Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S]
APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary arte...
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doaj-1a8746f8461148e69d310f27b1354f872021-04-29T04:38:57ZengElsevierJournal of Lipid Research0022-22752020-02-01612244251Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S]Mark Castleberry0Xenia Davis1Min Liu2Thomas B. Thompson3Patrick Tso4W. Sean Davidson5Departments of Molecular Genetics, Biochemistry, and MicrobiologyUniversity of Cincinnati College of Medicine, Cincinnati, OHPathology and Laboratory Medicine,University of Cincinnati College of Medicine, Cincinnati, OHPathology and Laboratory Medicine,University of Cincinnati College of Medicine, Cincinnati, OHDepartments of Molecular Genetics, Biochemistry, and MicrobiologyUniversity of Cincinnati College of Medicine, Cincinnati, OHPathology and Laboratory Medicine,University of Cincinnati College of Medicine, Cincinnati, OHTo whom correspondence should be addressed davidswm@ucmail.uc.edu; Pathology and Laboratory Medicine,University of Cincinnati College of Medicine, Cincinnati, OH; To whom correspondence should be addressed davidswm@ucmail.uc.eduAPOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.http://www.sciencedirect.com/science/article/pii/S0022227520435400lipoproteinslipid and lipoprotein metabolismtriglyceridesdyslipidemias |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mark Castleberry Xenia Davis Min Liu Thomas B. Thompson Patrick Tso W. Sean Davidson |
spellingShingle |
Mark Castleberry Xenia Davis Min Liu Thomas B. Thompson Patrick Tso W. Sean Davidson Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S] Journal of Lipid Research lipoproteins lipid and lipoprotein metabolism triglycerides dyslipidemias |
author_facet |
Mark Castleberry Xenia Davis Min Liu Thomas B. Thompson Patrick Tso W. Sean Davidson |
author_sort |
Mark Castleberry |
title |
Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S] |
title_short |
Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S] |
title_full |
Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S] |
title_fullStr |
Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S] |
title_full_unstemmed |
Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH[S] |
title_sort |
functional recombinant apolipoprotein a5 that is stable at high concentrations at physiological ph[s] |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
2020-02-01 |
description |
APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid. |
topic |
lipoproteins lipid and lipoprotein metabolism triglycerides dyslipidemias |
url |
http://www.sciencedirect.com/science/article/pii/S0022227520435400 |
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