title: Expression and characterization of rat surfactant protein A synthesized in Chinese hamster ovary cells.
abstract: 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.
authors.affiliation: Lord and Taylor Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO.
authors.forename: F X
authors.initials: FX
authors.lastname: McCormack
authors.forename: J H
authors.initials: JH
authors.lastname: Fisher
authors.forename: A
authors.initials: A
authors.lastname: Suwabe
authors.forename: D L
authors.initials: DL
authors.lastname: Smith
authors.forename: J M
authors.initials: JM
authors.lastname: Shannon
authors.forename: D R
authors.initials: DR
authors.lastname: Voelker
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D002460:Cell Line; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D015870:Gene Expression; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase; D008727:Methotrexate / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D010957:Plasmids; D011510:Proteolipids / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic
chemical_list: D005839:Gentamicins; D010743:Phospholipids; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; C010680:antibiotic G 418; D013762:Tetrahydrofolate Dehydrogenase; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D008727:Methotrexate