Expression and Purification of a Recombinant ...

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Original Paper Intervirology 2008;51:189–195 DOI: 10.1159/000151532

Received: January 30, 2008 Accepted after revision: July 7, 2008 Published online: August 25, 2008

Expression and Purification of a Recombinant Adenovirus Fiber Knob in a Baculovirus System Luis E. Farinha-Arcieri a Bruna M. Porchia a Cassiano Carromeu a Fernando M. Simabuco a Rodrigo E. Tamura a Luis C.S. Ferreira a Luiz F. Zerbini b Armando M. Ventura a a b

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil; BIDMC, Harvard Institutes of Medicine, Boston, Mass., USA

Key Words Adenovirus ⴢ Fiber knob ⴢ Baculovirus ⴢ Ni-NTA purification ⴢ Sf 9 insect cells

in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright © 2008 S. Karger AG, Basel

© 2008 S. Karger AG, Basel 0300–5526/08/0513–0189$24.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/int

Introduction

Adenoviruses (Ads) are DNA viruses which infect vertebrates, from fish to humans. In humans 51 serotypes were characterized, which can cause respiratory diseases, conjunctivitis and infantile gastroenteritis [1]. Ads are nonenveloped, icosahedral particles of approximately 90 nm in diameter. Virions consist of a protein shell (capsid) that contains a linear, double-stranded DNA (dsDNA) genome. The capsid is composed of three different proteins: the hexon, forming most of the capsid surface; the penton, located at each vertex of the icosahedron, and the fiber, projecting from each vertex [1]. Ad fiber is a trimeric protein and it is organized in three well-defined regions: the N-terminal ‘tail’ that interacts with the penton base; a central shaft of variable length, and a globular C-terminal knob (fiber knob). The fiber knob binds the Armando Morais Ventura Departamento de Microbiologia, Instituto de Ciências Biomédicas Universidade de São Paulo, Avenida Professor Lineu Prestes 1374 São Paulo, SP 05508-900 (Brazil) Tel. +55 11 3091 7276, Fax +55 11 3091 7354, E-Mail [email protected]

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Abstract Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf 9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced

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Materials and Methods Cell Culture Spodoptera frugiperda 9 insect cells (Sf 9) were grown at 27 ° as monolayer cultures in Grace’s insect cell culture medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 100 ␮g/ml gentamicin (Cultilab) as described [14]. HEK293 cells were cultured at 37 ° in a 5% CO2 atmosphere in minimum essential medium (MEM; Cultilab) supplemented with 10% FBS and 100 ␮g/ml gentamicin (Cultilab). Cloning of Fiber Knob Gene and Baculovirus Construction Cloning of the fiber knob sequence was accomplished by PCR amplification using the pFibPac plasmid [15] as template and specific oligonucleotides designed for cloning the PCR products into a baculovirus transfer vector. The sequences of the oligonucleotides were GCA ATG GCC ATT ACA ATA GGA AAC (5ⴕ oligonucleotide) and TTC CTG GGC AAT GTA GGA GAA (3ⴕ oligonucleotide), which amplify the region corresponding to the fiber knob domain of the Ad2 fiber protein (nucleotides 1162–1746 of the fiber gene, corresponding to residues 388–582 of the Fiber protein). After PCR amplification, the products were cloned into the transfer vector pBluebac4.5/V5-His from the pBlueBac4.5/ V5-his TOPO-TA Expression Kit (Invitrogen). This expression vector contains the polyhedrin promoter and allows the expression of the gene products fused with a C-terminal V5 epitope and a histidine tag. Escherichia coli DH5␣ strain was used as the host strain. Recombinant plasmids were identified by restriction enzyme digestion. DNA sequencing was made using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and ABI PRISM쏐 3100 Genetic Analyzer (Applied Biosystems). For the production of the recombinant baculoviruses, Sf 9 cells were cultured in 25-cm2 flasks at 50% confluence and transfected with the recombinant plasmid together with the Bac-N-Blue DNA (Invitrogen) following manufacturer’s instructions. The Bac-NBlue DNA and the transfer vector possess homologous regions that allow their recombination in insect cells and the generation of recombinant baculoviruses expressing the cloned genes. After the transfection procedure, cells were incubated until 100% lysis was obtained. Culture supernatants were collected and recombinant baculoviruses were isolated by plaque assay and amplified for several rounds in Sf 9 cells. Viral DNA was extracted from culture media and analyzed by PCR with the oligonucleotides AAA TGA TAA CCA TCT CGC (5ⴕ oligonucleotide) and CAA CAA CGC ACA GAA TCT AGC (3ⴕ oligonucleotide). Positive clones were analyzed for protein expression in Sf 9 cells (see below). Viral titers were determined by plaque assay [14] and viral stocks were kept at 4 ° . Expression and Purification of the Fiber Knob In preliminary assays, Sf 9 cells cultured in a 6-well plate (1 ! 106 cells/well) were infected with the recombinant baculovirus at a multiplicity of infection (MOI) of 5 or greater, and protein expression was analyzed at 72, 96 and 120 h after infection. At the end of each period, cells were suspended in the culture supernatant and the samples were centrifuged at 9,500 g for 10 min. Culture supernatants were transferred to fresh tubes and stored for further analysis. Cells were suspended in 100 ␮l of lysis buffer (0.1% Triton X-100 in PBS supplemented with Protease Inhibitor

