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)

We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.
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PMID:Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C. 247 19

We have developed a host cell/vector system based on the use of adenovirus-transformed cells and a promoter, designated GBMT, capable of being activated by the Ela tumor antigen produced in these cells. GBMT-based vectors were constructed with hygromycin phosphotransferase and murine dihydrofolate reductase as selective markers. We demonstrate their utility in two adenovirus-transformed cell lines, human kidney 293 and Syrian hamster AV12-664. Further, we describe methods and conditions for the direct adaptation of isolated recombinant clones to serum-free suspension growth conditions. For exemplary purposes, we describe the generation of stable recombinant 293 cell lines with single-copy integrated vectors secreting the highly complex clotting factor human protein C at levels as high as 20 mg/l in serum-free suspension culture. In addition, using the AV12-664 cell line with GBMT and direct dominant selection of the dhfr gene, we have isolated clones secreting a tissue plasminogen activator derivative at levels of about 40 mg/l under serum-free suspension conditions. The distinct advantages of this vector/host cell system are 1) the direct selection of stable clones expressing relatively high levels of recombinant protein, eliminating the need for the tedious stepwise gene amplification process and 2) the direct adaptation to serum-free suspension culture.
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PMID:High-level expression of secreted proteins from cells adapted to serum-free suspension culture. 833 65

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.
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PMID:Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. 1517 95