Expression of parasite antigens in Escherichia coli

P. McIntyre, R. L. Coppel, D. B. Smith, H. D. Stahl, L. M. Corcoran, C. J. Langford, J. M. Favaloro, P. E. Crewther, G. V. Brown, G. F. Mitchell, R. F. Anders, D. J. Kemp

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Abstract

Immunological detection of cloned parasite antigens in E. coli offers an efficient means of identifying parasite antigens which may be relevant to immunity. Moreover, this technique enables the sequence of an antigen to be determined and also enables purification of the recombinant protein from bacterial lysates. The variety of different vectors available means that specific vectors are available for most purposes. Useful vectors are available which have not yet been employed to study parasites. Lambda phage has been extensively used while other phage vector systems have not, even though they could offer advantages over existing systems. Cloning in the M13 vector for example, allows direct sequencing of cloned parasite genes or gene fragments from expressing clones (Messing, 1983). A series of M13 vectors has been constructed with a Hind 111 cloning site in all three reading frames specifically for this purpose (Lathe et al., 1984). Some plasmid vectors which could be used rely on expression of hybrid fusion proteins. The pUR vectors 278, 279, 289, 290, 291 and 292 (Ruther & Muller-Hill, 1983) have a full length lac Z gene with synthetic polylinkers at the 5' end in all three phases so that a hybrid β-1 fusion protein can be produced under the control of the lac UV5 promotor. The pEX 1-3 vectors (Stanley et al., 1984) have lambda Pl promotor and N-terminus linked to β-galactosidase and contain a 5' synthetic polylinker in three translational phases. This vector was designed for the cloning of short regions of foreign DNA which can be probed with antibodies to define an epitope. This approach to dissecting antigens has great potential for the analysis of complex antigens like those of P. falciparum The interest in expressing foreign genes in bacteria ensures that host-vector systems will continue to improve. Although prokaryotic expression systems have obviously worked successfully for the expression of many foreign genes, problems still exist where carbohydrate addition or other eukaryotic post-translational modifications are important in the generation of epitopes. One approach to solving these problems associated with heterologous expression of proteins, is to use eukaryotic host-vector systems such as yeast or vaccinia virus infected mammalian cells (Mackett et al., 1984). The vaccinia virus system has been used to express the P. knowlesi sporozoite antigen (Smith et al., 1984) and a hybrid form of the normally secreted P. falciparum S antigen has been expressed on the surface of vaccinia virus infected cells, anchored in the cell membrane (Langford et al., 1986). These live, infectious viruses can be used as live vaccines. Special problems exist with these systems and it is still not possible to generate the large libraries possible using E. coil. Consequently, it is not possible to routinely detect rare sequences in eukaryotic libraries as it is in E. coli. The ability to generate large amounts of parasite fusion proteins will allow production of reagents for detecting infections. Furthermore, it should soon be possible to study parasitic diseases that hitherto could not be studied and to isolate defined elements that can be used to vaccinate against parasitic disease.

Original languageEnglish
Pages (from-to)59-67
Number of pages9
JournalInternational Journal for Parasitology
Volume17
Issue number1
DOIs
Publication statusPublished - 1 Jan 1987
Externally publishedYes

