Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts

被引:30
作者
Massou, S
Puech, V
Talmont, F
Demange, P
Lindley, ND
Tropis, M
Milon, A
机构
[1] CNRS, IPBS, F-31077 Toulouse, France
[2] CNRS, UMR INSA, Lab Biotechnol Bioprocedes, F-31077 Toulouse 4, France
[3] INSA, UR INRA, F-31077 Toulouse 4, France
关键词
carbohydrates; deuterium labelling of lipids; human mu-opiate receptor; membrane proteins; Pichia angusta; Pichia pastoris;
D O I
10.1023/A:1008371118772
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
Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human mu-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%-90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water.
引用
收藏
页码:231 / 239
页数:9
相关论文
共 70 条
[1]   2-DIMENSIONAL NMR-STUDIES OF THE INTERACTIONS BETWEEN A PEPTIDE OF CHOLERA-TOXIN AND MONOCLONAL-ANTIBODIES [J].
ANGLISTER, J ;
SCHERF, T ;
ZILBER, B ;
LEVY, R .
BIOPOLYMERS, 1995, 37 (06) :383-389
[2]   Interaction of a type II myosin with biological membranes studied by 2H solid state NMR [J].
Arêas, JAG ;
Gröbner, G ;
Glaubitz, C ;
Watts, A .
BIOCHEMISTRY, 1998, 37 (16) :5582-5588
[3]   DEUTERIUM NMR ANALYSIS OF THE STRUCTURE AND DYNAMICS OF MEMBRANE-COMPONENTS [J].
AUGE, S ;
MARSAN, MP ;
CZAPLICKI, J ;
DEMANGE, P ;
MULLER, I ;
TROPIS, M ;
MILON, A .
JOURNAL DE CHIMIE PHYSIQUE ET DE PHYSICO-CHIMIE BIOLOGIQUE, 1995, 92 (10) :1715-1720
[4]   Membrane structure and dynamics as viewed by solid-state NMR spectroscopy [J].
Auger, M .
BIOPHYSICAL CHEMISTRY, 1997, 68 (1-3) :233-241
[5]   PREPARATION OF ISOTOPICALLY LABELED RIBONUCLEOTIDES FOR MULTIDIMENSIONAL NMR-SPECTROSCOPY OF RNA [J].
BATEY, RT ;
INADA, M ;
KUJAWINSKI, E ;
PUGLISI, JD ;
WILLIAMSON, JR .
NUCLEIC ACIDS RESEARCH, 1992, 20 (17) :4515-4523
[6]   Improved large scale culture of Methylophilus methylotrophus for C-13/N-15 labeling and random fractional deuteration of ribonucleotides [J].
Batey, RT ;
Cloutier, N ;
Mao, HY ;
Williamson, JR .
NUCLEIC ACIDS RESEARCH, 1996, 24 (23) :4836-4837
[7]  
Befort K, 1996, MOL PHARMACOL, V49, P216
[8]  
BERSCH B, 1993, J BIOMOL NMR, V3, P443
[9]  
Blake AD, 1997, RECEPTOR CHANNEL, V5, P231
[10]   Characterization of NADP(+) binding to perdeuterated MurB: Backbone atom NMR assignments and chemical-shift changes [J].
Constantine, KL ;
Mueller, L ;
Goldfarb, V ;
Wittekind, M ;
Metzler, WJ ;
Yanchunas, J ;
Robertson, JG ;
Malley, MF ;
Friedrichs, MS ;
Farmer, BT .
JOURNAL OF MOLECULAR BIOLOGY, 1997, 267 (05) :1223-1246