Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Separation of micro-organism from culture media
Reexamination Certificate
1995-11-28
2001-12-18
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Separation of micro-organism from culture media
C210S406000, C422S105000, C422S105000, C435S259000, C435S283100, C435S308100
Reexamination Certificate
active
06331431
ABSTRACT:
Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
The present invention relates generally to the expression and isolation of proteins expressed in the periplasmic space of bacteria and, more particularly, to methods of simultaneously isolating periplasmic fractions from multiple samples of host cells.
With the advent of recombinant DNA technology the demand for rapid and efficient methods of recombinant protein expression has increased steadily over the years. This demand has resulted in a detailed understanding of essential elements and requirements for expression in both procaryotic and eukaryotic systems. It is now routine for one skilled in the art to express essentially any desired protein coding sequence as a recombinant protein in various different hosts including bacteria, yeast, insect cells and mammalian cells.
The available systems for recombinant expression have similarly evolved to keep pace with the increasing demand. For example, there are numerous vectors available for efficient expression of protein coding sequences, such as cDNA, as soluble fusion proteins or authentic sequences without a fusion partner in
E. coli
or other bacteria. Specific examples of such expression vectors include the T7, tac or Lac UV5 vectors. Many vectors also include characteristics which enable the enrichment or purification of the recombinant protein. Most of these modifications fuse an affinity tag to the recombinant sequence for subsequent purification by affinity chromatography of the cell extract or of the secreted protein into the growth medium. Some vectors also include protease cleavage sites to remove the fusion partner from the recombinant sequence. Many other attributes have also been incorporated into various expression vectors and systems to meet the desired needs of a particular application. Moreover, one skilled in the art can routinely engineer and construct various modifications to these systems, or construct novel systems tailored to the needs and desired outcome for a particular application.
Expression vectors and systems have also been devised which direct expression of recombinant proteins to specific locations. Directed expression to particular locations offers specific advantages over expression of soluble fusion proteins described above. For example, systems have been devised which allow for the surface expression of recombinant proteins on the inner membrane of
E. coli
, to the periplasmic space of bacteria, or on the coat of bacteriophage. Expression in the periplasmic space allows for a enriched source of starting material which is partially free of contaminating cellular material. The advantage of surface expression is that it allows for the direct and simultaneous isolation of an unknown protein and its encoding nucleotide sequence (Chang et al.,
J. Immunol.
147:3610-3614 (
1991)
; Gram et al.,
Proc. Natl. Acad. Sci. USA
89:3576 (1992); Hawkins et al.,
J. Mol. Biol.
226:889 (
1992)
; and Huse et al.,
J. Immunology
149:3914 (1992)).
Systems similar to those described above for recombinant expression in procaryotic systems have also been developed for systems which include species as diverse as insect cells to mammalian cells. Moreover, recombinant protein expression has also been, and is expected to continue to be a major component in the commercial development of new biological-based drugs. The most successful recombinant proteins being used as therapeutics include erythropoietin, G-CSF, interferons, growth factors, interleukin
1
and monoclonal antibodies. In addition to the use of recombinant proteins as therapeutics, essentially all biotechnology companies and most pharmaceutical companies rely heavily on the use of recombinant expression within their research and development and product development programs. Such uses range from protein expression for antibody production and functional studies to the creation of mutagenized species for identifying enhanced functional characteristics to the generation of large libraries of peptides and antibodies for drug screening and genes for reconstructing biosynthetic pathways.
Regardless of the advances of recombinant expression, there remains one major disadvantage associated with all of these systems and especially those utilized for drug discovery of biopharmaceuticals. This disadvantage is the need to identify and characterize the active components present in the cell extract. For example, identification of lead proteins requires the preparation of sufficient quantities of active protein samples for analysis by a variety of techniques such as binding assays, enzyme kinetics, inhibition assays, growth assays and signal transduction. It is the screening of large libraries that can be labor intensive. Purification methods usually rely on standard biochemical or immunoaffinity techniques which are labor intensive and require multiple time consuming steps. Such drawbacks rapidly become burdensome when recombinant expression and purification is coupled to a high throughput process such as those necessary in the biotechnology and drug development industries.
Thus, there exists a need for the efficient and simultaneous isolation of recombinant proteins from multiple samples of host cells. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The invention provides a high throughput device for simultaneously isolating periplasmic fractions from multiple samples of host cells. The high throughput device consists of a vacuum chamber having one end open, a means for supporting a collection chamber and a vacuum aperture. The open end of the vacuum chamber contains a means for supporting a sample chamber which can be sealed under vacuum. The device can be formatted to operate more than one in series. The invention also provides a device for simultaneously culturing of a plurality of samples. This culture device consists of a bottom component having a means for supporting a plurality of multi-well culture chambers, a top component attached to the bottom component by substantially vertical supports and a means for attaching the culture device to a platform of a shaker or orbital incubator. Also provided is a method for simultaneously isolating periplasmic fractions from multiple samples of host cells. The method consists of: (a) providing a high throughput device for simultaneously isolating periplasmic fractions from multiple samples of host cells having a vacuum chamber containing one end open, a means for supporting a collection chamber and a vacuum aperture, the open end of the vacuum chamber having a means for supporting a sample chamber which can be sealed under vacuum; (b) attaching the sample chamber containing a sample of host cells in media to the vacuum chamber; (c) applying a vacuum to the vacuum chamber to remove the media; (d) removing the outer membrane of the host cells; (e) attaching a collection chamber to the vacuum chamber; and (f) applying a vacuum to the vacuum chamber to collect the periplasmic fraction from the host cells. A workstation is also provided. The workstation consists of one or more high throughput devices for simultaneously isolating periplasmic space fractions from a plurality of samples of host cells and a device for simultaneously culturing a plurality of samples.
REFERENCES:
patent: 4306514 (1981-12-01), Bouclier
patent: 4327661 (1982-05-01), Boeckel
patent: 5205989 (1993-04-01), Aysta
Zweibel et al, “Polycrystalline thin-film . . . ”, 1984, see abstract.*
FiltaPlates: Polyfiltronics, 150 Weymouth Street; Rockland, MA 02370.
Silent Monitor Loprodyne 1.2 m membrane-bottomed plates: Pall Corporation; Portsmouth, England.
Easy-Titer Elifa System: Pierce; 3747 N. Meridian Rd.; P.O. Box 117; Rockford, IL 61105.
Environmental Shaker: Baxter Diagnostics, Inc.; Scien
Glaser Scott M.
Huse William D.
MacConnell William P.
Campbell & Flores LLP
Ixsys Inc.
Naff David M.
Ware Deborah K.
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