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primary receptor on host cells, the plasma membrane protein Coxsackie B virus and Ad receptor (CAR) [2, 3]. Recombinant Ad vectors are widely used for gene transfer studies in vitro and in vivo and in gene therapy experiments [4, 5]. Modification of fiber proteins is a promising strategy for overcoming the limitations imposed by the CAR dependence of Ad infection, which allows the Ad tropism to be expanded or changed [4]. The choice of the site where these modifications can be made was facilitated after the structure of the fiber knob domain was determined [6, 7]. It was shown that foreign peptides can be introduced into de HI loop or the C terminus of the fiber knob without altering the characteristics of the fiber protein [8]. Ad vectors have also been employed successfully as genetic vaccines, as they rapidly evoke immune responses against the transgene product [9–12]. The capsid proteins can also act as immune adjuvants. Immunizations with Ad vectors which have been modified to include antigenic epitopes in the capsid proteins can induce strong immune responses to the foreign epitope. In this regard, it has been shown that the insertion of foreign epitopes in the fiber knob is the best strategy to induce strong humoral and cellular anti-epitope responses [13]. The fiber knob has been expressed successfully in insect cells and purified by means of ion-exchange chromatography [3, 6]. Other authors [5] have shown that the fiber protein can incorporate a histidine tag (His-tag) at the C terminus which would allow the purification of this protein by means of Ni-NTA purification. The incorporation of the His-tag in the fiber did not alter the protein’s biological properties. In the present report, we describe the construction of a novel recombinant baculovirus expressing a modified form of the fiber knob domain of human Ad type 2 (Ad2). The recombinant protein was expressed fused with a 31-amino acid peptide, including the V5 epitope and a polyhistidine tag in the C terminus, the latter allowing purification by Ni-NTA chromatography. The recombinant protein was produced in baculovirusinfected Sf 9 cells recovered from culture supernatants and purified by a combination of Ni-NTA and ion-exchange chromatography. We show that despite the addition of 31 amino acids at the C terminus, the recombinant protein maintains its ability to trimerize in solution. Finally, the recombinant fiber knob reduces the ability of a recombinant human Ad type 5 to infect HEK293 cells, showing that the recombinant Ad2 protein produced competes for the binding of the Ad cellular receptor CAR.

Western Blot Samples containing the fiber knob protein were resolved by SDS-PAGE by the Laemmli method. Proteins were electrophoretically transferred to a Hybond-ECL Nitrocellulose membrane (Amersham Biosciences). The membrane was incubated with blocking buffer (0.1% Tween and 5% non-fat dry milk in phosphate-buffered saline, PBS) overnight at 4 ° and then with the anti-knob monoclonal antibody 1D5 (provided by Dr. Jadwiga Chroboczek, Institute de Biologie Structurale, Grenoble, France) [17], diluted 1:6,000 in blocking buffer for 90 min. After 3 washes with washing solution (Tween 0.1% in PBS), the membrane was

