Biosci. Biotechnol. Biochem., 76 (4), 749–754, 2012
Attempts to Express the A1-GMCSF Immunotoxin in the Baculovirus Expression Vector System Ali JAHANIAN-N AJAFABADI,1 Saeid B OUZARI,1; y Mana O LOOMI,1; y Mehryar H ABIBI R OUDKENAR,2 and Lorenz M. M AYR3 1
Department of Molecular Biology, Pasteur Institute of Iran, Tehran 13164, Iran Research Center, Iranian Blood Transfusion Organization, Tehran 1157-14665, Iran 3 Novartis Institutes for BioMedical Research, NIBR/CPC/EPP, Fabrikstrasse-16.3.72, CH-4002 Basel, Switzerland 2
Received November 11, 2011; Accepted December 29, 2011; Online Publication, April 7, 2012 [doi:10.1271/bbb.110862]
Immunotoxins are fusion proteins consisting of two elements, a targeting and a toxin moiety, and are designed for specific elimination of tumor cells. Previously we expressed a recombinant fusion protein consisting of the toxic fragment of Shiga toxin (A1) and GMCSF (A1-GMCSF) in Escherichia coli, and evaluated its cytotoxic properties in acute myeloid leukemia and colon carcinoma cell lines. In view of the specific cytotoxic effects of this immunotoxin, further detailed in-vitro and preclinical studies were undertaken. Large amounts of the recombinant protein of high purity and free of unwanted side products, such as lipopolysaccharides (LPS), were required. Since GMCSF is of mammalian origin and it requires proper disulfide bond formation, we intended to use the baculovirus expression vector system (BEVS) for the expression of the recombinant fusion protein. However, despite previous reports on the expression of several other immunotoxins by this system, the A1 derived fusion proteins revealed an inhibitory effect on baculoviral particle formation and even caused cell death in insect cells. This observation was further pursued and confirmed by the use of other baculoviral specific promoters. The salient features of this finding are described below. Key words:
Shiga toxin; GMCSF; baculovirus; immunotoxin; pUltraBac1
The application of immunotoxins is one of the strategies for targeting cancer cells, especially in the case of drug resistance.1) Immunotoxins are fusion proteins composed of targeting and toxic moieties. The targeting fragment binds to a specific cell antigen, leading to internalization of the whole construct, allowing the toxin to migrate to the cytoplasm, where it executes its cytotoxic effects. The targeting moieties include monoclonal antibodies, antibody fragments, and growth factors.2) The common toxic moieties are plant or bacterial ribosome inactivating proteins such as ricin, abrin, diphtheria, and pseudomonas toxins.3) Another bacterial toxin that can be used for this purpose is Shiga toxin.4,5) One of the cell-surface y
receptors that can be used for targeting of immunotoxins to cancer cells is the receptor for granulocyte macrophage colony-stimulating factor (GM-CSF receptor, GMR). It has been found that expression of the GM-CSF receptor is increased on the surfaces of a majority of leukemic cells, while it is almost absent on the surfaces of the hematopoietic progenitors.6) In a previous study, we constructed recombinant immunotoxin A1-GMCSF by linking the coding sequence of GMCSF to the toxic fragment (the A1 subunit) of the Shiga toxin (StxA). In-vitro studies of this novel immunotoxin revealed specific cytotoxicity to GMRpositive hematologic cells, such as the HL-60 and U937 cell lines7) and also the colon tumor cell line LS174T.8) In view of these results, we decided to perform more detailed in-vivo and in-vitro pre-clinical studies, requiring large amounts of the recombinant fusion protein, purified to homogeneity and free of impurities such as lipopolysaccharides (LPS). Previous studies have found that the first 247 amino acids of the mature A1 protein has cytotoxic potency almost equal to the intact A1 fragment.9) Hence, the cloning and expression of the A247-GMCSF fusion protein was also included in the present study. The baculovirus expression vector system (BEVS) is widely used for high yield expression of heterogeneous recombinant proteins, especially those with eukaryotic sources.10,11) The BEVS has the advantage of performing most post-translational modifications such as glycosylation, phosphorylation, and acylation, over prokaryotic systems. LPS contamination, a special drawback related to prokaryotic expression systems such as E. coli, is not seen with the baculovirus expression vector system. In addition, among eukaryotic expression systems, insect cells are easier to work with, there are fewer infective agents that contaminate insect cells, and those cells do not produce any compound that is pathogenic or harmful to humans.12,13) Therefore, since recombinant human GMCSF14,15) and various recombinant immunotoxins had been successfully expressed in this system,16,17) we studied the expression of recombinant fusion proteins A254-GMCSF and A247-GMCSF by the baculovirus expression system.
