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
2000-02-18
2001-08-14
Schwartzman, Robert A. (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...
C435S069100, C435S069800, C435S243000, C435S252300, C536S023400, C536S023100
Reexamination Certificate
active
06274345
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to expression vectors comprising a gene encoding outer membrane protein C(“OmpC”) from
Escherichia coli
((“
E. coli
”) as a cell surface anchoring motif, more specifically, to expression vectors comprising a gene encoding OmpC which is designed to express a gene of foreign protein in fused form with OmpC on the cell surface of
E. coli
, and a method for displaying the desired protein on the surface of
E. coli
employing the OmpC as a cell surface anchoring motif.
BACKGROUND OF THE INVENTION
Bacteria have played a crucial role in the development of the biotechnology. Since the development of recombinant DNA technology, genetically engineered bacteria have been employed for the production of various recombinant proteins. Also, recombinant bacteria have been developed and used for a wide range of industrial applications such as biodegradable plastics production, heavy metal removal, sulfur removal, waste treatment, and food processing.
Recently, new advances in molecular biology and secretory expression of proteins made it possible to express foreign proteins at the outer surface of microorganisms by the technology called cell surface display.
Since the first development of surface-expression system by George P. Smith in the mid 1980 by expressing peptides or small proteins fused with pIII of the filamentous phage (see: Smith, G. P., Science, 228:1315-1317, 1985), various mechanisms of protein secretion in microorganisms have been extensively studied to develop new and better cell surface display systems by which proteins of interest can be expressed on the surface of the microorganisms. Cell surface display is a relatively new technology expressing proteins or peptides on the surface of the cell in a stable manner using the surface protein of bacteria, yeast, or even mammalian cells as a surface anchoring motif.
Before the cell surface display system was developed, phage system was used to express foreign protein on the surface of the phage, because the structure of the phage coat is simpler than that of bacteria. However, the size of foreign protein to be expressed on the surface of phage was limited. Therefore, the application of the phage surface display system has been limited. This is why new focus has been given to bacterial cell surface display system.
Gram-negative bacteria possess unique as well as complex cell envelope structure which consists of inner cellular membrane, periplasm, and outer cellular membrane. Therefore, surface anchoring motif is needed to efficiently transport foreign protein to the surface of the bacteria. For the expression of foreign proteins or peptides using the surface protein of the bacteria, appropriate bacterial surface protein has to be fused to the foreign protein of interest at the genetic level, and the expressed fusion protein has to be transported through the inner cellular membrane and outer membrane to the surface and be maintained on the surface of the bacteria.
Successful candidates for surface anchoring motif should have the following characteristics: The surface protein to be used as an anchoring motif should have efficient secretion signal sequences for facilitating the penetration of the foreign protein through the inner membrane of the cell, targeting signal for anchoring foreign protein to the surface of the cell in a stable manner, and capacity to accommodate foreign proteins or peptides of various sizes. Furthermore, it would be beneficial if the fusion protein can be expressed in large amounts.
A variety of cell surface display systems have been developed to date, which may be classified into three groups according to their recombinant profiles: C-terminal fusion, N-terminal fusion, and sandwich fusion. If a native surface protein has a discrete localization signal within its N-terminal portion, a C-terminal fusion strategy may be considered to fuse the foreign peptides to the C-terminal of that function portion. The Lpp-OmpA motif developed in
E. coli
is a good example of C-terminal fusion system (see: Georgiou, G., et al., Protein Eng., 9:239-247, 1996). Similarly, N-terminal fusion systems have been developed using
Staphylococcus aureus
protein A (see: Gunneriusson, E., et al., J. Bacteriol., 178:1341-1346, 1996),
Staphylococcus aureus
fibronectin binding protein B (see: Strauss, A., et al., Mol. Microbiol., 21:491-500, 1996), and
Streptococcus pyogenes
fibrillar M protein (see: Pozzi, G., et al., Infect. Immun., 60:1902-1907, 1992), all of which contain C-terminal sorting signals to target foreign proteins to the cell wall. However, many surface proteins do not have such anchoring regions, and thus the whole structure is required for the assembly. Therefore, a sandwich-fusion system in which a foreign protein of interest is inserted into the surface protein has also been developed. Several examples employing this system include
E. coli
PhoE (see: Agterberg, M., et al., Gene, 88:37-45, 1990), FimH (see: Pallesen, L., et al., Microbiology, 141:2839-2848, 1995), and PapA (see: Steidler, L., et al., J. Bacteriol., 175:7639-7643, 1993). However, it is generally believed that the exposed loops of outer membrane proteins (OMPs) can only accept insertions of 60-70 amino acids or less (see: Georgiou, G., et al., Nature Biotechnol., 15:29-34, 1997; Stahl, S., et al., Trends Biotechnol., 15:185-192, 1997).
Under the current circumstances, there are strong reasons for exploring and developing an alternative cell surface display system to allow expression and display of foreign proteins consisted of more amino acid residues.
The cell surface display can be employed for a wide range of biotechnological and industrial applications such as:
(1) Live vaccine development—to expose heterologous epitopes on human commensal or attenuated pathogenic bacterial cells to elicit antigen-specific antibody responses (see: Nguyen, T. N., et al., Gene, 128:89-94, 1993);
(2) Peptide libraries screening—to screen displayed peptide libraries by sequential binding and elution, or more efficiently, by fluorescence-activated cell sorting (see: Francisco, J. A., et al., Proc. Natl. Acad. Sci., USA., 90:10444-10448, 1993; Georgiou, G., WO9849286, 1998);
(3) Antibody production—to express surface antigens to raise polyclonal antibodies in animal (see: Charbit, A., et al., Gene, 70:181-189, 1988);
(4) Environmental bioadsorbents—to modify cell surface for the removal of harmful chemicals and heavymetals (see: Sousa, C., et al., J. Bacteriol., 180:2280-2284, 1998);
(5) Whole cell catalysts—to immobilize enzymes on the outmost layer of cells to catalyze biochemical reactions directly (see: Richins, R. D., et al., Nature Biotechnol., 15:984-987, 1997); and,
(6) Biosensor development—to anchor enzymes, receptors, or other signal-sensitive components on cell surface to develop novel biosensors for diagnostic, industrial or environmental purposes.
SUMMARY OF THE INVENTION
The present inventors have made an effort to develop a novel cell surface display system to allow expression of larger foreign proteins/peptides by employing outer membrane protein C(OmpC) of
E. coli
as a cell surface anchoring motif, and found that recombinant proteins can be expressed efficiently on the cell surface of recombinant
E. coli
transformed with a novel expression vector containing a gene encoding OmpC. In particular, they found that a transformant harboring a recombinant plasmid vector expressing poly-histidine linker peptide in fused form with OmpC on the cell surface of
E. coli
has outstanding potential to be used as heavy metal adsorbent.
The first object of the invention is, therefore, to provide novel expression vectors containing OmpC gene of
E. coli
as a cell surface anchoring motif to express foreign proteins on the surface of
E. coli.
The second object of the invention is to provide recombinant
E. coli
strains that are transformed with the said expression vectors.
The third object of the invention is to provide a method for manufacturing foreign proteins/peptides on the cell surface of
E. coli
,
Choi Jong Hyun
Lee Sang Yup
Xu Zhaohui
Knobbes Martens Olson & Bear LLP
Korea Advanced Institute of Science and Technology
Leffers, Jr. Gerald G.
Schwartzman Robert A.
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