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Trypanosoma brucei:Generation of Specific Antisera to Recombinant Variant Surface Glycoproteins
Experimental Parasitology 91, 199–202 (1999) Article ID expr.1998.4369, available online at http://www.idealibrary.com on
RESEARCH BRIEF Trypanosoma brucei: Generation of Specific Antisera to Recombinant Variant Surface Glycoproteins
Maarten Hoek, Hui Xu, and George A. M. Cross1 Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, U.S.A.
Hoek, M., Xu, H., and Cross, G. A. M. 1999. Trypanosoma brucei: Generation of specific antisera to recombinant variant surface glycoproteins. Experimental Parasitology 91, 199–202. 䉷 1999 Academic Press Index Descriptors and Abbreviations: CRD, cross-reacting determinant; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; VSG, variant surface glycoprotein; VSG, VSG gene; rVSG, recombinant VSG.
unified approach to producing antibodies to any VSG. We describe an approach to the expression and purification of recombinant VSGs that lack carboxy-terminal peptide and glycosylphosphatidylinositol crossreacting determinants (CRD) (Barbet and McGuire 1978; Carrington and Boothroyd 1996; Carrington et al. 1991; Cross 1979; Zamze et al. 1988). We subcloned PCR-generated fragments representing the aminoterminal domains of VSG 221 (MITat 1.2) and VSG 117 (MITat 1.4) into the T7 polymerase-based bacterial expression vector pET15b (Novagen), which encodes an amino-terminal His6 tag (Fig. 1A). BL21(DE3) cells containing the recombinant plasmid were induced with IPTG and recombinant VSG (rVSG) was purified over a Ni2⫹ column. In both cases, rVSG seems to run at a slightly higher than expected molecular weight when separated on 10% SDS–PAGE gels (Fig. 1B). VSG 221 was moderately soluble, whereas VSG 117 was present almost exclusively in the insoluble fraction (data not shown). VSG 117 was purified from the bacterial pellet by treatment with 6 M urea and passing the supernatant over the Ni2⫹ resin. Fractions containing high levels of rVSG 117 were pooled and dialyzed against Hepes-buffered saline (100 mM NaCl, 20 mM Hepes, pH 7.0), producing soluble protein (Fig. 1C). Antiserum generated against purified rVSG 117 reacted specifically, on Western blots, with lysates from bacteria expressing rVSG 117 but not with lysates from bacteria expressing rVSG 221. Anti-rVSG 117 reacted strongly with VSG from T. brucei expressing VSG 117 (Fig. 2A). There was no cross-reaction with lysates from trypanosomes expressing either VSG 118 or VSG 221, but there was a small consistently seen cross-reaction with clones expressing VSG 121 (MITat 1. 6). Overall cross-reactivity was similar to that of anti-VSG 221 that had been CRD-depleted. Anti-rVSG 117 also reacted specifically with VSG 117 in lysates from T. brucei cell lines engineered to express VSG 117 in conjunction with VSG 221 (Munoz-Jordan et al. 1996) (data not shown).
To escape the immune response, the African trypanosomes rely upon the sequential expression of immunodominant variant surface glycoproteins (VSGs), which display remarkable primary sequence diversity. In our studies of vsg switching, we were frustrated by the lack of antisera to newly expressed VSGs. Raising specific antisera to novel VSGs is not a trivial task, even when VSGs from antigenically homogeneous populations of virulent trypanosomes can be purified in large quantities (Cross 1984). The problems increase when working with less virulent pleomorphic strains or with species other than Trypanosoma brucei. Purification of native VSGs involves cloning trypanosomes, monitoring population homogeneity, and several chromatographic steps (Cross 1984). Some T. brucei clones that we and others study switch rapidly, meaning that homogeneous populations cannot be maintained (Davies et al. 1997; Horn and Cross 1997). Rabbit antibodies raised against native VSGs also show strong cross-reactions (Barbet and McGuire 1978; Cross 1979). To generate specific antibodies, rabbit polyclonal antisera have to be depleted of cross-reacting antibodies. For all of these reasons, we needed to develop a simple To whom correspondence should be addressed. Fax: ⫹1 212 327 7845. E-mail; [email protected]. 1
0014-4894/99 $30.00 Copyright 䉷 1999 by Academic Press All rights of reproduction in any form reserved.
