Expression, purification, and characterization of soluble K-Ras4B for structural analysis

被引：18

作者：

Abraham, Sherwin J. ^{[1
]}

Muhamed, Ismaeel ^{[1
]}

Nolet, Ryan ^{[1
]}

Yeung, Fung ^{[1
]}

Gaponenko, Vadim ^{[1
]}

机构：

[1] Univ Illinois, Dept Biochem & Mol Genet, Chicago, IL 60607 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2010年 / 73卷 / 02期

关键词：

K-Ras4B; NMR; SPR; Hypervariable region; Nanodiscs; K-RAS; H-RAS; ESCHERICHIA-COLI; N-RAS; PLASMA-MEMBRANE; PROTEIN; P21; ACTIVATION; GTPASE; DOMAIN;

D O I：

10.1016/j.pep.2010.05.015

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A p21 GTPase K-Ras4B plays an important role in human cancer and represents an excellent target for cancer therapeutics. Currently, there are no drugs directly targeting K-Ras4B. In part, this is due to the lack of structural information describing unique features of K-Ras4B. Here we describe a methodology allowing production of soluble, well-folded K-Ras4B for structural analysis. The key points in K-Ras4B preparation are low temperature expression and extraction of K-Ras4B from the insoluble fraction using a nucleotide loading procedure in the presence of Mg2+ and citrate, a low affinity chelator. Additionally, a significant amount of K-Ras4B could be extracted from the soluble fraction. We show that recombinant K-Ras4B is monomeric in solution. Excellent NMR signal dispersion suggests that the protein is well-folded and is amenable to solution structure determination. In addition, using phospholipid bilayer nanodiscs we show that recombinant K-Ras4B interacts with lipids and that this interaction is mediated by the C-terminal hypervariable region. (C) 2010 Elsevier Inc. All rights reserved.

引用

页码：125 / 131

页数：7

共 35 条

[1] The Hypervariable Region of K-Ras4B Is Responsible for Its Specific Interactions with Calmodulin [J].

Abraham, Sherwin J. ;

Nolet, Ryan P. ;

Calvert, Richard J. ;

Anderson, Lucy M. ;

Gaponenko, Vadim .

BIOCHEMISTRY, 2009, 48 (32) :7575-7583

[2]

Andres Douglas A, 2004, Methods Mol Biol, V284, P163

[3] Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli [J].

Bleimling, Nathalie ;

Alexandrov, Kirill ;

Goody, Roger ;

Itzen, Aymelt .

PROTEIN EXPRESSION AND PURIFICATION, 2009, 65 (02) :190-195

[4]

CAMPBELLBURK SL, 1995, METHOD ENZYMOL, V255, P3

[5] Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor [J].

Clyde-Smith, J ;

Silins, G ;

Gartside, M ;

Grimmond, S ;

Etheridge, M ;

Apolloni, A ;

Hayward, N ;

Hancock, JF .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (41) :32260-32267

[6] NMRPIPE - A MULTIDIMENSIONAL SPECTRAL PROCESSING SYSTEM BASED ON UNIX PIPES [J].

DELAGLIO, F ;

GRZESIEK, S ;

VUISTER, GW ;

ZHU, G ;

PFEIFER, J ;

BAX, A .

JOURNAL OF BIOMOLECULAR NMR, 1995, 6 (03) :277-293

[7] ISOLATION AND REFOLDING OF H-RAS FROM INCLUSION-BODIES OF ESCHERICHIA-COLI - REFOLD PROCEDURE AND COMPARISON OF REFOLDED AND SOLUBLE H-RAS [J].

DELOSKEY, RJ ;

VANDYK, DE ;

VANAKEN, TE ;

CAMPBELLBURK, S .

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1994, 311 (01) :72-78

[8]

FEUERSTEIN J, 1987, J BIOL CHEM, V262, P8455

[9] K-ras as a target for cancer therapy [J].

Friday, BB ;

Adjei, AA .

BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON CANCER, 2005, 1756 (02) :127-144

[10] Review on bacterial stress topics [J].

Giuliodori, Anna Maria ;

Gualerzi, Claudio O. ;

Soto, Sara ;

Vila, Jordi ;

Tavio, Maria M. .

STRESS RESPONSES IN BIOLOGY AND MEDICINE: STRESS OF LIFE IN MOLECULES, CELLS, ORGANISMS, AND PSYCHOSOCIAL COMMUNITIES, 2007, 1113 :95-104

← 1 2 3 4 →