To whom correspondence should be addressed. Saeid BOUZARI, Tel: +98-66953311 ext. 2223; Fax: +98-66492619; E-mail: saeidbouzari@ yahoo.com; Mana OLOOMI, Fax: +98-66492619; E-mail:
[email protected]
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A. JAHANIAN-NAJAFABADI et al. Table 1. Sequences of the Oligonucleotides Used in Amplification and Fusion of the Fragments Primer name ATFr A247Rv GM47Fr GMCSFRv GMFr
a
Primer featuresa
Primer sequence 0
0
5 -CGG GAT CCG ATG GAA TTT ACC TTA G-3 50 -CAGCGGGCGGGTGCTCGCGATGCATGATG-30 50 -CATGCATCGCGAGCACCCGCCCGCTGC-30 50 -CCGC TCG AGG GTC ACT CCT GGA CTG GCT CCC-30 50 -CGG GAT CCG ATG GCA CCC GCC-30
BamHI site Overlap sequence with the GMCSF Overlap sequence with the A247 XhoI site BamHI site
Primer features are represented in bold.
Materials and Methods Construction of coding sequences of A247-GMCSF and A254GMCSF fusion proteins. The coding sequences of the first 247 amino acids of mature Stx-A1 and the GMCSF fragments were obtained from our previous pBAD-A1-GMCSF construct7) by PCR and were fused through overlap PCR, as follows: First, the A247 fragment was amplified by ATFr and A247Rv primers and the GMCSF sequence was amplified using GM47Fr and GMCSFRv primers (Table 1). Following purification of the amplified fragments, a mixture of them was used as template for overlap PCR using the ATFr and GMCSFRv primers. All reactions were performed by Pfu DNA polymerase (Fermentas; Vilnius, Lithuania), and the PCR conditions included a primary denaturation time of 3 min at 94 C, followed by 10 cycles of 30 s at 94 C, 30 s at 45 C, and 1 min at 72 C, and then 20 cycles of 30 s at 55 C as annealing temperature and a final extension of 10 min at 72 C. Amplification of the A254-GMCSF fusion protein was also performed using the plasmid pBAD-A1-GMCSF as template. The reaction conditions were as described above, except that the annealing temperature was 55 C for all cycles and the ATFr and GMCSFRv primers were used for amplification of the fragments. The amplified fragments were cloned into the BamHI and XhoI restriction sites of the pFastBac HT-A (pFBH-A) transfer plasmid (Bac-to-Bac Baculovirus Expression System, Invitrogen, Carlsbad, CA). Finally, the fidelity of the cloned sequences was verified by DNA sequencing (Genfanavaran, Tehran, Iran). Baculovirus expression vector system. Recombinant baculoviral DNAs were generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen) following the manufacturer’s instructions. The pFastBac1-GUS plasmid, the positive control of the kit, was also included in the study. Insect cell culture and recombinant baculovirus production. Spodoptera frugiperda Sf9 cells (Invitrogen) were grown in supplemented Grace’s insect medium (Invitrogen) containing 10% (v/v) fetal bovine serum, 100 U of penicillin/mL, and 100 mg of streptomycin/ mL (Biosera, Tehran, Iran) at 27 C. Recombinant baculoviral constructs were used to transfect Sf9 cells using Cellfectin II transfection reagent (Invitrogen) following manufacturer’s instructions. Transfected cells were observed continuously for cytopathic effects (CPEs), and when occurred viral particles were harvested 8 d post transfection, and the viral stock was amplified through 3 consecutive rounds of Sf9 cell infection. Finally, the titer of the third generation of virus particles was determined by viral plaque assay as instructed by the manufacturer. Expression of recombinant proteins. Expression of the recombinant proteins was evaluated by infecting Sf9 cells with MOI (multiplicity of infection) of 5 and 10, and the transfected cells were harvested 48, 72, 96 h post infection. Finally, SDS–PAGE and Western blot analysis were performed on the lysed cells to detect recombinant protein expression. Polyclonal rabbit anti-human GMCSF antibody (Abcam, Cambridge, MA) and horse-radish peroxidase (HRP) conjugated polyclonal goat anti-rabbit antibody (Abcam) were used as primary and secondary antibodies in Western blot analysis.
