Modifications of Wheat Germ Cell-Free System for Functional Proteomics of Plant Membrane Proteins - Plant Proteomics: Methods and Protocols - page 258

a b

M TE T E

kDa

75

50

37

25
20

BR

Fig. 3 Purifi cation of bR. ( a ) Purifi cation of bR. His 6 -tagged bR synthesized in the
presence of 1 mM FC14 and 1.5 mM CHAPS was purifi ed using Ni-NTA-agarose, and
visualized by SDS-PAGE and CBB staining. Lane M, molecular size markers; lane T,
1 l of the imidazole-eluted fraction. ( b ) Photographs

of the translation mixture (T) and the imidazole-eluted fraction (E). The purple color
indicates that the protein was folded correctly (reproduced from ref. 3 )

μ l of translation mixture; lane E, 2 μ

3.10 Purifi cation
of bR Protein

1. Equilibrate 200 μ l of Ni-NTA-agarose with buffer A.

2. Incubate 200 l of the

equilibrated Ni-NTA-agarose and 4 ml of buffer A at 4 °C for
1 h with gentle shaking.
3. Load the Ni-NTA-agarose into a gravity-fl ow column. Save
the fl ow-through for purifi cation analysis.
4. Add 0.5 ml of buffer B to wash out nonspecifi cally bound pro-
teins. Repeat this step four times.
5. Add 0.2 ml of buffer C to the column.
6. Evaluate the purifi cation by SDS-PAGE and CBB staining
(Fig. 3 ).

μ l of the translation mixture with 200 μ

N -myristoylation is a co-translational modifi cation of N -terminal
glycine residues. This modifi cation is catalyzed by myristoyl-CoA:
protein N -myristoyltransferase, and involves the covalent attach-
ment of myristic acid to the N -terminal Gly that is exposed during
co-translational elimination of the initiating Met by methionine
aminopeptidase [ 6 , 7 ]. N -myristoylation facilitates the interaction
of target proteins with membranes by anchoring the proteins
through miristoyl group. Recently, we reported the consensus
motif for N -myristoylation in a WGCF system [ 8 ]. In that publica-
tion, a method for synthesis of N -myristoylated proteins by the
WGCF system was established. In the following Subheadings 3.11 -
3.14 , this method using AtCBL1 as a model protein is described.
AtCBL1 is a calcium-binding protein belonging to calcineurin

3.11 Batch-Mode
Synthesis of
Myristoylated AtCBL1
in the Presence of
[ 14 C]Myristic Acid