High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli
Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lac...
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doaj-93d182fbdc2a450cb9cc608277ac48e82021-04-28T06:05:33ZengElsevierJournal of Lipid Research0022-22752012-08-0153817081715High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coliLoren E. Smith0Jun Yang1Leah Goodman2Xinqi Huang3Rong Huang4James Dressman5Jamie Morris6R. A. Gangani D. Silva7W. Sean Davidson8Giorgio Cavigiolio9Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andDepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andthe Children's Hospital Oakland Research Institute, Oakland, CA 94609the Children's Hospital Oakland Research Institute, Oakland, CA 94609Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andDepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andDepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andDepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andTo whom correspondence should be addressed. e-mail: gcavigiolio@chori.org; parafilm@tiscali.it; or davidswm@ucmail.uc.edu.; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273 andTo whom correspondence should be addressed. e-mail: gcavigiolio@chori.org; parafilm@tiscali.it; or davidswm@ucmail.uc.edu.; the Children's Hospital Oakland Research Institute, Oakland, CA 94609Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.http://www.sciencedirect.com/science/article/pii/S0022227520418759inteinfusion proteinaffinity tagbacteriainexpensivelow temperature cleavage |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Loren E. Smith Jun Yang Leah Goodman Xinqi Huang Rong Huang James Dressman Jamie Morris R. A. Gangani D. Silva W. Sean Davidson Giorgio Cavigiolio |
spellingShingle |
Loren E. Smith Jun Yang Leah Goodman Xinqi Huang Rong Huang James Dressman Jamie Morris R. A. Gangani D. Silva W. Sean Davidson Giorgio Cavigiolio High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli Journal of Lipid Research intein fusion protein affinity tag bacteria inexpensive low temperature cleavage |
author_facet |
Loren E. Smith Jun Yang Leah Goodman Xinqi Huang Rong Huang James Dressman Jamie Morris R. A. Gangani D. Silva W. Sean Davidson Giorgio Cavigiolio |
author_sort |
Loren E. Smith |
title |
High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli |
title_short |
High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli |
title_full |
High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli |
title_fullStr |
High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli |
title_full_unstemmed |
High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli |
title_sort |
high yield expression and purification of recombinant human apolipoprotein a-ii in escherichia coli |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
2012-08-01 |
description |
Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies. |
topic |
intein fusion protein affinity tag bacteria inexpensive low temperature cleavage |
url |
http://www.sciencedirect.com/science/article/pii/S0022227520418759 |
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