Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-31
2004-12-07
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S501000, C530S350000, C536S023100
Reexamination Certificate
active
06828099
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to protein complementation assays (PCA) and more specifically to PCA assays based on the
E. coli
TEM-1 &bgr;-Lactamase for the detection of protein-protein, protein-small molecule and protein nucleic acid interactions.
BACKGROUND OF THE INVENTION
Applicants' have previously described oligomerization domain-assisted complementation of enzyme fragments as a general strategy for detecting protein-protein, protein small molecule and protein nucleic acid interactions (ref. 9). In the present invention, we describe assays based on the
E. coli
TEM-1 &bgr;-Lactamase (Accession number: AAB59737). In the present invention, applicants' disclose three assays in mammalian cells: (1), an in vitro colorimetric assay using the substrate nitrocefin, and (2) an in vivo positive
egative fluorescence assay using the substrate CCF2/AM. The invention is also directed to positive and negative survival assays using cephalosporin-cytotoxic pro-drug conjugates, as well as a series of &bgr;-lactamase point mutations that would be predicted to enhance the efficiency of the &bgr;-lactamase PCA.
The TEM-1 &bgr;-lactamase is a member of a family of bacterial enzymes that hydrolyze antibiotics of the penicillin and cephalosporin class, thus imparting resistance to bacteria expressing these enzymes. TEM-1 &bgr;-lactamase is the standard ampicillin resistance gene included in most plasmids used in molecular biology. The three-dimensional structure, proposed catalytic mechanism and optimal substrates and inhibitors have been well documented. TEM-1 &bgr;-Lactamase is a small (29 kiloDaltons) and monomeric protein consisting of 286 amino acids. The first 23 amino acids constitute a secretory signal peptide. &bgr;-lactamases catalyses the irreversible hydrolysis of the amide bond of &bgr;-lactam rings in penicillin or cephalosporin compounds. &bgr;-lactamases are secreted into the periplasmic space of gram-negative strains or into the outer media by their gram-positive counterparts where they normally act. However, they will accumulate in the cytoplasm when expressed in
E. coli
or other prokaryotic or eukaryotic cells if the secreting signal peptide is genetically deleted, without effecting catalytic activity.
TEM-1 &bgr;-lactamase meets all of the essential criteria to be an excellent candidate for a PCA strategy. Specifically, TEM-1 &bgr;-lactamase is a relatively small, monomeric protein and is well characterized both structurally and functionally. TEM-1 &bgr;-lactamase can be expressed in and is not toxic to prokaryotes and eukaryotes. In addition to these, unique features include that: First, &bgr;-lactamase is strictly a bacterial enzyme and has been genetically deleted from many standard
E. coli
strains. It is not present at all in eukaryotes. Thus, a &bgr;-lactamase PCA could be used universally in eukaryotic cells and many prokaryotes, without any intrinsic background. Second, assays are based on catalytic turnover of substrates with rapid accumulation of product. This enzymatic amplification should allow for relatively weak molecular interactions to be observed. Finally, the assay can be performed simultaneously or serially in a number of modes, such as in vitro colorimetric or fluorometric assays, or in vivo fluorescence or survival assays. Assays can be performed independent of the measurement platform and can easily be adapted to high-throughput formats requiring only one pipetting step.
The PCA strategy of the present invention is based on the reassembly of two rationally designed complementary fragments of TEM-1 &bgr;-lactamase. Crystal structures of TEM-1 suggest that residues 196-200 form a loop situated outside of the core of the protein and distal to the enzymatic pocket (FIG.
1
). This loop is not implicated in the catalytic machinery and seems not to be important for catalysis (ref. 4). For these reasons, this site was selected to generate the two fragments. We chose to cut in the middle of the loop between residues Glu197 and Leu198. In addition, the secreting signal peptide of 23 amino acids was deleted to leave only the functional enzyme. Thus fragment [1] (BLF[1]) consists of residues 24 to 197 and fragment [2] (BLF[2]) of residues 198-286. Each of these fragments are linked to interacting domains (GCN 4 leucine Zipper or the pair of rapamycin inducible interacting proteins FKBP/FRB domain) by a linker of 15 amino acids (Gly-Gly-Gly-Gly-Ser)
3
.
OBJECTS OF THE INVENTION
A primary object of the present invention are protein complementation assays (PCA) based on the
E. coli
TEM-1 &bgr;-lactamase.
Another object of the present invention is an in vitro colorimetric PCA assay in mammalian cells using the substrate nitrocefin.
A further object of the invention is an in vivo positive
egative fluorescence PCA assay in mammalian cells using the substrate CCF2/AM.
Still, another object of the invention is positive and negative survival assays using cephalosporin-cytotoxic pro-drug conjugates.
An additional object of the invention is positive and negative survival assays using a series of &bgr;-lactamase point mutations that would be predicted to enhance the efficiency of the &bgr;-lactamase PCA.
We have found that these objects and others are achieved by the use of
E. coli
TEM-1 &bgr;-Lactamase as further described below.
REFERENCES:
patent: 2002/0004202 (2002-01-01), Cornish
patent: 2004/0038317 (2004-02-01), Balint et al.
Galarneau André
Michnick Stephen William Watson
Angres Isaac A.
Fredman Jeffrey
Odyssey Thera Inc.
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