Results Cloning of fragments and preparation of recombinant baculoviral DNA The amplified GMCSF (381 bp) and A247 (741 bp) (Figs. 1A, 2, and 3 respectively) and their fusion
A
B
Fig. 1. Evaluation of PCR Amplified Fragments. A, Amplification and fusion of fragments. Amplification of the GMCSF and A247 fragments revealed two bands, of about 381 and 741 bp respectively (A2 and A3). Fusion of these fragments resulted in a band of about 1,122 bp (A1). B, Amplification of the A254GMCSF fragment resulted in a band of about 1,143 bp (B1). Mw, molecular weight marker.
products (1,122 bp) (Fig. 1A, 1) as well as a band of about 1,143 bp corresponding to amplification of the A254-GMCSF fragment (Fig. 1B) were detected via gel electrophoresis. The authenticity of the fragments was verified by sequencing of the recombinant pFBH-A247GMCSF and pFBH-A254-GMCSF plasmids. Finally, the recombinant baculoviruses were constructed and evaluated via PCR using M13 forward and reverse primers which resulted in fragments of about 3,552 and 3,573 in the case of the A247-GMCSF and the pFBHA254-GMCSF bacmids respectively (Fig. 2). Transfection of Sf9 cells The confirmed recombinant pFastBac constructs were used to transfect the Sf9 cells, and the transfected cells were investigated for the occurrence of cytopathic effects indicative of baculovirus infection. Compared to the non-transfected control cells (Fig. 3B), the cells transfected with the pFastBac1-GUS derived bacmids showed typical CPE of nucleus enlargement 2 d post transfection (Fig. 3A) and cell detachment over the following days, but these effects were not observed when cells were transfected with the pFBH-A254GMCSFand pFBH-A247-GMCSF derived bacmids (Fig. 3C and D respectively) and the growth pattern of the cells was similar to that of the normal cells. The transfection experiment was repeated several times, but the outcome was the same. Our inability to obtain viral particles led us to theorize that A1 fusions are toxic for Sf9 cells, or prevent progression of the viral infection. To verify the accuracy of the experimental procedure, the 381-bp coding sequence of GMCSF, the non-toxic part of the fusion proteins, was amplified with primers GMFr and GMCSFRv (Table 1) and cloned in the
Attempts to Express the A1-GMCSF Immunotoxin
Fig. 2. PCR Analysis of Transposition Fidelity. PCR using the M13 primers revealed bands of about (1) 4,300 bp in the case of GUS; (2) 3,573 in the case of A254-GMCSF and (3) 3,552 bp in the case of A247-GMCSF confirming correct transposition of the fragments of the corresponding pFastBac plasmids into the recombinant bacmid DNAs. Mw, molecular weight marker.
Fig. 3. Appearance of CPEs Following Transfection of Sf9 Cells by the Recombinant Bacmid DNA. Enlargement of the nucleus and growth cessation was observed in the cells transfected with the pFastBac1-GUS and pFBH-GMCSF derived bacmids (A and E respectively) compared to the controls (B) from 2 d post-transfection on. However, the Sf9 cells transfected with the pFBH-A254-GMCSF and pFBH-A247-GMCSF derived bacmids did not show any CPEs (C and D respectively).
pFastBacHT-A plasmid. Preparation of recombinant bacmid DNA and transfection of Sf9 cells were performed as previously described for the fusion constructs. Similarly to the results obtained for pFastBac1GUS derived bacmids, cytopathic effects were also observed in the cells transfected with the recombinant baculoviruses harboring the GMCSF fragment (Fig. 3E). Expression of GMCSF was detected in the transfected cells after consecutive amplification of the harvested GMCSF-baculoviruses and final infection of the Sf9 cells at MOI of 5 and 10 (Fig. 4). These results confirmed the accuracy of the experimental procedures. Afterwards, in order to verify our hypothesis concerning inhibition of baculoviral particle formation or the toxicity of A1 derived proteins to the Sf9 cells, we received the plasmid pUltraBac1 as a gift from Novartis Institutes for BioMedical Research (NIBR; Basel, Switzerland). This plasmid carries basic and polyhedrin promoters, which are late and very late promoters respectively (Fig. 5). The Basic promoter is activated earlier (about 6 h post-infection) than the polyhedrin promoter (PPH ), which is activated at about 20 h post-
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Fig. 4. Western Blot Analysis of Expression of GMCSF. Sf9 cells were infected with different MOI of 5 and 10, and were harvested after 72 h. GMCSF expression was confirmed by antihuman GMCSF antibody, revealing three bands of about 18 to 20 kDs (2 and 3 respectively), which may have contributed to different glycosylated forms of the expressed GMCSF. Lane 1 represents cells transfected with empty baculovirus lacking any cloned gene.
