Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-12
2004-01-20
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100
Reexamination Certificate
active
06680179
ABSTRACT:
BACKGROUND OF THE INVENTION
Rhodospirillum rubrum
is a facultatively phototrophic purple nonsulfur bacterium. Under reduced oxygen concentration, this organism forms an intracytoplasmic membrane (ICM) that is the site of the photosynthetic apparatus (Collins, M. L. P., and C. C. Remsen,
The purple phototrophic bacteria
, p. 49-77, In J. F. Stolz (ed.), Structure of Phototrophic Procaryotes. CRC Press, Boca Raton Fla., 1990; Crook, S. M., et al.,
J. Bacteriol
. 167:89-95, 1986; Hessner, M. J., et al.,
J. Bacteriol
. 173:5712-5722, 1991). This apparatus consists of the light-harvesting antenna (LH) and the photochemical reaction center (RC). The pigment-binding proteins, the LH &agr; and &bgr; and the RC-L and -M, are encoded by the puf operon while RC-H is encoded by puhA. The nucleotide sequences of puhA and the puf operon have been determined in
R. rubrum
(Bélanger, G., et al.,
J. Biol. Chem
. 263:7632-7638, 1988; Bérard, J., et al.,
J. Biol. Chem
. 261:82-87, 1986; Bérard, J., and G. Gingras,
Biochem. Cell Biol
. 69:122-131, 1991) and related bacteria (Donohue, T. J., et al.,
J. Bacteriol
. 168:953-961, 1986; Kiley, P. J., et al.,
J. Bacteriol
. 169:742-750, 1987; Michel, H., et al.,
EMBO J
. 5:1149-1158, 1986; Michel, H., et al.,
EMBO J
. 4:1667-1672, 1985; Weissner, C., et al.,
J. Bacteriol
. 172:2877-2887, 1990; Williams, J. C., et al.,
Proc. Natl. Acad. Sci
. 81:7303-7307, 1984; Williams, J. C., et al.,
Proc. Natl. Acad. Sci
. 80:6505-6509, 1983; Youvan, D. C., et al.,
Proc. Natl. Acad. Sci
. 81:189-192, 1984; Youvan, D. C., et al.,
Cell
37:949-957, 1984).
R. rubrum
may grow phototrophically under anaerobic light conditions or by respiration under aerobic or anaerobic conditions in the dark. Because
R. rubrum
is capable of growth under conditions for which the photosynthetic apparatus is not required, and because the photosynthetic apparatus and the ICM may be induced by laboratory manipulation of oxygen concentration, this is an excellent organism in which to study membrane formation (Collins, M. L. P., and C. C. Remsen, supra, 1990; Crook, S. M., et al., supra, 1986).
In previous studies, the puf region was cloned and interposon mutations within this region were constructed (Hessner, M. J., et al., supra, 1991).
R. rubrum
P5, in which most of the puf genes were deleted, was shown to be incapable of phototrophic growth and ICM formation. P5 was restored to phototrophic growth and ICM formation by complementation with puf in trans (Hessner, M. J., et al., supra, 1991; Lee, I. Y., and M. L. P. Collins,
Curr. Microbiol
. 27:85-90, 1993). These results imply that in
R. rubrum
the puf gene products are required for ICM formation. These results differ from those obtained with a puf interposon mutant of
Rhodobacter sphaeroides
(Davis, J., et al.,
J. Bacteriol
. 170:320-329, 1988) which was phototrophically incompetent but was still capable of ICM formation (Kiley, P. J., and S. Kaplan,
Microbiol. Rev
. 52:50-69, 1988). In the case of
R. sphaeroides
, the formation of ICM in the absence of the puf products may be attributable to the presence of an accessory light-harvesting component (LHII) encoded by puc (Hunter, C. N., et al.,
Biochem
. 27:3459-3467, 1988). This implies that
R. rubrum
is a simpler model for studies of membrane formation.
Because the puf-encoded proteins are required for ICM formation in
R. rubrum
and because the RC is assembled from puf and puhA products, it is important to evaluate the role of puhA-encoded RC-H in RC assembly and ICM formation in
R. rubrum.
Cheng, et al.,
J. Bacteriol
. 182(5):1200-1207, 2000 and Yongjian S. Cheng, “Molecular Analysis of Biochemical Intracytoplasmic Membrane Proteins,” PhD thesis, UW-Milwaukee, August, 1998 describe the cloning, mutation, and complementation of the puhA region of
R. rubrum
. (Both of these documents are incorporated herein by reference.) The present application proposes a model for the preparation of proteins, preferably membrane proteins.
SUMMARY OF THE INVENTION
In one embodiment, the present invention is a method of expressing protein comprising the steps of placing a DNA sequence encoding a protein or peptide in an expression vector that contains a regulatable promoter expressible in
Rhodospirillum rubrum
and expressing the protein within a bacterial host, wherein the host has extra capacity for membrane formation and wherein the host is a member of the genus Rhodospirillum.
In a preferred embodiment of the present invention, the protein or peptide is a membrane protein or peptide and/or the protein or peptide is a heterologous protein or peptide.
In another preferred form of the present invention, the host is
Rhodospirillum rubrum.
In another embodiment, the present invention is a protein expression system. In one embodiment, the protein expression system encompasses a vector comprising a DNA molecule encoding the protein or peptide in an expression vector containing a regulatable promoter expressible in
R. rubrum
. The vector is contained within a host, preferably
R. rubrum
with extra capacity for membrane formation.
REFERENCES:
Grunwald et al. J. Bacteriol. 1995, vol. 177, No. 3, pp. 628-635.*
J. Bérard, et al., “Mapping of the puh Messenger RNAs fromRhodospirillum rubrum,” J. Biol. Chem.264(18):10897-10903, 1989.
J. Bérard and G. Gingras, “The puh Structural Gene Coding for the H Subunit of theRhodospirillum rubrumPhotoreaction Center,”Biochem. Cell. Biol.69:122-131, 1991.
J.M. Blatny, et al., “Construction and Use of a Versatile Set of Broad-Host-Range Cloning and Expression Vectors Based on the RK2 Replicon,”Appl. Env. Microbiol.63(2):370-379, 1997.
Y.S. Cheng, “Molecular Analysis of Bacterial Intracytoplasmic Membrane Proteins,” pp. 1-97, 1998 (Thesis).
Y.S. Cheng, et al., “Role of the H Protein in Assembly of the Photochemical Reaction Center and Intracytoplasmic Membrane inRhodospirillum rubrum,” J. Bacteriol.182(5):1200-1207, 2000.
M.J. Hessner, et al., “Construction, Characterization, and Complementation ofRhodospirillum rubrumpuf Region Mutants,”J. Bacteriol.173(18):5712-5722, 1991.
B.C. Jester, “Construction and Characterization of a puf Null Mutant ofRhodospirillum rubrum,” pp. 1-45, 1998 (Thesis).
N.T. Keen, et al., “Improved Broad-Host-Range Plasmids for DNA Cloning in Gram-negative Bacteria,”Gene70:191-197, 1988.
W.R. Jones, et al., “Mutants ofRhodobacter sphaeroidesLacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes,”Molec. Microbiol6(9):1173-1184, 1992.
I.Y. Lee and M.L.P. Collins, “Identification and Partial Sequence of the BchA Gene ofRhodospirillum rubrum,” Curr. Microbiol.27:85-90, 1993.
Cheng Yongjian S.
Perille-Collins Mary Lynne
Low Christopher S. F.
Quarles & Brady LLP
WiSYS Technology Foundation, Inc.
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