Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1997-09-29
2001-07-03
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C435S006120, C435S007100, C435S810000, C435S975000, C530S327000, C530S403000
Reexamination Certificate
active
06255462
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of immunodetection and immunopurification and to the incorporation of an immunogenic peptide from a
Varicella Zoster
Virus protein into a protein.
BACKGROUND OF THE INVENTION
Inserting a peptide tag into a protein facilitates the characterization of that protein when antibodies to the protein are not available. Recent techniques incorporate a peptide tag into a recombinant protein to aid protein purification or isolation of that protein. Antibodies recognizing the peptide tag facilitate purification and/or isolation (Brizzard, et al.
BioTechniques
16:730-735, 1994; Fulton, et al.
Eur. J. Immunol
. 25:2069-2076, 1995; Olah, et al.,
Anal. Biochem
. 221:94-102, 1994; Prickett, K. S., et al.
BioTechniques
7:580-589, 1989). These methods usually depend upon the availability of unique restriction endonuclease recognition sites for the insertion of the antigenic peptide tag.
There are two basic strategies for incorporating an antigenic peptide inframe into a protein. In one method, the nucleic acid sequence encoding the peptide is added to the N-terminus equivalent of nucleic acid that encodes a protein. If the N-terminus contains a signal peptide that is cleaved when the protein enters the endoplasmic reticulum, then a tag inserted at this site will be cleaved during protein maturation. The peptide tag will not be available for mature protein isolation (Walter, P. et al.
Annu. Rev. Cell Biol
. 10:87-119, 1994).
C-terminal tagging techniques are also available (Olah Z., et al.
Anal. Biochem
221:94-102, 1994 and Prickett, et al.
BioTechniques
7:580-9, 1989). These techniques can be used when the addition of the tag to the C-terninus of a protein does not negatively affect the conformation of that protein. There are proteins where N-terminal and C-terminal tagging methods are not useful strategies for incorporating antigenic tags into a protein because, for example, the N-terminus is cleaved during protein maturation or additions to the C-terminus interfere with protein folding.
Reliance on restriction endonuclease recognition sites is not always practical and the addition of a peptide tag to the N-terninus or the C-terminus of a protein may not be useful. There is a need for a strategy to circumvent the limitations created by relying on restriction recognition sites and the limitations created when a tag is merely added to a terminal portion of a protein.
Varicella Zoster
Virus (VZV) is a member of the Herpesvirus family. The VZV virion is formed as an icosahedral nucleocapsid surrounded by a lipid envelope containing a number of viral glycoproteins. The VZV envelope glycoproteins include gE, gB, gH, gI, gC and gL. gE is the most abundant protein in the virion envelope (Grose, et al.
Infect. Immun
. 40:381-388, 1983) and is encoded by VZV gene 68. The mature protein is about 98 kDa and is about 623 amino acid residues in length. Glycoprotein gE is the predominant immunogen of VZV and was formerly called gpI or gp98 (Grose, C.
Annu. Rev. Microbiol
. 44:59-80, 1990; Montalvo, E. A., et al.
J. Virol
. 53:761-770, 1985; and Yao, Z., et al.
J. Virol
. 67:305-314, 1993). gE induces neutralizing antibodies and the most antigenic fragment within gE is reported to be between residues 1-134. (Fowler, et al.
Virology
214:531-540, 1995).
SUMMARY OF THE INVENTION
The present invention identifies a linear antigenic fragment from gE for use as a tag for protein immunopurification and isolation. Recombinant polymerase chain reaction (PCR) was used to incorporate the linear antigenic peptide from gE into any desired location within an open reading frame of a protein, independent of restriction endonuclease recognition sites. The combination of the peptide (SEQ ID NO:1) in a protein to produce a chimeric protein and antibody recognizing the peptide is used to immunopurify and/or immunlocalize the chimeric protein.
