Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1995-01-03
1997-12-16
Feisee, Lila
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 693, 4353201, 4241861, 4241931, 4241941, 536 2372, C12N 1511, A61K 3576
Patent
active
056984243
DESCRIPTION:
BRIEF SUMMARY
This application claims priority under 35 USC 371 from International Application PCT/GB92/01159, filed Jun. 26, 1992 and priority under 35 USC 119 from United Kingdom Application 9114003.8, filed Jun. 28, 1991.
This invention relates to a chimaeric protein and particularly directed to virus proteins containing foreign inserts, the preparation of such proteins and their use, for example as vaccines or as carriers for presenting molecular species such as targeting moieties.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is illustrated by the accompanying drawings, in which:
FIG. 1 is an SDS-PAGE tracing of MS2 capsid proteins;
FIG. 2 is an electromicrograph of cys-14 modified capsids assembled into particles;
FIG. 3 is a graph used to determine the extent of reaction of free cysteine with Ellman's reagant; and
FIG. 4 is a graph showing the reaction of cysteine and activated galactose.
Epitopes inserted insertion in an identified class of virus protein carriers can be specifically directed so that foreign epitopes are reliably presented on the surface of the protein capsid assembly after the expression of the chimaeric protein in a bacterial host.
It was by no means predictable that such a result could be achieved. Not all viruses possess coat proteins which will self assemble: furthermore it is not possible to predict such self assembly. Even further, unlike animal viruses, bacterial viruses cannot naturally be expected to possess immunodominant regions and there is thus no guidance as to which region of a bacterial virus coat protein (if any) could be modified in the reasonable expectation of inducing an immune response.
One example of a chimeric protein is capable of forming part of a capsid assembly and comprises the amino acid sequence of the coat protein of phage MS-2, or a conservatively modified variant thereof, or sufficient of said sequence or variant to retain capsid-forming capability, which amino acid sequence has been modified by insertion of a foreign epitope in the region of the protein corresponding to an N-terminal .beta.-hairpin, as determined by X-ray crystallography of the whole phage particle.
Surprisingly, it was found that such a chimaeric protein can be expressed in a suitable bacterial host to yield capsids empty of the phage RNA and largely free of other nucleic acid contaminants.
While it is contemplated that the Insertion of a foreign epitope which is a linear polypeptide of comparatively short length, for example up to about 30 amino acid residues, can be accomplished, it will be appreciated that the presentation of non-linear epitopes by simple insertion in the .beta.-hairpin may not be feasible. Thus many epitopes do not consist of simple linear fragments of polypeptide: instead they are made up of several such fragments spatially related by the overall three-dimensional folding of the native protein antigen, i.e. discontinuous epitopes. It is clearly desirable to be able to present such epitopes at the surface of the phage capsid.
It has now been found that the coat protein of MS-2 can be employed with a view to presenting a range of epitopes or other molecular species such as targeting moieties at the capsid surface by removing the cysteine residues which occur at sites not included within the N-terminal .beta.-hairpin and introducing a cysteine residue within the .beta.-hairpin region, which cysteine residue is then available for linking with desired antigens or other molecular species, suitably via an appropriate spacer.
Therefore in accordance with the present invention there is provided a chimaeric protein capable of forming part of a capsid assembly and comprising the amino acid sequence of the coat protein of phage MS-2, or a conservatively modified variant thereof, or sufficient of said sequence or variant to retain the capability of forming a capsid assembly, which amino acid sequence has been modified by removal of the cysteine residues present externally of the N-terminal protruberant .beta.-hairpin of the coat protein and insertion of a cysteine residue
REFERENCES:
Fieb et al. Nature 1976 vol. 260:500-507.
Peabody J. Biol. Chem 5 Apr. 1990 vol. 265 No. 10 5684-5689.
Reisner et al Thrombosis & Haemastasis vol. 58 No. 1 p. 58 1987.
Rohrmann et al. Biochem Biophys Res Com. 1970 vol. 38 No. 3: 406-413.
Y.M. Berzin et al, Biological Abstracts vol. 74 (1982) Abstract No. 51034.
Mastico Robert Allan
Stockley Peter George
British Technology Group Ltd.
Feisee Lila
Reeves Julie E.
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