Preparation of Isotopically Labeled Recombinant Fragments of Fibronectin for Functional and Structural Study by Heteronuclear Nuclear Magnetic Resonance Spectroscopy - Methods in Molecular Biology: Extracellular Matrix Protocols - page 57

mechanisms, and protein-ligand interactions. The introduction of isotope-
labeling of proteins in conjunction with heteronuclear experiments has
increased the upper limit for three-dimensional protein structure determination
using NMR, from ~100 residues (for unlabeled proteins) to around 300 resi-
dues (for 15 N / 13 C-labeled proteins) (13 , 14) .
Labeling of proteins with 15 N and/or 13 C allows the extension of NMR
experiments into three and four dimensions, reducing the overlap of signals
and facilitating the extraction of the interproton distance information used in
structure calculations. Isotope labeling also allows additional information to
be obtained, e.g., about protein backbone dynamics (15) . These advantages
can only be exploited if isotope-labeling can be performed cost-effectively,
especially for labeling with the relatively expensive isotope 13 C.
Escherichia coli has been used extensively for the production of recombi-
nant isotopically labeled proteins (16) and has been used to express recombi-
nant fibronectin fragments, although the recombinant material often needs to
be exposed to an involved refolding and purification procedure (17) . We have
found that Pichia pastoris can secrete recombinant fibronectin fragments very
efficiently, without the need for refolding, but to date there are only two reports
of isotope-labeling in this system (18 - 19C) . We have developed a procedure
for the cost-effective labeling of recombinant fibronectin fragments by grow-
ing P. pastoris strains in isotopically enriched media in a small fermentor.
The preparation of an isotopically labeled NMR sample is a multistep pro-
cedure. Typically, this involves the design, generation, and screening of
recombinant clones, their growth in isotopically enriched media, purification
of the recombinant product, and assessment of its purity and integrity by meth-
ods such as N-terminal peptide sequencing, mass spectrometry and 1-dimen-
sional NMR. Coverage of all of these areas is beyond the scope of this chapter;
here we describe a procedure for expression of isotope-labeled fibronectin
modules and an initial purification step that we have found well suited to this
system. Methodologies for the design, generation, and screening of P. pastoris
strains use well-established molecular biological techniques and are described
in detail elsewhere (20-24) . Procedures for the final stages of protein purifica-
tion have to be tailored to the individual protein and these procedures and those
for NMR sample preparation are also described elsewhere (25 , 26) .
The small fermentor used in this procedure is described in detail below; the
strategy we employ uses media adapted from (27) and has been developed to
make relatively small quantities of recombinant proteins reproducibly, using
cost-effective amounts of labeled substrates ( Fig. 1 ). However, it is a “first-
shot” approach, which should be tried with unlabeled reagents to assess the
performance of a given clone; the scale of the culture and/or the feed times can
be adjusted to compensate for clones that perform poorly. Fermentation of