Fig. 5. Map of the pUltraBac1 Plasmid. In this construct, the EGFP reporter gene is expressed under the control of a late promoter, pBasic, while the gene of interest was expressed under the control of the strong baculoviral polyhedrin promoter.
infection.18) In the pUltraBac1 plasmid, the coding sequence for EGFP (enhanced green fluorescence protein) was cloned downstream of the basic promoter, and hence expression of the EGFP protein occurred prior to that of the gene cloned downstream of the PPH promoter. To verify our hypothesis, the coding sequences of the A247-GMCSF, A254-GMCSF, and GMCSF proteins were cloned in the pUltraBac1 plasmid. All the cloning and recombinant virus production steps were carried out as described above. Following transfection of Sf9 cells with the pUltraBac1 derived bacmids (pUB-A247-GMCSF, pUB-A254-GMCSF, and pUB-GMCSF), expression of EGFP was investigated by florescence microscopy. Twenty-four to 30 h post-transfection, the cells transfected with the pUB-GMCSF bacmid showed an intensive green fluorescence signal (Fig. 6A), whereas the pUB-A254-GMCSF or pUB1A247-GMCSF bacmid transfected cells were faintly fluorescent (Fig. 6B and C). The cells were observed continuously at 24-h intervals, and on the 5th day posttransfection, almost all the cells transfected with the pUB-GMCSF bacmid showed expression of the EGFP protein (Fig. 6D), a consequence of the infection of nontransfected cells by the released baculoviral particles. In this case, the expression of EGFP was concurrent with
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A. JAHANIAN-NAJAFABADI et al.
Fig. 6. Evaluation of EGFP Expression by the Sf9 Cells Transfected with Various Recombinant Bacmids Derived from Various pUltraBac1 Constructs. A, B, and C, Twenty-four to 30 h after transfection of Sf9 cells with the pUB-GMCSF, pUB-A254-GMCSF, and pUB-A247-GMCSF bacmid respectively (image C captured at high ISO to better detect the fluorescence signal). D, E, and F, Five d after transfection of Sf9 cells with the pUB-GMCSF, pUB-A254-GMCSF and pUB-A247-GMCSF bacmid respectively. As shown, release of baculoviral particles from the cells transfected with the pUB-GMCSF bacmids resulted in the expression of EGFP by adjacent cells. However, this phenomenon was not observed in the case of the cells transfected with the pUB-A254-GMCSF and pUB-A247-GMCSF bacmid.
the occurrence of cytopathic effects in the Sf9 cells. However, in case of the cells transfected with the pUBA254-GMCSF and the pUB-A247-GMCSF bacmids, no green fluorescence signals was detected (Fig. 6E and F, respectively).
Discussion We have reported the expression of recombinant immunotoxin A1-GMCSF.7) This fusion protein, which harbors first 254 amino acids of the toxic subunit of Shiga toxin (Stx-A1), and the human GMCSF fragment showed specific cytotoxic effects against tumor cell lines that carry large numbers of GMCSF receptors, such as the HL-60 and LS174T cell lines.7,8) Previous studies to identify the minimum active domains of the Stx-A1 subunit indicate that a fragment lacking seven amino acids of the C-terminus produce cytotoxic effects very similar to the intact A1 fragment.9) Therefore, considering the smaller size but full toxicity of this fragment, which promises less complicated immunological reactions or manipulation procedures, we also included
expression of the A247-GMCSF fusion protein in the present study. However, for further evaluation of the specific and non-specific cytotoxic effects of these proteins, and to perform pre-clinical and animal studies, huge amounts of the proteins are required. Considering previous studies of the expression of recombinant human GMCSF by the baculovirus expression vector system (BEVS),14,15) and also the successful expression of the recombinant diphtheria toxin (DT)-GMCSF immunotoxin by this system, we selected the baculovirus expression system for the production of large amounts of the recombinant A254-GMCSF and A247GMCSF immunotoxins. However, despite a report on the expression of recombinant DT-GMCSF immunotoxin by the BEVS, the recombinant A1 derived fusion proteins could not be expressed by this system. This might have been due to the difference in the mechanisms of action of the two bacterial toxin conjugates, since the diphtheria toxin acts on eukaryotic elongation factor 2,3) whereas the Shiga toxin directly inhibits the formation of eukaryotic ribosomal assembly by removing the glycoside group of A4324 on the 28s rRNA.19) However,
Attempts to Express the A1-GMCSF Immunotoxin
