Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.
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PMID:Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. 977 Apr 53

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.
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PMID:An in vivo library-versus-library selection of optimized protein-protein interactions. 1040 52

The protein NPR1/NIM1 is required for the induction of systemic acquired resistance (SAR) in plants and has been shown to interact with members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors. However, to date, there is no method available to monitor such interactions in plant cells. We report here an in vivo protein fragment complementation assay (PCA), based on association of reconstituted murine dihydrofolate reductase (mDHFR) with a fluorescent probe to detect protein-protein interaction in planta. We demonstrate that the interaction between Arabidopsis NPR1/NIM1 and the bZIP factor TGA2 is induced by the regulators of SAR, salicylic acid (SA), and its analog 2,6-dichloroisonicotinic acid (INA) with distinct species-specific responses. Furthermore, the induced interaction is localized predominantly in the nucleus. Protein fragment complementation assays could be of value to agricultural research by providing a system for high-throughput biochemical pathway mapping and for screening of small molecules that modulate protein interactions.
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PMID:Direct visualization of protein interactions in plant cells. 1147 72

Recombinant antibodies are increasingly used as therapeutics for a wide variety of diseases. Generation of cell lines expressing high levels of recombinant antibody typically requires labor-intensive cloning and screening steps. We describe a mammalian expression system for the high-level production of full-length antibody molecules. It has been shown that the dihydrofolate reductase (DHFR) selectable marker can be divided into two fragments that, with the aid of a leucine zipper, can re-associate to form an active molecule. Using bicistronic vectors, we linked the expression of each antibody chain to the expression of a DHFR fragment. Survival in selective media requires expression of both DHFR fragments that, by virtue of these vectors, also selects for the expression of both antibody chains. Initial pools produced 5 microg of Ab/10(6) cells/d (qP = microg/10(6) cells/d). Expression of each antibody chain in conjunction with a portion of DHFR also leads to concurrent amplification of both antibody chains in the presence of methotrexate, a DHFR inhibitor, and results in a two- to fivefold increase in antibody production with basal qPs ranging from 10-25 ug/10(6) cells/d. Shake-flask cultures of amplified pools produced up to 600 mg/L of antibody in 7 days. This system allows for rapid generation of antibodies without cloning and greatly simplifies selection of cell lines for the production of potential antibody therapeutics.
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PMID:High-level expression of full-length antibodies using trans-complementing expression vectors. 1457 1