Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-05-14
2004-08-24
Wessendorf, T. D. (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S006120, C435S005000, C435S194000, C435S320100, C435S069100, C530S350000, C536S023400
Reexamination Certificate
active
06780599
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to the reassembly of fusion peptides into a functionally active protein complex. Specifically, the present invention provides a method of forming peptide complexes that associate through the combination of helical domains to form an antiparallel leucine zipper. The present invention is also related to the use of assays to investigate protein-protein interactions. The assays of the present invention involve the association of fusion proteins comprising GFP fragments and heterologous polypeptides into functionally active GFP that exhibits fluorescence.
BACKGROUND OF THE INVENTION
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Green Fluorescent Protein
Green fluorescent protein (GFP), a relatively small protein comprising 238 amino acids, is the ultimate source of fluorescent light emission in the jellyfish
Aequorea victoria.
The gene for GFP was first cloned by Prasher et al. (1992, Gene, 111:229-233), and cDNA for the protein produces a fluorescent product identical to that of native protein when expressed in prokaryotic (
E. coli
) and eucaryotic (
C. elegans
) cells (Chalfie et al., 1994, Science, 263, 802-805).
The GFP excitation spectrum shows an absorption band (blue light) maximally at 395 nm with a minor peak at 470 nm, and an emission peak (green light) at 509 nm. The longer-wavelength excitation peak has greater photostability than the shorter peak, but is relatively low in amplitude (Chalfie et al., 1994,
Science,
263: 802-805). The crystal structure of the protein and of several point mutants has been solved (Ormo et al., 1996,
Science
273, 1392; Yang et al.,
Nature Biotechnol.
14, 1246). The fluorophore, consisting of a tripeptide at residues 65-67, is buried inside a relatively rigid beta-can structure, where it is almost completely protected from solvent access. The GFP absorption bands and emission peak arise from an internal p-hydroxybenzylideneimidazolidinone chromophore, which is generated by cyclization and oxidation of the tripeptide sequence Ser-Tyr-Gly sequence at residues 65-67 (Cody et al., 1993,
Biochemistry
32: 1212-1218).
GFP fluorescence in procaryotic and eucaryotic cells does not require exogenous substrates and cofactors. Accordingly, GFP is considered to have tremendous potential in methods to monitor gene expression, cell development, or as an in situ tag for fusion proteins (Heim et al., 1994, P.N.A.S. USA, 91,12501-12504). Chalfie and Prasher, WO 95/07463 (Mar. 16, 1995), describe various uses of GFP, including a method of examining gene expression and protein localization in living cells. Methods are described wherein: 1) a DNA molecule is introduced into a cell, said DNA molecule having DNA sequence of a particular gene linked to DNA sequence encoding GFP such that the regulatory element of the gene will control expression of GFP; 2) the cell is cultured in conditions permitting the expression of the fused protein; and 3) detection of expression of GFP in the cell, thereby indicating the expression of the gene in the cell. Methods such as those described by Chalfie and Prasher are advantageous compared to previously reported methods which utilized &bgr;-galactosidase fusion proteins (Silhavy and Beckwith, 1985, Microbiol. Rev., 49, 398; Gould and Subramani, 1988
, Anal. Biochem.,
175, 5; Stewart and Williams, 1992
, J. Gen. Microbiol.,
138,1289) or luciferases, in that the need to fix cell preparations and/or add exogenous substrates and cofactors is eliminated.
GFP is a valuable marker for intracellular protein localization. However, the fusion of GFP with structural proteins can alter their properties, resulting in loss of fusion protein localization, decreased GFP fluorescence or both. The fluorescence of this protein is sensitive to a number of point mutations (Phillips, G. N., 1997
, Curr. Opin. Struct. Biol.
7, 821-27). The fluorescence appears to be a sensitive indication of the preservation of the native structure of the protein, since any disruption of the structure allowing solvent access to the fluorophoric tripeptide will quench the fluorescence. Abedi et al. (1998
, Nucleic Acids Res.,
26, 623-30) have inserted peptides between residues contained in several GFP loops. Inserts of the short sequence LEEFGS (SEQ ID NO: 9) between adjacent residues at 10 internal insertion sites were tried. Of these, inserts at three sites, between residues 157-158, 172-173 and 194-195 gave fluorescence of at least 1% of that of wild type GFP. Only inserts between residues 157-158 and 172-173 had fluorescence of at least 10% of wild type GFP.