The nucleic acid encoding the linear amino acid sequence QRQYGDVFKGD (SEQ ID NO:1) was incorporated into nucleic acid encoding a protein to produce a chimeric protein containing a heterologous linear amino acid sequence QRQYGDVFKGD (SEQ ID NO:1). The chimeric protein was recognized by antibody binding to the heterologous amino acid sequence QRQYGDVFKGD (SEQ ID NO:1). In a preferred embodiment the antibody is the monoclonal antibody 3B3.
In another aspect of this invention a method is disclosed for identifying a recombinant chimeric protein comprising the steps of: obtaining a vector capable of directing expression of a protein in a cell wherein the vector comprises a first DNA sequence encoding a protein; incorporating a second DNA sequence encoding the peptide QRQYGDVFKGD (SEQ ID NO:1) in frame into the first DNA sequence; expressing a chimeric protein encoded by the product of the first DNA and second DNA sequence from a cell; and identifying the chimeric protein, using an antibody binding to the peptide. In one method the cell of the expressing step is prokaryotic and in another the cell of the expressing step is eukaryotic. Preferably the antibody of the identifying step is a monoclonal antibody and in one embodiment the monoclonal antibody is 3B3. In a preferred aspect of this embodiment, the identifying step is immunoaffinity column chromatography, in another the identifying step uses immunofluorescence and in another the identifying step is a method using flow cytometry.
In another aspect of this invention a kit is disclosed to purify a protein comprising two overlapping PCR primers selected from an antibiotic resistance gene and a monoclonal antibody recognizing the peptide fragment QRQYGDVFKGD (SEQ ID NO:1) and a protein including the amino acid sequence QRQYGDVFKGD (SEQ ID NO:1). In a preferred aspect of this embodiment the kit additionally comprises a protein including the amino acid sequence QRQYGDVFKGD (SEQ ID NO:1). In one embodiment the monoclonal antibody is 3B3. In another preferred aspect of this invention the kit additionally comprises a vector having at least one restriction endonuclease recognition site positioned to receive a DNA fragment encoding protein and in a preferred embodiment the expression vector contains an antibiotic resistance gene.
In yet another aspect of this invention a method is disclosed for identifying antibody in a sample, preferably antibody to VZV, comprising the steps of: creating a chimeric protein comprising a heterologous peptide fragment QRQYGDVFKGD (SEQ ID NO:1); adhering the chimeric protein to a surface; contacting the chimeric protein with a sample; and detecting antibodies binding to QRQYGDVFKGD (SEQ ID NO:1). In a preferred aspect of this embodiment the adhering step comprises adhering the chimeric protein to a solid surface using an antibody to the chimeric protein and the solid surface is an ELISA multiwell plate. In another embodiment the solid surface is a suspendable particle and in another embodiment the solid surface is a planar membrane.
In yet another aspect of this invention a method is disclosed for identifying a chimeric protein comprising a heterologous a peptide fragment QRQYGDVFKGD (SEQ ID NO:1) comprising the steps of exposing the chimeric protein to a monoclonal antibody recognizing the peptide fragment QRQYGDVFKGD (SEQ ID NO:1); and detecting protein bound to the antibody. In one embodiment the method additionally comprises isolating the chimeric protein. In one embodiment the method employs affinity chromatography and in another the method employs immunofluorescence.
REFERENCES:
patent: 4812559 (1989-03-01), Ellis et al.
patent: 4950595 (1990-08-01), Masuho et al.
patent: 4952674 (1990-08-01), Kellet et al.
patent: 5700487 (1997-12-01), Gerber et al.
patent: 0 321 249 A2 (1989-06-01), None
patent: 0 482 671 A1 (1992-04-01), None
patent: WO 95/04080 (1995-02-01), None
patent: WO 96/01900 (1996-05-01), None
B.L. Brizzard et al., “Immunoaffinity Purification of FLAG® Epitope-Tagged Bacterial Alkaline Phosphatase Using a Novel Monoclonal Antibody and Peptide Elution”, BioTechniques 16 730-735 (1994).
A.J. Davison et al.; “The Complete DNA Se
Mueting Raasch & Gebhardt, P.A.
Nolan Patrick J.
University of Iowa Research Foundation
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