Recombinant Adenovirus Fiber Knob in a Baculovirus System

incubated with an anti-mouse peroxidase-conjugated antibody (KPL), diluted 1:1,000 in blocking buffer for 90 min. The membrane was washed again 3 times with washing solution, treated with SuperSignal West Pico Chemiluminescent Substrate (Pierce) for 5 min, and exposed to Hyperfilm (Amersham Biosciences) for 20 min. For V5 epitope detection, a commercial anti-V5 monoclonal antibody was used (Invitrogen). Inhibition of Adenovirus Infection HEK293 cells were cultured in a 24-well plate at approximately 80% confluence for 16 h. After this period, cells were infected with Ad-XPA-EGFP [18], a human Ad type 5 based vector, at a MOI of 5 in the presence of increasing amounts of the purified fiber knob protein (5, 100 and 200 ng of the purified protein) or with control bovine serum albumin (BSA). For this purpose, 6 ␮l of Ad were mixed with the corresponding proteins in 1.5-ml tubes containing 250 ␮l of culture medium (MEM supplemented with 2% FBS). After mixing, the culture medium from the cells was replaced with the Ad/protein solutions and the cells were incubated for 2 h at 37 ° . The virus-containing medium was replaced with fresh growth medium and 16 h after infection, the expression of the enhanced green fluorescent protein (EGFP) was visualized in a Zeiss fluorescence microscope.

Results and Discussion

Expression of the Recombinant Fiber Knob The knob domain of the Ad2 fiber protein (195 amino acids in length) was cloned into the pBlueBac vector in frame with a C-terminal fragment containing the V5 epitope and a histidine tag, which added 31 amino acids to the protein. The resulting vector was denominated pBBFK. The fiber knob was cloned under the control of the late polyhedrin promoter which induces high levels of protein expression. The recombinant baculovirus bearing the fiber knob gene was obtained by recombination of the pBB-FK vector with a baculovirus genome in Sf 9 cells. The presence of the fiber knob gene in the resulting baculovirus vector was determined by PCR analysis (data not shown). After obtaining the recombinant viruses, we performed a small-scale time-course analysis of the fiber knob expression in Sf 9 cells. Cells were infected with a MOI of 5 and harvested at 72, 96 and 120 h after infection. Intracellular proteins and culture supernatants were resolved in a 12% SDS-PAGE and analyzed by Western blot with a monoclonal antibody specific to the knob region of the fiber protein [17]. The fiber knob protein was detected intracellularly (data not shown) and in the culture supernatants throughout the period of study (fig. 1B) as a band of approximately 22 kDa. The recombinant protein from culture supernatants was easily visualized in Coomassie brilliant blue-stained gels (fig. 1 A). Intervirology 2008;51:189–195

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Cocktail Set III (Calbiochem) and 100 ␮g/ml PMSF). After incubation for 30 min at 4 ° the samples were centrifuged at 9,500 g for 10 min. The soluble fraction was transferred to a fresh tube. After treatment, all samples were stored at –20 ° . Culture supernatants, soluble and insoluble fractions were analyzed by SDS-PAGE and Western blot. For purification procedures, the fiber knob was produced in Sf 9 cells cultured in 75-cm2 flasks (5 ! 106 cells/flask). Culture supernatants were collected 120 h after infection. Proteins from the culture supernatants were precipitated by addition of ammonium sulfate at 100% saturation. After incubation at 4 ° for 30 min, the suspension was centrifuged at 6,000 g for 15 min and the protein pellet was resuspended in Ni-NTA buffer A (0.1 M TrisHCl, 0.1 M NaCl, 0.02 M imidazole, pH 8.5). The solution was dialyzed twice against 2 liters of Ni-NTA buffer A at room temperature. Protein purification was performed through a 3-step procedure. In the first step, the fiber knob protein was purified with a 1-ml HisTrapTM HP Nickel column (GE Healthcare) using an ÄKTA chromatography system. First, the column was equilibrated with Ni-NTA buffer A, and after protein binding, fiber knob was eluted with a linear gradient of Ni-NTA buffer B (0.1 M TrisHCl, 0.1 M NaCl, 1 M imidazole, pH 8.5). Fractions containing the fiber knob protein were pooled and dialyzed overnight against ion-exchange buffer A (50 mM Tris-HCl, 5 mM NaCl, 1 mM EDTA, pH 7.0). In the second purification step, the fiber knob protein was purified with a 1-ml HiTrapTM SP-XL column (GE Healthcare). The column was equilibrated with ion-exchange buffer A and afterwards the protein solution was injected. Fiber knob was eluted with a linear gradient of ion-exchange buffer B (50 m M Tris-HCl, 1 M NaCl, 1 mM EDTA, pH 7.0). Protein-containing fractions were pooled and dialyzed against ion-exchange buffer C (50 mM TrisHCl, 5 mM NaCl, 1 mM EDTA, pH 8.0). In the final purification step, the fiber knob protein was passed through a 1-ml HiTrapTM DEAE FF column (GE Healthcare) in order to eliminate contaminants. After column equilibration with ion-exchange buffer C, the protein solution was injected into the column. However, this time the column flow through was collected because at pH 8.0 the fiber knob did not bind to the DEAEsepharose resin. Purification fractions were analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue or analyzed by Western blot. For oligomerization assays, the samples were not boiled and a different sample buffer was used (sample buffer contained 0.1% SDS and no ␤-mercaptoethanol) [15, 16]. For His-tag detection, gels were stained with InVisionTM His-tag In-gel Stain (Invitrogen).