199
200
HOEK, XU, AND CROSS
FIG. 1. (A) Diagram of the two expression constructs used in this study. Fragments encoding the vsg sequence, excluding the conserved Cterminal domain and the signal sequence, were PCR-amplified with BamHI linkers introduced through the primers. The amplified fragment encoding rVSG 117 began at Ala34 and ended at Glu384: the fragment encoding rVSG 221 began at Gln35 and ended at Ser370. The sequences were amplified from plasmid pH30 (vsg 117) and pNEG (vsg 221), using the Expand Long Template PCR kit from Boehringer-Mannheim. The upstream and downstream primers for the amplification of vsg 117 were 5⬘-CGCGGATCCGGCCAAAGAAGCCCTTGAATA-3⬘ and 5⬘-TCGGATCCTACTCGCTTTCAGGGGATTTGC-3⬘, respectively, and the upstream and downstream primers used for the amplification of vsg 221 were 5⬘-TCGGGATCCGCAAGCTTTTTGGCAACCTCTT-3⬘ and 5⬘-CGCGGATCCTAAGCTGCCTTCTGTTTCTGCTGC-3⬘, respectively. The PCR products were digested with BamHI and inserted into similarly digested pET15b to generate in-frame fusions with the N-terminal His6 sequence encoded by the plasmid. All final constructs were sequenced. The calculated molecular weight of rVSG 117 is 43.8 kDa and of rVSG 221 is 42.6 kDa, although the apparent molecular weights on SDS–PAGE were somewhat higher: rVSG 117 generally migrated at 49 kDa and rVSG 221 migrated at approximately 45 kDa. (B) Coomassie and Western analyses of rVSG-expressing cell lines. BL21 cells expressing T7 polymerase from a lysogen
ANTISERA TO VSGs
201
FIG. 2. (A) Western blot of total cell lysates from 2 ⫻ 105 trypanosomes run on 10% SDS-PAGE gels. Antiserum to purified rVSG 117 was raised in rabbits using the standard protocol supplied by Covance Research Products which produced the antisera. Briefly, two New Zealand White rabbits were inoculated intradermally with 500 g of reconstituted rVSG 117 in Freund’s complete adjuvant, followed by three subcutaneous booster injections with 500 g of antigen and two boosts with 250 g of antigen, all in Freund’s incomplete adjuvant, at 21-day intervals. Blots were probed either with rabbit anti-rVSG 117 (antiserum 553) or with CRD-depleted rabbit anti-native VSG 221 at a 1:10,000 dilution. Blots were probed with secondary antibody and developed as in Fig. 1B. VSG 121 cell lysates were run in a separate experiment, using similar conditions. (B) Indirect immunofluorescence analysis of 117- and 221-expressing cell lines probed with either rat anti-native 221 or rabbit anti-rVSG 117 antibodies. Cells were spun down, fixed in 4% paraformaldehyde, resuspended in phosphate-buffered saline, 1% bovine serum albumin, and airdried for several hours. After rehydration in phosphate-buffered saline, cells were blocked with 1% bovine serum albumin and incubated with a 1:300 dilution of the respective antibody in the blocking solution. Cells were subsequently stained with a cocktail of rhodamine-conjugated goat anti-rat and fluorescein-conjugated goat anti-rabbit (Pierce) also at 1:300 in order to differentially identify VSG 221- or 117-expressing cells in the same field. Cells were observed with a Nikon Epifluorescence microscope using the 20X Fluor objective and the appropriate rhodamine or fluorescein filter sets. Images were captured with a Sony DKC5000 CCD camera at ISO 100 using a 0.5-s integration time and imported directly into Adobe Photoshop 4.0. (DE3) and containing the respective plasmid were grown overnight from a fresh streak, diluted 1:100 in fresh LB-carbenicillin, grown to an OD600 of 0.6, and induced for 2 h with 1 mM IPTG. Bacterial lysates were run on 10% SDS–PAGE gels and were processed for either Coomassie staining or Western blotting. Western blots were probed with CRD-depleted anti-native 221 (1:10,000) or anti-native 117 (1:2,000) and secondarily probed with HRP-conjugated anti-rabbit (Amersham) (1:5,000). Blots were developed using Pierce SuperSignal substrate according to the manufacturer’s directions. Arrows indicate the band containing rVSG in the Coomassie-stained gels. (C) Purification scheme for generating soluble rVSG117 and a Coomassie-stained 10% SDS–PAGE gel showing the purity of the final preparation.
