A facile method for isolation of recombinant human apolipoprotein A-I from E. coli.
Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609, USA.
Nikita
N
Ikon
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609, USA.
Jennifer
J
Shearer
The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720, USA.
Jianfang
J
Liu
Department of Chemistry and Biochemistry, California State University, Long Beach 1250, Bellflower Boulevard, Long Beach, CA 90840, USA.
Jesse J
JJ
Tran
Research Institute of the McGill University Health Centre, Glen Site, EM1.2220, 1001 Boul Decarie, Montreal, QCH4A 3J1 Canada.
ShiBo
S
Feng
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609, USA.
Ayako
A
Kamei
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609, USA.
Jennifer A
JA
Beckstead
Research Institute of the McGill University Health Centre, Glen Site, EM1.2220, 1001 Boul Decarie, Montreal, QCH4A 3J1 Canada.
Robert S
RS
Kiss
Department of Chemistry and Biochemistry, California State University, Long Beach 1250, Bellflower Boulevard, Long Beach, CA 90840, USA.
Paul M
PM
Weers
The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720, USA.
Gang
G
Ren
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609, USA. Electronic address: rryan@chori.org.
Robert O
RO
Ryan
D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D002784:Cholesterol
Apolipoprotein A-I; Cholesterol efflux; Circular dichroism; E. coli; High density lipoprotein; Reversed phase HPLC; Thermal denaturation