Fig. 1. Time-course expression of the re-

combinant fiber knob protein in Sf 9 cells analyzed by SDS-PAGE and Coomassie brilliant blue staining (A) or by Western Blot (B). Sf 9 cells were infected with recombinant baculovirus at a MOI of 5 and culture supernatants were recovered at 72 (lane 1), 96 (lane 2) and 120 h after infection (lane 3). Media from uninfected Sf 9 cells was used as negative control (lane 4). Recombinant fiber knob, indicated by an arrow, was detected throughout the period of study as a band of approximately 22 kDa.

1

2

3

4

1

A

2

3

4

B

1

2

M

M

3

4

48.8 kDa

Fig. 2. SDS-PAGE analysis of purification fractions. Fractions ob-

Table 1. Purification of fiber knob from culture medium of Sf9 cells (1.25 ! 108 cells)

Total proteina Fiber knob Purity b ␮g ␮g % Ni-NTA eluate SP-XL eluate DEAE-FF flow-through

500 250 170

366 202 166

73.2 80.9 96.5

a

Determined by Bradford assay. Estimated by band intensity in SDS-PAGE (fig. 2A, B) using the software ImageJ (National Institutes of Health). b

Purification of the Fiber Knob For purification, the fiber knob protein was produced in adherent cultures grown in 75-cm2 flasks. 120 h after infection, culture supernatants were collected and the 192

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25.9 kDa 19.4 kDa

proteins were precipitated with ammonium sulfate, in order to reduce the volume of the sample. This procedure was successful as no recombinant protein was detected in the supernatant (data not shown). After dialysis of the redissolved pellet against Ni-NTA buffer A, the protein was purified in a Ni-NTA column, with maximum elution peak at 0.2 M imidazole. Western blot analysis confirmed the identity of the purified protein (data not shown). No protein was detected in the flow-through fractions (fig. 2, lane 1). The analysis of the fractions containing the fiber knob revealed that other proteins coeluted with the recombinant protein (fig. 2, lane 2). At this point, we were able to recover approximately 366 ␮g of the fiber knob protein from 25 culture flasks of 75 cm2 (1.25 ! 108 cells). In order to remove the contaminant proteins, a twostep ion-exchange purification procedure was used. In the first step, after dialysis of the Ni-NTA purified proFarinha-Arcieri et al.

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tained during purification of the fiber knob from culture media were resolved by 12% SDS-PAGE and stained for protein with Coomassie brilliant blue. Lane 1 = Flow through from Ni-NTA purification; lane 2 = Ni-NTA Eluate; lane 3 = SP-XL column eluate; lane 4 = DEAE FF column flow through; lane M = Benchmark Pre-Stained Protein Ladder (Invitrogen). Some of the positions of the bands of the standard proteins are indicated. Fiber knob is indicated by an arrow on the right side of the figure.

D

Fig. 3. Analysis of fiber knob trimer for-

D

N

D

N

48.8 kDa

25.9 kDa A

tein with ion-exchange buffer A to remove imidazole and NaCl, the fiber knob was purified with a HiTrapTM SP-XL column. Maximum protein elution was obtained at 0.2 M NaCl (fig. 2, lane 3). In the second step, after dialysis of the protein-containing fractions with ion-exchange buffer A for removal of NaCl, the protein was passed through a HiTrapTM DEAE FF column for final removal of contaminants. At this point the protein was recovered from the column flow-through, since at pH 8.0 the protein has a slightly positive charge and should not bind the DEAE resin (fig. 2, lane 4). This strategy was successful as we obtained a purity of 96% of the fiber knob (fig. 2, lane 4; table 1). However, there was some protein loss in the process, since the fiber knob was detected in the column eluate among the contaminants (data not shown). At the end of the process we were able to obtain 170 ␮g of purified protein of Sf 9 cells seeded at 50% confluence (5 ! 106 cells per flask) from 25 flasks of 75 cm2. Functional Analysis of the Fiber Knob Ad fiber is known to be a trimeric protein [2] and the fiber knob by itself has been shown to trimerize [3]. To determine whether our purified fiber knob could trimerize as well, the recombinant protein was analyzed by SDS-PAGE under low reducing conditions. For this, protein samples were mixed with a sample buffer containing low concentrations of SDS (0.1%), no ␤-mercaptoethanol and they were not boiled. The analysis with an anti-fiber knob antibody showed a band of 22 kDa in the reduced and boiled samples (fig. 3A, sample D), and a band of approximately 50 kDa in the non-reduced and non-boiled sample (fig. 3A, sample N). These results are in agreeRecombinant Adenovirus Fiber Knob in a Baculovirus System