the susceptibility of insect cells to the toxicity induced by diphtheria toxin has been confirmed by Valdizan et al.20) and Dai et al.21) The first group showed that ectopic expression of diphtheria toxin receptor on the surface of Sf9 insect cells followed by exposure of them to intact diphtheria toxin resulted in cell death, and the second group found that cytosolic expression of recombinant diphtheria toxin in insect cells resulted in cell death. But the ability to express DT-GMCSF immunotoxin in insect cells might be due to the difference in the molecular structures of the DT-GMCSF and A1GMCSF fusion proteins. The DT-GMCSF fusion protein contains the diphtheria toxin translocation domain as well as its catalytic domain, which might inhibit the cytotoxic activity of the protein before the dissociation of these two domains, whereas in the A1-GMCSF the toxic fragment is directly fused to the GMCSF, which not suppress its cytotoxic effect. Therefore, in the final phases of viral infection, which is concurrent with activation of the viral very late polyhedrin promoter, the accumulated fusion protein might inhibit viral particle formation or result in cell death. In this case, those cells which are not transfected continue their natural growth cycles and fill the cell culture well. In order to evaluate this hypothesis, and to verify whether any transfection occurred, a baculovirus construct expressing a reporter gene much earlier than the gene of interest can be used. In this regard, the reporter gene should be expressed under the control of an early or late promoter, one activated much earlier than the polyhedrin promoter, which is a very late promoter. Philipps et al.22) developed a baculovirus transfer vector (pUltraBac1) that contains the EGFP reporter gene driven by the late promoter sequence (pBasic) and the very late polyhedrin promoter, downstream of which the gene of interest can be cloned. In this case, expression of EGFP starts at about 6 h post-infection while expression of the gene of interest starts at about 20 h post-infection.18) Hence, we cloned the toxic fusion proteins downstream of the polyhedrin promoter and evaluated the expression of EGFP by fluorescence microscopy. The cells transfected with bacmids containing the A254-GMCSF and A247GMCSF coding sequences revealed faint green fluorescence signals (Fig. 6) that were not distributed to other cells over time, as was observed in the case of intensive signals by the cells transfected with bacmids containing the GMCSF sequence. This experiment not only confirmed that transfection occurred, but also showed the inhibitory effects of A1 derived fragments on the formation of baculoviral particles. Recently, it was found that cytosolic expression of the diphteria toxin A (DT-A) fragment resulted in tumor cell death.23,24) Even cytosolic expression of the DT-A fragment mediated by a baculovirus gene therapy vector inhibited the proliferation of malignant glioma cells.25) Furthermore, the A1 fragment has been found to exert antiviral effects on various ruminant viruses such as bovine leukemia virus (BLV), bovine retroviruses, and bovine immunodeficiency virus (BIV),26–29) similarly to its inhibitory effects on baculoviruses, observed in the present study. Therefore, considering the strong cytotoxic and antiviral effects of the A1 fragment when it is expressed cytosolically, it appears that this bacterial toxin can be used in gene and antiviral therapy applications.
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In this study, we expressed human GMCSF using a baculovirus expression system. The hGMCSF protein contains 127 amino acids and has a molecular weight of about 14.5 to 32 kD, depending on the degree of glycosylation.30) It has shown different molecular weights when expressed by various invertebrate expression systems. Chen et al.30) expressed hGMCSF by silkworm pupae. This resulted in the production of a 29-kD protein. Expression of hGMCSF by the baculovirus expression system and Sf9 cells revealed three bands, of about 14.5, 15.5, and 16.5 kDs, in which the two heavier molecules were found to be glycosylated.15) Here we detected three major bands of about 17.5, 18.5, and 19.5 kDs, by Western blotting using anti-human GMCSF antibody. Considering that 3 kDs were added by upstream elements of the pFastBac HT A vector, the observed bands might represent the unglycosylated and the two glycosylated forms of the expressed hGMCSF. In conclusion, we found that cytosolic expression of the A1 derived fusion proteins resulted in inhibition of baculoviral particle formation, or even cell death. It appears that this strong toxic effect of the Stx-A1 fragment on eukaryotic cells, when expressed in the cytosol, might be useful in gene therapy applications. Moreover, from this study it can be concluded that the baculovirus insect cell expression system is not a suitable vehicle for expression of A1-derived fusion proteins. However, using a non-viral secretory insect cell expression system that encodes fusion proteins under an early promoter, one is activated independently of viral infection, and most importantly, which is weaker than very late promoters such as PPH , can be an effective strategy for successful expression of A1-GMCSF.
Acknowledgment We would like to express our gratitude to the Novartis Institutes for BioMedical Research (NIBR; Basel, Switzerland) for the generous gift of plasmid pUltraBac1, and for their help with and advice as to the system. We are also grateful to Professor George Rohrmann (Center for Genome Research and Biocomputing, Oregon State University) and Professor David Theilmann (Pacific Agri-Food Research Centre, Summerland, B.C., Canada) for invaluable comments. This work was supported financially by the Pasteur Institute of Iran.
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