Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat surfactant protein A (SP-A) was expressed in a Chinese hamster ovary (CHO-K1) cell line and characterized for biologic activity using assays for receptor binding and modulation of phospholipid secretion from isolated type II cells. The CHO-K1 cell line was cotransfected with separate plasmids encoding for the rat SP-A, dihydrofolate reductase and neomycin phosphotransferase, respectively. Antibiotic (Geneticin-G418)-resistant transformants were screened by ELISA for the secretion of recombinant SP-A into the media. Northern analysis of the transfected cell lines demonstrated the expression of both 1.6 kb and 0.9 kb mRNA species for SP-A, consistent with the proposed differential polyadenylation of the primary transcript. Amplification with methotrexate resulted in a dose-dependent increase in mRNA for SP-A and a 20-fold increase in the production of recombinant SP-A relative to untreated cells. Maximum production of SP-A was 370 micrograms of SP-A/l of media in a 4-day incubation. Recombinant SP-A was purified from the serum-free media of large scale cultures of transfected, amplified CHO cells by affinity chromatography on mannose-Sepharose. The recombinant SP-A migrated similarly to native SP-A by NaDodSO4-PAGE analysis under reducing and nonreducing conditions and under reducing conditions after digestion with N-glycanase. Recombinant SP-A effectively competed with 125I-native SP-A for binding to the high affinity receptor for SP-A on isolated plasma membranes from rat alveolar type II cells. The recombinant SP-A was as effective as native SP-A in the inhibition of secretion of phospholipid from isolated type II cells. We conclude that recombinant rat SP-A produced in Chinese hamster ovary cells is physically and functionally similar to native rat SP-A.
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PMID:Expression and characterization of rat surfactant protein A synthesized in Chinese hamster ovary cells. 217 80