Protein Reassembly using Leucine Zipper
The unassisted reconstitution of proteins from peptide fragments has been demonstrated for several proteins; including ribonuclease (Richards et al., 1959
, J. Biol. Chem.
234, 1459-1465), chymotrypsin inhibitor-2 (Gay et al., 1994
, Biochemistry,
33, 7957-7963), tRNA synthetases (Shiba et al., 1992
, Proc. Natl. Acad. Sci. U.S.A.,
89, 1880-1884), and inteins (Southworth, et al., 1998
, EMBO J.,
17, 918-926). Protein reassembly has thus become an important avenue for understanding enzyme catalysis (Richards et al., 1959
, J. Biol. Chem.
234, 1459-1465), protein folding (Gay et al., 1994
, Biochemistry,
33, 7957-7963), and protein evolution (Shiba et al., 1992
, Proc. Natl. Acad. Sci. U.S.A.,
89, 1880-1884). Recently, assisted protein reassembly or “fragment complementation” has been applied to the in vivo detection of protein-protein interactions in such systems as dihydrofolate reductase (DHFR) (Pelletier et al., 1998
, Proc. Natl. Acad. Sci. U.S.A.,
95, 12141-12146; Remy et al., 1999
, Proc. Natl. Acad. Sci. U.S.A.,
96, 5394-5399; Pelletier et al., 1999
, Nat. Biotechnol.,
17, 683-690), ubiquitin (Karimova et al., 1998
, Proc. Natl. Acad. Sci. U.S.A.,
95, 5752-5756; Johnsson et al., 1994
, Proc. Natl. Acad. Sci. U.S.A.,
91, 10340-10344), and &bgr;-galactosidase (Rossi et al., 1997
, Proc. Natl. Acad. Sci. U.S.A.,
94, 8405-8410). These reassembly processes are contingent upon the proper choice of a dissection site within a protein and can be aided by techniques such as limited proteolysis, circular permutation (Baird et al., 1999
, Proc. Natl. Acad. Sci. U.S.A.,
96, 11241-11246; Topell et al., 1999
, FEBS Lett.,
457, 283-289; Zhang et al., 1993
, Biochemistry,
32, 12311-12318; Regan, L., 1999
, Curr. Opin. Struc. Biol.,
9, 494-499) and loop insertions (Abedi et al., 1998
, Nucleic Acid Res.,
26, 623-630; Nobuhide et al., 1999
, FEBS Lett.,
453, 305-307).
The dissection and subsequent reassembly of a protein from peptidic fragments provide an avenue for controlling its tertiary structure and hence its function. Although a majority of leucine zippers associate in a parallel fashion, recent examples of both naturally occurring and designed antiparallel leucine zippers have appeared in the literature (Lupas, A., 1996,
Trends Biochem. Sc.
21, 375-382; Kohn, W. D. et al., 1997,
S. J. Biol. Chem.
272, 2583-2586; Bryson, J. W. et al., 1995,
Science,
270, 935-941; Oakley M. G. et al., 1998,
Biochemistry,
37, 12603-12610, Oakley, M. G. et al., 1997,
Biochemistry,
36, 2544-2548). However, the prior art does not disclose the attachment of antiparallel leucine zippers to polypeptide fragments to form fusion proteins for reassembling the polypeptide fragments into functional proteins.
In contrast to parallel zippers, the antiparallel zippers are oriented in an opposite direction. Antiparallel Zippers have the advantage of occurring less frequently in natural proteins. Thus, antiparallel leucine zippers will interfere to a lesser extent with natural cellular proteins than parallel leucine zippers. Antiparallel attachment of leucine zippers to protein fragments (between a dissected peptide bond of the parent protein) requires a shorter amino acid linker region. As shown by the inventors of the present inventi
Ghosh Indraneel
Hamilton Andrew D.
Regan Lynne
Birch, Stesart, Kolasch & Birch, LLP
Wessendorf T. D.
Yale University
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