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Title: 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. In conclusion, purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux frommore » cholesterol loaded J774 macrophages.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [4] ; ORCiD logo [1] ;  [1] ;  [4] ;  [3] ;  [2] ;  [1]
  1. Children's Hospital Oakland Research Inst., Oakland CA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry
  3. California State Univ. (CalState), Long Beach, CA (United States). Dept. of Chemistry and Biochemistry
  4. McGill Univ. Health Centre, Montreal, QC (Canada). Research Inst.
Publication Date:
Grant/Contract Number:
AC02-05CH11231; T32 DK061918; R37 HL-64159
Type:
Accepted Manuscript
Journal Name:
Protein Expression and Purification
Additional Journal Information:
Journal Volume: 134; Journal Issue: C; Journal ID: ISSN 1046-5928
Publisher:
Elsevier
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Apolipoprotein A-I; High density lipoprotein; Thermal denaturation; Cholesterol efflux; Circular dichroism; E. coli; Reversed phase HPLC
OSTI Identifier:
1379871
Alternate Identifier(s):
OSTI ID: 1416242