Chemistry: molecular biology and microbiology – Vector – per se
Patent
1996-04-08
2000-10-10
Hutzell, Paula K.
Chemistry: molecular biology and microbiology
Vector, per se
435 691, 435 697, 435 711, 4352523, 43525233, 43525234, 4352528, 4352533, 435875, 435849, 435471, 435481, 435488, 530350, C12N 1500
Patent
active
061300853
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a recombinant vector containing a sequence of a structural lipoprotein gene for the expression of nucleotide sequences.
One of the aims of the invention is to propose new means for preparing, by genetic engineering techniques, proteins, polypeptides or peptides, in the form of products (optionally of fusion products) capable of being exposed at the level of the external membrane of the cell which produces them. In other words, the invention provides vectors for the expression, by recombinant cells, of extracellular proteins anchored in the membrane of these cells.
There are known in the state of the art various vectors which make it possible to express, in recombinant cell hosts, amino acid sequences and which allow, where appropriate, the exposure of these proteins at the surface of the cell host. Plasmids or phages have for example been used in the state of the art to express polypeptide in Gram-negative bacteria and to expose them at their surface.
Thus, Francisco J. A. et al. (Bio/Technology vol. 11, April 1993, p. 491-495) have expressed in E. coli an exoglucanase in the form of a hybrid protein (fusion protein) with the transmembrane domain of the OmpA protein naturally contained in the external membrane of E. coli, itself fused with the first nine amino acids of the E. coli major lipoprotein (also called Braun's lipoprotein (Lpp), expressed at the level of the external membrane). According to Francisco et al., the fusion protein thus obtained is anchored at the surface of the external membrane of E. coli.
Francisco et al. used a fusion product involving the OMPA protein which allows, within this hybrid construct, the exposure of exoglucanase at the surface of E. coli.
Other authors, Fuchs P. et al. (Bio/Technology Vol. 9, December 1991, p. 1369-1372) have described the expression, in E. coli, of a fusion protein formed by the variable domains of the heavy chains and light chains of antibodies, fused with the peptidoglycan-associated lipoprotein (PAL) of E. coli. In order to obtain a fusion protein which is accessible at the surface of E. coli cells, a sequence for the export of a pectin lyase was used and the N-terminal cysteine of PAL was converted to glycine, thus preventing the attachment of the lipids of lipids of the external cell membrane to the cysteine normally present in PAL.
The lipoproteins used by Fuchs et al. according to the abovementioned publication are lipoproteins belonging to the cytoplasmic membrane of the cell.
The inventors of the present application focused their attention on other lipoproteins, especially on structural lipoproteins analogous to the E. coli Braun's lipoprotein insofar as they have the capacity to become attached to the external cell membrane, on the one hand, and in some cases to the peptidoglycans, on the other hand. These lipoproteins were used within the framework of the invention to prepare recombinant vectors intended especially for the expression and export, at the surface of the external membrane of recombinant cells, of amino acid sequences.
Preferably, the lipoprotein used is not an E. coli lipoprotein.
The subject of the invention is a recombinant vector for the cloning and/or expression and/or transfer, into a cell host, of a heterologous nucleotide sequence, characterized in that it comprises, at a site not essential for its replication, the gene encoding a lipoprotein different from the E. coli lipoproteins, or part of this gene which contains the components necessary for controlling the expression of this lipoprotein and for its exposure at the surface of the external cell membrane of the host, such that the heterologous nucleotide sequence is capable of being introduced into the gene or the part of the gene encoding the lipoprotein, under conditions allowing the expression of this heterologous sequence and the exposure of the polypeptide obtained at the surface of the cell host.
The expression "heterologous nucleotide sequence" is understood to mean any nucleotide sequence which is not naturally contained in the nonrecom
REFERENCES:
patent: 5348867 (1994-09-01), Georgiou
patent: 5583038 (1996-12-01), Stover
Boss et al, Nucleic Acids Research 12 (9):3791-3806, 1984.
Georgiou et al., Trends in Biotechnology 11(1):6-10, Jan. 1993.
Georgiou et al., "Practical Applications of Engineering Gram-Negative Bacterial Cell Surfaces," Tibtech, vol. 11, pp. 6-10 (1993).
Laukkanen et al., "Lipid-tagged Antibodies," Protein Engineering, vol. 6, No. 4, pp. 449-454 (1993).
Biological Abstracts, vol. 88, No. 084626 (1989).
Gbrayeb et al., The Journal of Biological Chemistry, vol. 269, pp. 463-467 (Jan. 1984).
Francisco et al., Proc. Natl. Acad. Sci. USA, vol. 89, pp. 2713-2717 (Apr. 1992).
Cornelis Pierre
Hamers Raymond
Hutzell Paula K.
Lazar-Wesley Eliane
LandOfFree
Recombinant vector containing a sequence of a lipoprotein gene f does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant vector containing a sequence of a lipoprotein gene f, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant vector containing a sequence of a lipoprotein gene f will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2255445