202 We have consistently employed these antisera, at dilutions of 1:10,000, to visualize VSG on Western blots from lysates of 2 ⫻ 105 cells. Given an estimated value of 800 g of VSG per 109 cells (Cross 1975), we are easily able to detect approximately 160 ng of protein at this dilution. This approaches the sensitivity seen with the best currently available antisera against VSG 221. We have also used these antisera to detect VSG by indirect immunofluorescence analysis, using a dilution of 1:300 of the primary antibody with similar results (Fig. 2B). We anticipate that these antisera will react with native VSG, although we have not explicitly tested this. These results indicate that rVSG is an excellent immunogen and yields antisera that are highly reactive in the two most commonly used assays for VSG expression: immunofluorescence and Western blotting. (We thank Elizabeth Wirtz for helpful discussions and Claudia Ochatt for help with the control cell lines. This work was supported by the National Institutes of Health Grant AI 21729 and Predoctoral Training Grant GM 07982.)
REFERENCES
Barbet, A. F., and McGuire, T. C. 1978. Crossreacting determinants in variant-specific surface antigens of African trypanosomes. Proceedings of the National Academy of Sciences of the United States of America 75, 1989–1993. Carrington, M., and Boothroyd, J. 1996. Implications of conserved structural motifs in disparate trypanosome surface proteins. Molecular and Biochemical Parasitology 81, 119–126.
HOEK, XU, AND CROSS
Carrington, M., Miller, N., Blum, M., Roditi, I., Wiley, D., and Turner, M. J. 1991. Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. Journal of Molecular Biology 221, 823– 835. Cross, G. A. M. 1975. Identification, purification and properties of variant-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology 71, 393–417. Cross, G. A. M. 1979. Cross-reacting determinants in the C-terminal region of trypanosome variant surface antigens. Nature 277, 310– 312. Cross, G. A. M. 1984. Release and purification of Trypanosoma brucei variant surface glycoprotein. Journal of Cellular Biochemistry 24, 79–90. Davies, K. P., Carruthers, V. B. and Cross G. A. M., 1997. Manipulation of the vsg co-transposed region increases expression-site switching in Trypanosoma brucei. Molecular and Biochemical Parasitology 86, 163–177. Horn, D., and Cross, G. A. M. 1997. Position-dependent and promoterspecific regulation of gene expression in Trypanosoma brucei. EMBO Journal 16, 7422–7431. Munoz-Jordan, J. L., Davies, K. P., and Cross, G. A. M. 1996. Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei. Science 272, 1795–1797. Zamze, S. E., Ferguson, M. A. J., Collins, R., Dwek, R. A., and Rademacher, T. W. 1988. Characterization of the cross-reacting determinant (CRD) of the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein. European Journal of Biochemistry 176, 527–534. Received 9 June 1998; accepted with revision 29 September 1998
RESEARCH BRIEF Trypanosoma brucei: Generation of Specific Antisera to Recombinant Variant Surface Glycoproteins
Maarten Hoek, Hui Xu, and George A. M. Cross1 Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, U.S.A.
Hoek, M., Xu, H., and Cross, G. A. M. 1999. Trypanosoma brucei: Generation of specific antisera to recombinant variant surface glycoproteins. Experimental Parasitology 91, 199–202. 䉷 1999 Academic Press Index Descriptors and Abbreviations: CRD, cross-reacting determinant; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; VSG, variant surface glycoprotein; VSG, VSG gene; rVSG, recombinant VSG.
unified approach to producing antibodies to any VSG. We describe an approach to the expression and purification of recombinant VSGs that lack carboxy-terminal peptide and glycosylphosphatidylinositol crossreacting determinants (CRD) (Barbet and McGuire 1978; Carrington and Boothroyd 1996; Carrington et al. 1991; Cross 1979; Zamze et al. 1988). We subcloned PCR-generated fragments representing the aminoterminal domains of VSG 221 (MITat 1.2) and VSG 117 (MITat 1.4) into the T7 polymerase-based bacterial expression vector pET15b (Novagen), which encodes an amino-terminal His6 tag (Fig. 1A). BL21(DE3) cells containing the recombinant plasmid were induced with IPTG and recombinant VSG (rVSG) was purified over a Ni2⫹ column. In both cases, rVSG seems to run at a slightly higher than expected molecular weight when separated on 10% SDS–PAGE gels (Fig. 1B). VSG 221 was moderately soluble, whereas VSG 117 was present almost exclusively in the insoluble fraction (data not shown). VSG 117 was purified from the bacterial pellet by treatment with 6 M urea and passing the supernatant over the Ni2⫹ resin. Fractions containing high levels of rVSG 117 were pooled and dialyzed against Hepes-buffered saline (100 mM NaCl, 20 mM Hepes, pH 7.0), producing soluble protein (Fig. 1C). Antiserum generated against purified rVSG 117 reacted specifically, on Western blots, with lysates from bacteria expressing rVSG 117 but not with lysates from bacteria expressing rVSG 221. Anti-rVSG 117 reacted strongly with VSG from T. brucei expressing VSG 117 (Fig. 2A). There was no cross-reaction with lysates from trypanosomes expressing either VSG 118 or VSG 221, but there was a small consistently seen cross-reaction with clones expressing VSG 121 (MITat 1. 6). Overall cross-reactivity was similar to that of anti-VSG 221 that had been CRD-depleted. Anti-rVSG 117 also reacted specifically with VSG 117 in lysates from T. brucei cell lines engineered to express VSG 117 in conjunction with VSG 221 (Munoz-Jordan et al. 1996) (data not shown).
