文档介绍：BioNMR in Drug Research. Edited by Oliver Zerbe 1
Copyright © 2002 Wiley-VCH Verlag GmbH & Co. KGaA
ISBNs: 3-527-30465-7 (Hardback); 3-527-60066-3 (Electronic)
1
Modern Methods for the Expression of Proteins
in Isotopically Enriched Form
Heiko Patzelt, Natalie Goto, Hideo Iwai, h Lundstrom,
and Erhard Fernholz
Introduction
The introduction of stable isotopes into proteins has significantly reduced the time re-
quirements for structure elucidation of biomolecules. Moreover, structural studies of pro-
teins with molecular weights exceeding the 10 kDa limit are usually not possible without
uniform isotope labeling because of severe resonance overlap and inefficient coherence
transfer along the rather small 3J 1H-1H couplings. Nowadays, efficient expression of re-
combinant proteins is a prerequisite for many techniques used in structural biology, but
the requirement for isotope labeling in particular often precludes NMR (nuclear mag-
netic resonance) structure determination of proteins isolated from natural sources. Speci-
fically, proteins that have been uniformly labeled with 15N and/or 13C monly re-
quired for NMR spectroscopy, especially for backbone chemical shift assignment proce-
dures, which are greatly facilitated by the use of a series of rather sensitive multi-dimen-
sional triple-resonance NMR experiments (see Chapt. 4) [1], in a process that can also be
automated with good ess [2]. Moreover, the random replacement of nonexchangeable
protons by deuterons reduces 1H-1H dipolar interactions and scalar couplings, thereby re-
ducing peak line widths considerably and allowing structure elucidation of proteins ex-
ceeding 30 kDa [3, 4]. Random fractionally deuterated protein samples also permit the
use of longer mixing times in NOESY (nuclear Overhauser effect spectroscopy) experi-
ments, since spin-diffusion pathways are largely eliminated. In addition, transverse-re-
laxation optimized spectroscopy (TROSY [5], see also Chapt. 1