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
2001-03-19
2004-10-12
McKelvey, Terry (Department: 1636)
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
active
06803210
ABSTRACT:
The present invention relates generally to improved methods for the expression of recombinant protein products under the transcriptional control of an inducible promoter, such as the araB promoter, in bacterial host cells that are deficient in one or more of the active transport systems for an inducer. In the case of the araB promoter, the inducer is L-arabinose. The present invention also relates to improved bacterial host cells that are deficient in one or more of the active transport systems for an inducer, such as L-arabinose, and that contain an expression vector encoding a recombinant protein product under the transcriptional control of an inducible promoter, such as the araB promoter.
BACKGROUND OF THE INVENTION
Although many systems have been described for expression of recombinant proteins, including peptides and polypeptides, in microbial systems, most gene expression systems in gram negative bacteria such as
Escherichia coli
have relied exclusively on a limited set of bacterial promoters. The most widely used bacterial promoters have included the lactose [lac] (Yanisch-Perron et al., 1985,
Gene
33: 103-109), and the tryptophan [trp] (Goeddel et al., 1980,
Nature
(London) 287: 411-416) promoters, and the hybrid promoters derived from these two [tac and trc] (Brosius, 1984,
Gene
27: 161-172; and Amanna and Brosius, 1985,
Gene
40: 183-190). Other commonly used bacterial promoters include the phage lambda promoters P
L
and P
R
(Elvin et al., 1990,
Gene
37: 123-126), the phage T7 promoter (Tabor and Richardson, 1998,
Proc. Natl. Acad. Sci. U.S.A.
82: 1074-1078), and the alkaline phosphatase promoter [pho] (Chang et al., 1986,
Gene
44: 121-125). Each of these promoters has desirable features. However, the ideal promoter for expression of a wide variety of recombinant proteins would offer certain features not found in these commonly used systems. For example, many recombinant products can be toxic to the expression host. Therefore, it is often preferable for the promoter to tightly regulate gene expression during culture propagation when gene expression is undesirable. In contrast, when gene expression is desired, the promoter must be easily controlled and a high expression level is often preferred. The agent or environmental condition that initiates gene expression should be easy to use and ideally of low cost. In general, a tightly regulated system is most desirable. Features of a promoter and general expression system that are most preferred include tightly repressed gene expression in the absence of inducer and highly derepressed gene expression in the presence of inducer. Also desirable would be a system that will allow the expression level of a recombinant product to be proportional to the amount of inducing agent added into the cell culture.
One bacterial promoter system that has proven to be particularly advantageous for providing tightly repressed gene expression in the absence of the inducer arabinose and highly derepressed gene expression in the presence of the inducer arabinose is the araB promoter of the Enterobacteriaceae family. Of interest is U.S. Pat. No. 5,028,530, which is hereby incorporated by reference, which describes the use of the araB expression system for production of polypeptides, including cecropins, by microbiological techniques. Two features of the ara system have made it particularly well-suited for expression of recombinant products in bacteria such as
E. coli
. First, it is simple to exploit because the control elements of the araB promoter are conveniently contained within an approximately 300 base pair regulatory region and only a functional coding sequence for the araC gene is additionally needed. Second, regulation of the system has proven to be particularly tight, i.e., the ratio of the amount of product in the induced state (with arabinose) relative to that in the repressed state (without arabinose) from the araB promoter on multicopy expression plasmids is relatively high, most frequently in the range from >200-75,000 (Better et al., 1999, in
Gene Expression Systems: Using Nature for the Art of Expression
, Academic Press, New York, pp. 95-107). Additionally, the uninduced level of protein expression from the ara system is very low. This feature is particularly important and useful when protein products, including recombinant peptides and polypeptides, that are toxic to the host are to be expressed.
Bacteria such as
E. coli
have two known systems for active transport of arabinose into the cell. The first of these systems is an inducible, energy-dependent accumulation process catalyzed by the product of the araE gene. The araE gene product is an approximately 52,000 Da, membrane-associated protein that constitutes the low affinity arabinose transport system (Maiden et al., 1988,
Journal of Biol. Chem.
263: 8003-8101). The second L-arabinose transport system has a greater affinity for L-arabinose and is dependent on the activity of the L-arabinose binding protein, AraF. The locus encoding this arabinose binding protein, araF, is part of an operon with araG and araH. The protein products from these three genes, araFGH, make up the high affinity L-arabinose transport system (Horazdovsky and Hogg, 1989,
Jour. of Bacter.
171: 3053-3059). Arabinose transport-deficient mutant
E. coli
strains have been prepared (see, e.g., Maiden et al., supra; Harazdovsky and Hogg, supra).
Of interest are the disclosures of the following references which relate to use of the araB promoter for expression of polypeptides in bacteria.
Johnston et al., 1985,
Gene
34: 137-145, described the vector pING1 which contained the ara regulatory region, the complete araC gene and a portion of the araB gene from
S. typhimurium
. Restriction sites were introduced into the coding region of araB gene so that a gene fusion or a multigene transcription unit could be expressed under arabinose control. This system was used to express homologous (bacterial) proteins which normally are expressed in
E. coli
under certain circumstances, namely the M13 gene II (Johnson et al., 1985) and gene 8 proteins (Kuhn and Wickner, 1985,
J. Biol. Chem.
260: 15907-15918), and a similar vector were used to express the RepE protein from the
E. coli
F plasmid (Masson et al., 1986,
Nucleic Acids Res.
14: 5693-5711). Each of these proteins was produced in the cytoplasm of
E. coli
cells, and at least some of the expressed protein was soluble and could be detected in an active form either in vivo or in cell extracts.
Better et al., 1988,
Science
240: 1041-1043, described an araB expression system derived from pING1 that was subsequently engineered to regulate the expression of heterologous (non-bacterial) recombinant proteins. Heterologous proteins successfully produced with the araB expression system in
E. coli
include immunoglobulin Fab domains. In this case, the araB expression system was used to direct the production of polypeptides directly linked to hydrophobic signal sequences through the bacterial cytoplasmic membrane where Fab accumulated in the correctly folded, fully active configuration and could be recovered directly from the culture supernatant. Expression of Fab domains under the transcriptional control of the araB promoter was the first demonstration that a heterodimeric, heterologous protein could be produced in
E. coli
. The initially reported expression level was approximately 1-2 &mgr;g/mL, but in subsequent studies the level of protein expression could be increased nearly 1000-fold by growing the bacteria to a high cell density in a fermentor (Better et al., 1990,
ICSU Short Rep.
10: 105). In contrast, Clark et al., 1997,
Immunotechnology
3: 217-226, found that Fab genes under lac control can inhibit bacterial growth, and also that Fab expression from P
BAD
was more tightly repressed than that from P
lac
.
Better et al., 1992,
J. Biol. Chem.
267: 16712-16718; Nolan et al., 1993,
Gene
134: 223-227; and Bernhard et al., 1994,
Bioconjugate Chem.
5: 126-132, showed that the araB expre
McKelvey Terry
XOMA Technology Ltd.
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