Cite this

McIntyre, P., Coppel, R. L., Smith, D. B., Stahl, H. D., Corcoran, L. M., Langford, C. J., ... Kemp, D. J. (1987). Expression of parasite antigens in Escherichia coli. International Journal for Parasitology, 17(1), 59-67. https://doi.org/10.1016/0020-7519(87)90027-0
McIntyre, P. ; Coppel, R. L. ; Smith, D. B. ; Stahl, H. D. ; Corcoran, L. M. ; Langford, C. J. ; Favaloro, J. M. ; Crewther, P. E. ; Brown, G. V. ; Mitchell, G. F. ; Anders, R. F. ; Kemp, D. J. / Expression of parasite antigens in Escherichia coli. In: International Journal for Parasitology. 1987 ; Vol. 17, No. 1. pp. 59-67.
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abstract = "Immunological detection of cloned parasite antigens in E. coli offers an efficient means of identifying parasite antigens which may be relevant to immunity. Moreover, this technique enables the sequence of an antigen to be determined and also enables purification of the recombinant protein from bacterial lysates. The variety of different vectors available means that specific vectors are available for most purposes. Useful vectors are available which have not yet been employed to study parasites. Lambda phage has been extensively used while other phage vector systems have not, even though they could offer advantages over existing systems. Cloning in the M13 vector for example, allows direct sequencing of cloned parasite genes or gene fragments from expressing clones (Messing, 1983). A series of M13 vectors has been constructed with a Hind 111 cloning site in all three reading frames specifically for this purpose (Lathe et al., 1984). Some plasmid vectors which could be used rely on expression of hybrid fusion proteins. The pUR vectors 278, 279, 289, 290, 291 and 292 (Ruther & Muller-Hill, 1983) have a full length lac Z gene with synthetic polylinkers at the 5' end in all three phases so that a hybrid β-1 fusion protein can be produced under the control of the lac UV5 promotor. The pEX 1-3 vectors (Stanley et al., 1984) have lambda Pl promotor and N-terminus linked to β-galactosidase and contain a 5' synthetic polylinker in three translational phases. This vector was designed for the cloning of short regions of foreign DNA which can be probed with antibodies to define an epitope. This approach to dissecting antigens has great potential for the analysis of complex antigens like those of P. falciparum The interest in expressing foreign genes in bacteria ensures that host-vector systems will continue to improve. Although prokaryotic expression systems have obviously worked successfully for the expression of many foreign genes, problems still exist where carbohydrate addition or other eukaryotic post-translational modifications are important in the generation of epitopes. One approach to solving these problems associated with heterologous expression of proteins, is to use eukaryotic host-vector systems such as yeast or vaccinia virus infected mammalian cells (Mackett et al., 1984). The vaccinia virus system has been used to express the P. knowlesi sporozoite antigen (Smith et al., 1984) and a hybrid form of the normally secreted P. falciparum S antigen has been expressed on the surface of vaccinia virus infected cells, anchored in the cell membrane (Langford et al., 1986). These live, infectious viruses can be used as live vaccines. Special problems exist with these systems and it is still not possible to generate the large libraries possible using E. coil. Consequently, it is not possible to routinely detect rare sequences in eukaryotic libraries as it is in E. coli. The ability to generate large amounts of parasite fusion proteins will allow production of reagents for detecting infections. Furthermore, it should soon be possible to study parasitic diseases that hitherto could not be studied and to isolate defined elements that can be used to vaccinate against parasitic disease.",
author = "P. McIntyre and Coppel, {R. L.} and Smith, {D. B.} and Stahl, {H. D.} and Corcoran, {L. M.} and Langford, {C. J.} and Favaloro, {J. M.} and Crewther, {P. E.} and Brown, {G. V.} and Mitchell, {G. F.} and Anders, {R. F.} and Kemp, {D. J.}",
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McIntyre, P, Coppel, RL, Smith, DB, Stahl, HD, Corcoran, LM, Langford, CJ, Favaloro, JM, Crewther, PE, Brown, GV, Mitchell, GF, Anders, RF & Kemp, DJ 1987, 'Expression of parasite antigens in Escherichia coli', International Journal for Parasitology, vol. 17, no. 1, pp. 59-67. https://doi.org/10.1016/0020-7519(87)90027-0

Expression of parasite antigens in Escherichia coli. / McIntyre, P.; Coppel, R. L.; Smith, D. B.; Stahl, H. D.; Corcoran, L. M.; Langford, C. J.; Favaloro, J. M.; Crewther, P. E.; Brown, G. V.; Mitchell, G. F.; Anders, R. F.; Kemp, D. J.

In: International Journal for Parasitology, Vol. 17, No. 1, 01.01.1987, p. 59-67.

Research output: Contribution to journalArticleResearchpeer-review

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N2 - Immunological detection of cloned parasite antigens in E. coli offers an efficient means of identifying parasite antigens which may be relevant to immunity. Moreover, this technique enables the sequence of an antigen to be determined and also enables purification of the recombinant protein from bacterial lysates. The variety of different vectors available means that specific vectors are available for most purposes. Useful vectors are available which have not yet been employed to study parasites. Lambda phage has been extensively used while other phage vector systems have not, even though they could offer advantages over existing systems. Cloning in the M13 vector for example, allows direct sequencing of cloned parasite genes or gene fragments from expressing clones (Messing, 1983). A series of M13 vectors has been constructed with a Hind 111 cloning site in all three reading frames specifically for this purpose (Lathe et al., 1984). Some plasmid vectors which could be used rely on expression of hybrid fusion proteins. The pUR vectors 278, 279, 289, 290, 291 and 292 (Ruther & Muller-Hill, 1983) have a full length lac Z gene with synthetic polylinkers at the 5' end in all three phases so that a hybrid β-1 fusion protein can be produced under the control of the lac UV5 promotor. The pEX 1-3 vectors (Stanley et al., 1984) have lambda Pl promotor and N-terminus linked to β-galactosidase and contain a 5' synthetic polylinker in three translational phases. This vector was designed for the cloning of short regions of foreign DNA which can be probed with antibodies to define an epitope. This approach to dissecting antigens has great potential for the analysis of complex antigens like those of P. falciparum The interest in expressing foreign genes in bacteria ensures that host-vector systems will continue to improve. Although prokaryotic expression systems have obviously worked successfully for the expression of many foreign genes, problems still exist where carbohydrate addition or other eukaryotic post-translational modifications are important in the generation of epitopes. One approach to solving these problems associated with heterologous expression of proteins, is to use eukaryotic host-vector systems such as yeast or vaccinia virus infected mammalian cells (Mackett et al., 1984). The vaccinia virus system has been used to express the P. knowlesi sporozoite antigen (Smith et al., 1984) and a hybrid form of the normally secreted P. falciparum S antigen has been expressed on the surface of vaccinia virus infected cells, anchored in the cell membrane (Langford et al., 1986). These live, infectious viruses can be used as live vaccines. Special problems exist with these systems and it is still not possible to generate the large libraries possible using E. coil. Consequently, it is not possible to routinely detect rare sequences in eukaryotic libraries as it is in E. coli. The ability to generate large amounts of parasite fusion proteins will allow production of reagents for detecting infections. Furthermore, it should soon be possible to study parasitic diseases that hitherto could not be studied and to isolate defined elements that can be used to vaccinate against parasitic disease.

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