Human low density lipoprotein receptor fragment - Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli

被引:38
作者
Simmons, T [1 ]
Newhouse, YM [1 ]
Arnold, KS [1 ]
Innerarity, TL [1 ]
Weisgraber, KH [1 ]
机构
[1] UNIV CALIF SAN FRANCISCO,GLADSTONE INST CARDIOVASC DIS,CARDIOVASC RES INST,DEPT PATHOL,SAN FRANCISCO,CA 94141
关键词
D O I
10.1074/jbc.272.41.25531
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of Lipoprotein particles by high affinity binding to its ligands, apolipoprotein (ape) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds, As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the Ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion, Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions, We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency, The active fragment was isolated and purified with an LDL affinity column, The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing, The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked, In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of I-125-LDL to fibroblast LDL receptors, We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.
引用
收藏
页码:25531 / 25536
页数:6
相关论文
共 40 条
[1]  
ARNOLD KS, 1992, LIPOPROTEIN ANAL PRA, P145
[2]  
AUSUBEL FM, 1997, CURRENT PROTOCOLS MO, V2
[3]  
BEISIEGEL U, 1981, J BIOL CHEM, V256, P1923
[4]   Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL receptor [J].
Blacklow, SC ;
Kim, PS .
NATURE STRUCTURAL BIOLOGY, 1996, 3 (09) :758-762
[5]   A RECEPTOR-MEDIATED PATHWAY FOR CHOLESTEROL HOMEOSTASIS [J].
BROWN, MS ;
GOLDSTEIN, JL .
SCIENCE, 1986, 232 (4746) :34-47
[6]   HOW LDL RECEPTORS INFLUENCE CHOLESTEROL AND ATHEROSCLEROSIS [J].
BROWN, MS ;
GOLDSTEIN, JL .
SCIENTIFIC AMERICAN, 1984, 251 (05) :58-&
[7]   How important is the molten globule for correct protein folding? [J].
Creighton, TE .
TRENDS IN BIOCHEMICAL SCIENCES, 1997, 22 (01) :6-10
[8]   3-DIMENSIONAL STRUCTURE OF THE 2ND CYSTEINE-RICH REPEAT FROM THE HUMAN LOW-DENSITY-LIPOPROTEIN RECEPTOR [J].
DALY, NL ;
DJORDJEVIC, JT ;
KROON, PA ;
SMITH, R .
BIOCHEMISTRY, 1995, 34 (44) :14474-14481
[9]   3-DIMENSIONAL STRUCTURE OF A CYSTEINE-RICH REPEAT FROM THE LOW-DENSITY-LIPOPROTEIN RECEPTOR [J].
DALY, NL ;
SCANLON, MJ ;
DJORDJEVIC, JT ;
KROON, PA ;
SMITH, R .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (14) :6334-6338
[10]  
Goldstein JL, 1989, METABOLIC BASIS INHE, P1215