Faculty Collaboration Database

High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression. J Lipid Res 1996 Mar;37(3):673-83

Date

03/01/1996

Pubmed ID

8728328

Scopus ID

2-s2.0-0029916540 (requires institutional sign-in at Scopus site)   43 Citations

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.

Author List

Sorci-Thomas MG, Parks JS, Kearns MW, Pate GN, Zhang C, Thomas MJ

Authors

Mary Sorci Thomas PhD Professor in the Medicine department at Medical College of Wisconsin
Michael J. Thomas PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Apolipoprotein A-I
Apolipoproteins A
Baculoviridae
Cell Line
Circular Dichroism
Cloning, Molecular
Gene Expression
Genetic Vectors
Humans
Lipoproteins, HDL
Mass Spectrometry
Molecular Weight
Mutation
Phosphatidylcholine-Sterol O-Acyltransferase
Protein Precursors
Protein Structure, Secondary
Recombinant Proteins
Transfection