To escape the immune response, the African trypanosomes rely upon the sequential expression of immunodominant variant surface glycoproteins (VSGs), which display remarkable primary sequence diversity. In our studies of vsg switching, we were frustrated by the lack of antisera to newly expressed VSGs. Raising specific antisera to novel VSGs is not a trivial task, even when VSGs from antigenically homogeneous populations of virulent trypanosomes can be purified in large quantities (Cross 1984). The problems increase when working with less virulent pleomorphic strains or with species other than Trypanosoma brucei. Purification of native VSGs involves cloning trypanosomes, monitoring population homogeneity, and several chromatographic steps (Cross 1984). Some T. brucei clones that we and others study switch rapidly, meaning that homogeneous populations cannot be maintained (Davies et al. 1997; Horn and Cross 1997). Rabbit antibodies raised against native VSGs also show strong cross-reactions (Barbet and McGuire 1978; Cross 1979). To generate specific antibodies, rabbit polyclonal antisera have to be depleted of cross-reacting antibodies. For all of these reasons, we needed to develop a simple To whom correspondence should be addressed. Fax: ⫹1 212 327 7845. E-mail; [email protected]. 1
0014-4894/99 $30.00 Copyright 䉷 1999 by Academic Press All rights of reproduction in any form reserved.
199
200
HOEK, XU, AND CROSS
FIG. 1. (A) Diagram of the two expression constructs used in this study. Fragments encoding the vsg sequence, excluding the conserved Cterminal domain and the signal sequence, were PCR-amplified with BamHI linkers introduced through the primers. The amplified fragment encoding rVSG 117 began at Ala34 and ended at Glu384: the fragment encoding rVSG 221 began at Gln35 and ended at Ser370. The sequences were amplified from plasmid pH30 (vsg 117) and pNEG (vsg 221), using the Expand Long Template PCR kit from Boehringer-Mannheim. The upstream and downstream primers for the amplification of vsg 117 were 5⬘-CGCGGATCCGGCCAAAGAAGCCCTTGAATA-3⬘ and 5⬘-TCGGATCCTACTCGCTTTCAGGGGATTTGC-3⬘, respectively, and the upstream and downstream primers used for the amplification of vsg 221 were 5⬘-TCGGGATCCGCAAGCTTTTTGGCAACCTCTT-3⬘ and 5⬘-CGCGGATCCTAAGCTGCCTTCTGTTTCTGCTGC-3⬘, respectively. The PCR products were digested with BamHI and inserted into similarly digested pET15b to generate in-frame fusions with the N-terminal His6 sequence encoded by the plasmid. All final constructs were sequenced. The calculated molecular weight of rVSG 117 is 43.8 kDa and of rVSG 221 is 42.6 kDa, although the apparent molecular weights on SDS–PAGE were somewhat higher: rVSG 117 generally migrated at 49 kDa and rVSG 221 migrated at approximately 45 kDa. (B) Coomassie and Western analyses of rVSG-expressing cell lines. BL21 cells expressing T7 polymerase from a lysogen
ANTISERA TO VSGs
201
FIG. 2. (A) Western blot of total cell lysates from 2 ⫻ 105 trypanosomes run on 10% SDS-PAGE gels. Antiserum to purified rVSG 117 was raised in rabbits using the standard protocol supplied by Covance Research Products which produced the antisera. Briefly, two New Zealand White rabbits were inoculated intradermally with 500 g of reconstituted rVSG 117 in Freund’s complete adjuvant, followed by three subcutaneous booster injections with 500 g of antigen and two boosts with 250 g of antigen, all in Freund’s incomplete adjuvant, at 21-day intervals. Blots were probed either with rabbit anti-rVSG 117 (antiserum 553) or with CRD-depleted rabbit anti-native VSG 221 at a 1:10,000 dilution. Blots were probed with secondary antibody and developed as in Fig. 1B. VSG 121 cell lysates were run in a separate experiment, using similar conditions. (B) Indirect immunofluorescence analysis of 117- and 221-expressing cell lines probed with either rat anti-native 221 or rabbit anti-rVSG 117 antibodies. Cells were spun down, fixed in 4% paraformaldehyde, resuspended in phosphate-buffered saline, 1% bovine serum albumin, and airdried for several hours. After rehydration in phosphate-buffered saline, cells were blocked with 1% bovine serum albumin and incubated with a 1:300 dilution of the respective antibody in the blocking solution. Cells were subsequently stained with a cocktail of rhodamine-conjugated goat anti-rat and fluorescein-conjugated goat anti-rabbit (Pierce) also at 1:300 in order to differentially identify VSG 221- or 117-expressing cells in the same field. Cells were observed with a Nikon Epifluorescence microscope using the 20X Fluor objective and the appropriate rhodamine or fluorescein filter sets. Images were captured with a Sony DKC5000 CCD camera at ISO 100 using a 0.5-s integration time and imported directly into Adobe Photoshop 4.0. (DE3) and containing the respective plasmid were grown overnight from a fresh streak, diluted 1:100 in fresh LB-carbenicillin, grown to an OD600 of 0.6, and induced for 2 h with 1 mM IPTG. Bacterial lysates were run on 10% SDS–PAGE gels and were processed for either Coomassie staining or Western blotting. Western blots were probed with CRD-depleted anti-native 221 (1:10,000) or anti-native 117 (1:2,000) and secondarily probed with HRP-conjugated anti-rabbit (Amersham) (1:5,000). Blots were developed using Pierce SuperSignal substrate according to the manufacturer’s directions. Arrows indicate the band containing rVSG in the Coomassie-stained gels. (C) Purification scheme for generating soluble rVSG117 and a Coomassie-stained 10% SDS–PAGE gel showing the purity of the final preparation.
202 We have consistently employed these antisera, at dilutions of 1:10,000, to visualize VSG on Western blots from lysates of 2 ⫻ 105 cells. Given an estimated value of 800 g of VSG per 109 cells (Cross 1975), we are easily able to detect approximately 160 ng of protein at this dilution. This approaches the sensitivity seen with the best currently available antisera against VSG 221. We have also used these antisera to detect VSG by indirect immunofluorescence analysis, using a dilution of 1:300 of the primary antibody with similar results (Fig. 2B). We anticipate that these antisera will react with native VSG, although we have not explicitly tested this. These results indicate that rVSG is an excellent immunogen and yields antisera that are highly reactive in the two most commonly used assays for VSG expression: immunofluorescence and Western blotting. (We thank Elizabeth Wirtz for helpful discussions and Claudia Ochatt for help with the control cell lines. This work was supported by the National Institutes of Health Grant AI 21729 and Predoctoral Training Grant GM 07982.)
REFERENCES
Barbet, A. F., and McGuire, T. C. 1978. Crossreacting determinants in variant-specific surface antigens of African trypanosomes. Proceedings of the National Academy of Sciences of the United States of America 75, 1989–1993. Carrington, M., and Boothroyd, J. 1996. Implications of conserved structural motifs in disparate trypanosome surface proteins. Molecular and Biochemical Parasitology 81, 119–126.
HOEK, XU, AND CROSS
Carrington, M., Miller, N., Blum, M., Roditi, I., Wiley, D., and Turner, M. J. 1991. Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. Journal of Molecular Biology 221, 823– 835. Cross, G. A. M. 1975. Identification, purification and properties of variant-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology 71, 393–417. Cross, G. A. M. 1979. Cross-reacting determinants in the C-terminal region of trypanosome variant surface antigens. Nature 277, 310– 312. Cross, G. A. M. 1984. Release and purification of Trypanosoma brucei variant surface glycoprotein. Journal of Cellular Biochemistry 24, 79–90. Davies, K. P., Carruthers, V. B. and Cross G. A. M., 1997. Manipulation of the vsg co-transposed region increases expression-site switching in Trypanosoma brucei. Molecular and Biochemical Parasitology 86, 163–177. Horn, D., and Cross, G. A. M. 1997. Position-dependent and promoterspecific regulation of gene expression in Trypanosoma brucei. EMBO Journal 16, 7422–7431. Munoz-Jordan, J. L., Davies, K. P., and Cross, G. A. M. 1996. Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei. Science 272, 1795–1797. Zamze, S. E., Ferguson, M. A. J., Collins, R., Dwek, R. A., and Rademacher, T. W. 1988. Characterization of the cross-reacting determinant (CRD) of the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein. European Journal of Biochemistry 176, 527–534. Received 9 June 1998; accepted with revision 29 September 1998