Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

256 Li et al.
In order for these experiments to achieve common use, it is important to be
able to isotopically label a protein in an efficient and cost-effective manner and
to purify it in good yield, both uniformly and specifically. Practically, if the
protein under consideration for NMR studies can be cloned and expressed in
Escherichia coli, uniform labeling with 15 N is relatively straightforward and
inexpensive by using defined media containing [ 15 N, 99%] ammonium chloride
or ammonium sulfate as the nitrogen source. Uniform (>95%) labeling
with 13 C is also relatively straightforward by using defined media containing
[ 13 C 6, 99%] glucose. [ 13 C 6, 99%] glucose is the most reliable method in terms
of giving high yield and high 13 C-incorporation. It is also much less expensive
than commercially available 13 C-enriched media. Replacement of [ 13 C 6, 99%]
glucose by other reagents like [1,2– 13 C 2, 99%] acetate is possible (4). Partial or
complete aliphatic 2 H incorporation has been obtained by growth of E. coli in
defined media containing certain percentage of D 2O and backbone 1 H N can be
exchanged out by dissolving the sample in D 2O. In terms of efficient type-specific
labeling, the host bacteria can be grown on a defined medium supplemented with
one or more isotope-labeled amino acids or amino acid precursors.
In the past few years, we have enjoyed great success in isotope labeling the
Ca 2+ -binding protein, troponin C. Using the pET expression system and the
isopropyl β-D-thiogalactopyranoside (IPTG) induction protocol of Studier et al.
(5), we are able to efficiently produce tens to hundreds of milligrams of the
labeled protein from liter-scale growths of host E. coli. These labeled proteins
made it possible for us to determine the solution structures and dynamics of skeletal
and cardiac troponin C in a variety of states (6). We will summarize the procedures
in this chapter. The methods described here should be applicable to other
Ca 2+ -binding proteins with their proper expression system and bacterial host.
2. Materials
1. Expression Medium (Modified M9 Medium), (1 L):
a. NaH2PO4 (6 g)
b. K2HPO4 (3 g)
c. NaCl (0.5 g)
d. (NH4) 2SO4 (1 g) (see Notes 1 and 2)
e. D-glucose (2–10 g) (see Notes 3–5)
f. 1 M MgSO4 (4 mL) (see Note 6)
g. 1 mM FeSO4 (2 mL) (see Note 7)
h. Mineral Mixture (0.5 mL) (optional, see Note 8)
i. Vitamins and trace elements mixture (10 mL) (see Note 8)
j. Appropriate antibiotics
k. pH = 7.5
Dissolve NaH2PO4, K2HPO4, and NaCl in 1 L of double distilled (dd) H2O, adjust
pH to ~7.5, and divide into two 2-L flasks with 500 mL each. Stop flask with a