(7)-tacrine might activate α-secretase and/or inhibit BACE-1 activity. Furthermore, bis(7)-tacrine concentration-dependently inhibited BACE-1 activity in cultured cells, and also in recombinant human BACE-1 in a non-competitive manner with an IC50 of 7.5μM, but did not directly affect activities of BACE-2, Cathepsin D, α- or γ-secretase. Taken together, our results not only suggest that bis(7)-tacrine may reduce the biosynthesis of Aβ mainly by directly inhibiting BACE-1 activity, but also provide new insights into the rational design of novel anti-Alzheimer’s dimers that might have disease-modifying properties.

Inhibition of amyloid β‐peptide production by blockage of β‐secretase cleavage site of amyloid precursor protein

Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.

Lactacystin decreases amyloid‐β peptide production by inhibiting β‐secretase activity

The human amyloid precursor protein (APP) is processed by the nonamyloidogenic and the amyloidogenic catabolic pathways. The sequential cleavage of APP by the beta- and gamma-secretase activities, known as the amyloidogenic processing of APP, leads to the formation of the amyloid-beta peptide (Abeta). Abeta is the main constituent of the amyloid core of senile plaques, a typical hallmark of Alzheimer's disease. In addition to secretases, other cellular proteolytic activities, like the proteasome, might participate in the metabolism of APP. We investigated the consequence of proteasome inhibition on the amyloidogenic processing of human APP. CHO cells and primary cultures of rat cortical neurons expressing human APP or a protein corresponding to its beta-cleaved C-terminal fragment (C99) were treated with lactacystin, an irreversible inhibitor of the chymotrypsin-like activity of the proteasome. Lactacystin significantly decreased the level of Abeta produced from APP in both cellular models, whereas the production of Abeta from C99 was not affected. Lactacystin did not inhibit gamma-secretase activity but was found to inhibit the beta-cleavage of APP, leading to a proportional decrease in Abeta production. Although lactacystin did not inhibit the catalytic activity of recombinant BACE1, a decrease in neuronal beta-secretase activity was measured after treatment with lactacystin.

Heparin Activates β-Secretase (BACE1) of Alzheimer's Disease and Increases Autocatalysis of the Enzyme

BACE1 is an aspartic protease that generates the N-terminus of the beta-amyloid protein (Alphabeta) from the beta-amyloid precursor protein (APP). BACE1 is a key target for Alzheimer drug development. However, little is known about the physiological regulation of the enzyme. Heparin can promote beta-secretase cleavage of APP in neuroblastoma cells. However, heparin has also been reported to directly inhibit BACE1 activity in vitro. To clarify the role of heparin in regulating BACE1, we examined the effect of heparin on the activity of recombinant human BACE1 (rBACE1) in vitro. Low concentrations (1 microg/mL) of heparin were found to stimulate rBACE1, increasing enzyme V(max) and decreasing the K(M). In contrast, higher concentrations of heparin (10 or 100 microg/mL) were inhibitory. Heparin affinity chromatography demonstrated that heparin interacted strongly with the zymogen form of rBACE1 and bound to a peptide homologous to the N-terminal pro sequence of BACE1. Mature (pro sequence cleaved) enzyme lacked the capacity to be stimulated by heparin, indicating that the pro domain was necessary for the stimulation by heparin. Furthermore, in the presence of stimulatory concentrations of heparin, there was an increase in autocatalytic cleavage of the protease domain and a subsequent loss of enzyme activity in vitro. Our results strongly suggest that heparin stimulates the partially active BACE1 zymogen, and we propose that the activation is mediated by high-affinity binding of heparin to the pro domain. Our study provides evidence that heparan sulfate proteoglycans could regulate the rate of Alphabeta production in vivo.

β Subunits of Voltage-gated Sodium Channels Are Novel Substrates of β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE1) and γ-Secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

Biochemical and kinetic characterization of BACE1: investigation into the putative species‐specificity for β‐ and β′‐cleavage sites by human and murine BACE1

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.

Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.