N

B

C

ment with those previously reported by Louis et al. [3]. The authors showed by cross-linking experiments that fiber knob dimers migrate with an apparent molecular weight of around 33 kDa, and that trimers migrate with an apparent molecular weight of around 50 kDa. The accessibility of the V5 epitope in the knob trimer was analyzed with a similar approach, using a V5-specific antibody. As depicted in figure 3B, the V5 antibody also detected the fiber knob as a 22-kDa band in reducing conditions (sample D), and as a 50-kDa band under low reducing conditions (sample N). The same pattern was observed when staining the purified proteins with a Histag specific stain (fig. 3C). To evaluate whether the purified protein retained the ability to bind CAR, an inhibition of Ad infection assay was employed. In this assay, we used the Ad Ad-XPAEGFP [18], a type 5 Ad that expresses EGFP. HEK293 cells were infected for 2 h with a MOI of 5 in the presence of increasing amounts of the fiber knob protein. BSA was used as control protein and 24 h after infection the expression of EGFP was visualized under a fluorescence microscope. As shown in figure 4, the expression of EGFP was substantially reduced in HEK293 cells treated with the recombinant protein. The reduction was more than 90% when comparing 200 ng fiber knobtreated cell sample (fig. 4A) with the BSA-treated cell sample (fig. 4D). The expression of EGFP was reduced even when 5 ng of fiber knob were used ( fig. 4C). This result clearly shows that the recombinant protein produced in insect cells competes with type 5 Ad for the binding of the cellular receptor CAR, indicating that it might also compete with other serotypes of the subgroup C Ads. Intervirology 2008;51:189–195

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mation. Denatured (D) and Native (N) samples of the fiber knob protein were analyzed by Western blot (A , B) or with a Histag specific Stain (C). A Trimerization of the recombinant fiber knob analyzed with an anti-fiber knob antibody [17]. B Fiber knob samples analyzed with a V5 specific antibody. C Fiber knob samples were stained with the InVisionTM His-tag In-gel Stain (Invitrogen). Sample D = Fiber knob treated with sample buffer containing ␤mercaptoethanol, 1% SDS and boiled; sample N = fiber knob treated with nonreducing sample buffer and not boiled.

Color version available online

A

B

C

D

Fig. 4. Functional analysis of the purified fiber knob. HEK293 cells were infected with 5 MOI of adeno-XPAEGFP in the presence of the purified fiber knob (A–C) or with control BSA (D). A–C Cells treated with 200, 100 and 5 ng of fiber knob, respectively. D Cells treated with 200 ng of BSA.

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Acknowledgments This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; proc. No. 2003/02041-4, 06/59976-3) and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (proc. No. 479814/2004-0). L.E.F.A., F.M.S., C.C. and R.E.T. have doctoral degree fellowships from FAPESP. We are grateful to Joselma Siqueira and Fernanda Yeda for their expertise in handling insect cell culture, and to Camila Calderón and Rafael Ciro Marques Cavalcante for help with the protein purifications. We thank Dr. Carlos Menck (São Paulo University, Brazil) for DNA sequencing and fluorescence microscopy support.

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In summary, we have produced a recombinant baculovirus that expresses a modified form of the fiber knob that includes the V5 epitope and a His-tag at the C terminus. The inclusion of these sequences allows the purification of this protein by methods other than ion-exchange chromatography, such as Ni-NTA chromatography, reaching high levels of purification. The recombinant fiber knob was recovered from the culture supernatants of infected Sf 9 cells and was purified by a combination of Ni-NTA chromatography and ion-exchange chromatography. The addition of the extra 31 amino acids at the C terminus of the protein did not alter the trimerization properties of the fiber knob or the binding to its cellular receptor CAR. We now intend to introduce other foreign epitopes, in substitution to V5, to evaluate the potential of using the Ad fiber knob as a protein adjuvant.

References

Recombinant Adenovirus Fiber Knob in a Baculovirus System

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