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)

Several years ago, we proposed that polypeptide regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) target intracellular proteins for destruction (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). To test the PEST hypothesis, we have produced chimeric proteins in which the N or C terminus of mouse dihydrofolate reductase is extended by the PEST-containing C terminus of mouse ornithine decarboxylase. Oligonucleotides encoding the 37 C-terminal residues of mouse ornithine decarboxylase (mODC) or equivalent lengths of dissimilar amino acids were inserted at appropriate sites in a dihydrofolate reductase (DHFR) expression vector. The various fusion proteins were expressed in Escherichia coli and purified to homogeneity by enzyme affinity chromatography. All purified fusion proteins exhibited similar abilities to convert dihydrofolate to tetrahydrofolate, thereby demonstrating that the attachment of peptide extensions to either terminus did not prevent the proper folding of DHFR. Metabolic stabilities of the radioiodinated fusion proteins were assayed in rabbit reticulocyte lysate or Xenopus egg extract. Proteolysis was found to be energy-dependent with mODC-DHFR fusion proteins being degraded from 2 to almost 40-fold faster than the parental DHFR molecule or DHFR fusion proteins bearing non-PEST extensions. Deletion of most of the PEST region from the mODC extension resulted in a significantly more stable fusion protein. Rapid proteolysis of DHFR proteins containing intact mODC extensions provides support for the PEST hypothesis.
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PMID:The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the pest hypothesis. 204 Jun 28

We have developed a systematic and generic way to improve recombinant protein productivities in stable transfections by applying mRNA and protein destabilizing elements to reduce selection marker expression strength. Interferon-gamma (IFNgamma) expression vectors containing different combinations of AU-rich elements (ARE) and mouse ornithine decarboxylase (MODC) PEST region on the amplifiable dihydrofolate reductase (dhfr) selection marker were stably transfected into CHO-DG44 cells. Improvements in specific IFNgamma productivities were 1.7-, 6.6- and 13.3-fold with the application of ARE, MODC PEST, and both ARE and MODC PEST, respectively. To further enhance productivities, compatibility of the destabilizing sequences with methotrexate (MTX) amplification was validated by amplifying the transfected cells to 50nM MTX. A 14- to 27-fold increase in specific IFNgamma productivities were observed after amplification, indicating the compatibility of the two systems. A high specific IFNgamma productivity of 1.05pg/cell/day was also attained by the amplified cell pool with both ARE and MODC PEST.
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PMID:Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44. 1736 64

A method combining the use of a destabilized dihydrofolate reductase (DHFR) selection marker with methotrexate (MTX) amplification to generate high-expressing cells is described here. The selection marker expression is weakened with the use of the murine ornithine decarboxylase PEST region and AU-rich element to target the DHFR protein and mRNA, respectively, for degradation in the cell. Cells that produce higher levels of DHFR protein, and the adjoining recombinant protein gene, can compensate for the more rapid turnover of the DHFR protein and survive the selection process. This effect can complement MTX amplification to reduce the amount of MTX and shorten the time needed to generate a high-expressing clone. The gene of interest is first inserted into an expression vector that contains a destabilized DHFR selection marker. The resulting expression vector is then linearized and transfected into suspension CHO-DG44 cells. Selection is performed by culturing the cells in a selection medium lacking hypoxanthine and thymidine. Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.
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PMID:Generation of high-expressing cells by methotrexate amplification of destabilized dihydrofolate reductase selection marker. 2198 53