title: High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression.
abstract: 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.
authors.affiliation: Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1040, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Sorci-Thomas
authors.forename: J S
authors.initials: JS
authors.lastname: Parks
authors.forename: M W
authors.initials: MW
authors.lastname: Kearns
authors.forename: G N
authors.initials: GN
authors.lastname: Pate
authors.forename: C
authors.initials: C
authors.lastname: Zhang
authors.forename: M J
authors.initials: MJ
authors.lastname: Thomas
mesh_terms: D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008075:Lipoproteins, HDL / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D009154:Mutation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D011498:Protein Precursors; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase