High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression
| Autor: | Mary G. Sorci-Thomas, Michael J. Thomas, C Zhang, Mary W. Kearns, G N Pate, John S. Parks |
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| Rok vydání: | 1996 |
| Předmět: |
Mutant
Cell Wild type Phospholipid QD415-436 Cell Biology Biochemistry High-performance liquid chromatography law.invention chemistry.chemical_compound Endocrinology medicine.anatomical_structure chemistry law Recombinant DNA medicine lipids (amino acids peptides and proteins) Secretion Polyacrylamide gel electrophoresis |
| Zdroj: | Journal of Lipid Research, Vol 37, Iss 3, Pp 673-683 (1996) Scopus-Elsevier |
| ISSN: | 0022-2275 |
| DOI: | 10.1016/s0022-2275(20)37608-2 |
| Popis: | To facilitate the investigation of apoA-I structure:function relationships as they relate to LCAT activation and lipid binding, we have developed an apoA-I baculoviral expression and purification system that yields milligram quantities of wild-type or mutant proapoA-I. Baculovirus-infected Sf-9 cells, grown in suspension, were found to secrete high levels of human wild-type (40-50 mg/l) or mutant apoA-I protein (1-38 mg/l), which was determined to be > 95% pure following a two-step purification procedure. In the case of wild-type apoA-I, ELISA showed that approximately 13-18% of the total protein secreted into the culture medium was apoA-I. To isolate pure protein from culture medium, 72 h post-infection medium was subjected to preparative reverse phase high performance liquid chromatography (HPLC), followed by DEAE ion-exchange chromatography. Purity and molecular size determination of wild-type proapoA-I protein was verified by SDS polyacrylamide gel electrophoresis, electrospray mass spectrometry, and N-terminal sequencing. In addition, recombinant discoidal apoA-I:phospholipid complexes prepared from wild-type or plasma apoA-I showed similar particle size and LCAT activation properties. To fully characterize the utility of this expression system, the expression levels of various mutant apoA-I proteins were compared to wild-type. Despite a lower production level seen with selected apoA-I mutants, milligram quantities of these purified mutant proteins were also obtained. In summary, we show that baculovirus-derived wild-type proapoA-I shows properties similar to plasma apoA-I relative to recombinant HDL formation, LCAT reactivity, and alpha-helical content. In addition, we show that a variety of mutant forms of human proapoA-I can be expressed and purified in abundant quantity from baculoviral-infected Sf-9 cells. |
| Databáze: | OpenAIRE |
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