Page 47 - Macromolecular Crystallography

36 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 2.7 Expression of SeMet labelled proteins II

IPTG induction protocol using the ‘poisoning’ Autoinduction protocol using the ‘poisoning’
method method
This method follows the same protocol as for production of The method follows that for autoinduction using the
SeMet labelled protein using the auxotroph, however on auxotroph B834, however at Stage 4, 9 ml 10 mg/ml
addition of IPTG, the following chemicals are also added to selenomethionine is added along with the following
each 500 ml culture: amino acids:
2.5 ml 10 mg/ml SeMet 5 ml 10 mg/ml lysine
5 ml 10 mg/ml lysine 5 ml 10 mg/ml threonine
5 ml 10 mg/ml threonine 5 ml 10 mg/ml phenylalanine
5 ml 10 mg/ml phenylalanine 5 ml 5 mg/ml leucine
5 ml 5 mg/ml leucine 5 ml 5 mg/ml isoleucine
5 ml 5 mg/ml isoleucine 5 ml 5 mg/ml valine.
5 ml 5 mg/ml valine.

Protocol 2.8 Lysis using cell disruption or sonication
1. Cell pellets are thawed for 15 min at room temperature or
prior to resuspension. The lysate is sonicated on ice at 20–25% power, 9.9 sec
2. The cell pellets are resuspended in ∼30 ml per 10 g pulse on, 9.9 sec pulse off (500W) for ∼15 min. At this
pellet of Cell Lysis Buffer (50 mM Tris pH 7.5, 500 mM stage the lysate should no longer be viscous and the cells
NaCl, 20 mM imidazole, and 0.2% Tween) with protease fully lysed.
◦
inhibitors and DNaseI as required. 4. The lysate is spun at 4 C and 30,000 g for 30 min to
3. The lysate is passed through the basic Z cell disruptor at remove cell debris.
30 kpsi. At this stage the lysate should no longer be viscous 5. The cleared lysate is then filtered through a 45 µm
and the cells fully lysed. membrane before purification.

In this section the use of these systems for HTP affinity step usually follows a simple bind–elute
protein purification is described. protocol, this method both purifies and concen-
trates the protein. For example the target protein
2.4.4.1 Chromatography strategy is eluted from a 1-ml IMAC column within 2–4 ml
The chromatographic strategy is determined by the of buffer containing 0.5 M imidazole. Protocol 2.9
construct chosen during cloning and the number outlines a simple method for IMAC purification in
of chromatography steps desired (Fig. 2.3). In gen- which 20 mM imidazole is used in the binding and
eral, constructs designed for HTP projects contain wash buffers to reduce levels of proteins that bind
an affinity tag to aid in purification, as discussed in non-specifically.
Section 2.2.2.2. Therefore the first step in purification If the sample is of the purity required, only
of a recombinant protein is often affinity chromatog- buffer exchange is needed to give the final prod-
raphy as this is highly specific for the target protein. uct. If further purification is required, a size exclu-
The most common tag used is hexahistidine, which sion chromatography step (SEC) is usually carried
binds to immobilized metal affinity chromatogra- out. This step both removes contaminants from
phy (IMAC) beads, although other tags such as GST the sample, typically giving protein products of
and maltose binding protein are also used. As the >90% purity, and gives information regarding the