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
1999-04-16
2002-05-07
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S069800, C530S300000, C530S350000, C530S387300, C536S023100, C536S023400, C536S023700, C536S023720
Reexamination Certificate
active
06383777
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to compositions and methods for the high yield production of eukaryotic proteins and in particular membrane proteins, by expression of recombinant vectors designed for such high yield production in bacterial cells.
2. Background Art
Certain classes of eukaryotic, prokaryotic and viral proteins, including membrane proteins, needed in large quantities for therapeutic uses as well as for biochemical and structural studies, have proven difficult to express in recombinant systems in sufficient yields. This is particularly difficult for eukaryotic proteins with multiple membrane spanning regions including, but not limited to, G-protein coupled receptors (QPCRs) and ion channels derived from eukaryotic cells (Goeddel, 1990).
Eukaryotic membrane proteins have been expressed in a number of eukaryotic systems including mammalian cells, baculovirus systems [up to 55 pmol/mg of protein (125 &mgr;g/L of culture); Loisel et al., 1997] and yeast cells (up to 14 pmol/mg membrane protein; Sander et al., 1994). However, none of these approaches has proven successful for the production of large quantities of purified eukaryotic proteins.
Furthermore, although a number of reports in the literature describe expression of eukaryotic membrane proteins such as GPCRs in prokaryotic cells (e.g.,
E. coli
), none of these systems has proven capable of producing high levels of an intact eukaryotic protein (Table I). These bacterial cell systems have produced GPCRs in amounts of approximately several hundred receptor molecules per cell, with none of the systems producing greater than 300 receptors per cell, which corresponds to approximately 5 &mgr;g protein per liter of bacterial culture.
TABLE 1
Expression levels of &bgr; adrenergic receptor in
E. coli
Leader Sequence
Expression level
LamB
33 to 225 receptors/cell (Chapot et al. 1990)
&bgr;-galactosidase
25 receptors/cell (Marullo et al., 1988)
none
200 receptors/levels (Breyer et al 1990)
The present invention overcomes previous shortcomings associated with high yield production of eukaryotic proteins by providing compositions and methods for producing eukaryotic proteins and in particular, membrane proteins, in high yield (i.e., at least 100 &mgr;g protein/L of culture), for use in biochemical and structural studies and as therapeutic agents.
REFERENCES:
patent: 5665865 (1997-09-01), Lerner et al.
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Ramjeesingh et al. “A Novel Procedure for the Efficient Purification of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)”Biochem. J.327:17-21, 1997.
Loisel et al. “Recovery of Homogeneous and Functional &bgr;2-Adrenergic Receptors from Extracellular Baculovirus Particles”Nature Biotech.15:1300-1304, Nov. 1997.
Sander et al. “Expression of the Human D2SDopamine Receptor in the YeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombe:a comparative study”FEBS Letters334:41-46, 1994.
Breyer et al. “Mutational Analysis of Ligand Binding Activity of &bgr;2Adrenergic Receptor Expressed inEscherichia coli” EMBO J.9(9):2679-2684, 1990.
Chapot et al. “Localization and Characterization of Three Different &bgr;-Adrenergic Receptors Expressed inEscherichia coli” Eur. J. Biochem.187:137-144, 1990.
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Breyer Richard M.
Kennedy Chris
Ma Lijun
Brannock Michael
Eyler Yvonne
Needle & Rosenberg P.C.
Va{umlaut over (n)}derbilt University
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