Page 46 - Macromolecular Crystallography

HIGH-THROUGHPUT CLONING, EXPRESSION, AND PURIFICATION 35

incorporation (Sreenath et al., 2005). In contrast to frequency sound waves to agitate and lyse the cells.
E. coli expression systems, there are only a few The sound waves are emitted through a probe which
reports of biosynthetic labelling of proteins with is immersed in the cell suspension. As sonication
selenomethionine in eukaryotic cells (insect, mam- causes heating in the sample, the sound waves are
malian, or yeast) (Laurila et al., 2005; McWhirter appliedinshortburstsandthesampleisimmersedin
et al., 1999; Davis et al., 2001). an ice bath. The sample volume and acoustic power
applied are critical in the yield of protein recovered
2.4.3 Cell lysis and in minimizing heating of the sample, which
can lead to protein degradation (Feliu et al.,
At scale-up, cells are generally lysed by mechanical 1998).
means using either a French press or cell disrup-
tor or by sonication (Protocol 2.8). Before breakage, 2.4.4 Chromatography
the cell pellet is resuspended in buffer to give a
uniform cell suspension. The lysis buffer often con- The main challenge in protein purification for HTP
tains a small percentage of detergent which assists facilities is to combine generic multidimensional
with the lysis and aids solubilization of the protein chromatography for all target proteins with the par-
after it has been removed from the cell. In liquid allelization of purification runs. This requirement
homogenization using a French press or cell disrup- has been addressed by the commercial develop-
tor, cells are sheared by forcing the cell suspension ment of semiautomated chromatography systems,
throughanarrowapertureunderhighpressure. Son- notably the Äkta Explorer 3D and Äkta Xpress
ication is widely used in both HTP and non-HTP workstations, both available from GE Healthcare
laboratories for cell breakage and makes use of high (www.chromatography.amershambiosciences.com).

Protocol 2.6 Expression of SeMet-labelled proteins I
IPTG induction protocol with the methionine Autoinduction protocol with the methionine
auxotroph B834 auxotroph B834
1. A single colony is inoculated into 100 ml of SeMet media 1. A single colony is inoculated into 100 ml of glucose-free
containing antibiotics (Molecular Dimensions). The SeMet SeMet media (Molecular Dimensions) containing antibiotics
◦
media consists of 100 ml SeMet Base, 5 ml nutrient mix, and incubated at 37 C and 225 rpm overnight (∼16 h).
and 0.4 ml 10 mg/ml selenomethionine in a 250 ml 2. The culture is centrifuged at 4000 g for 5 min and the
shake-flask. The culture is incubated at 37 C and 225 rpm supernatant discarded.
◦
overnight (∼16 h). 3. The pellet is then resuspended in 25 ml of PBS and
2. The culture is centrifuged at 5000 g for 10 min and recentrifuged at 4000 g for 5 min.
the supernatant discarded. 4. The supernatant is removed and the pellet resuspended
3. The pellet is then resuspended in 50 ml of PBS and in 2 ml PBS before inoculation into 1 L of autoinduction
recentrifuged at 5000 g for 10 min. SeMet media (1 L SeMet Base, 20 ml OnEx Solution 1, 50 ml
4. The supernatant is removed and the pellet is inoculated OnEx Solution 2, 1 ml OnEx Solution 3, 50 ml SeMet
into 1 L of SeMet media (1 L SeMet Base, 50 ml nutrient glucose-free nutrient mix and 4 ml 10 mg/ml
mix, and 4 ml 10 mg/ml selenomethionine) which contains selenomethionine) which contains antibiotics.
antibiotics. 5. The 500 ml cultures in 2 L shake-flasks are incubated at
◦
5. The 500 ml cultures in 2 L shake-flasks are incubated 37 C and 225 rpm for 6 h.
◦
◦
at 37 C and 225 rpm until an OD 595 of ∼0.6 is reached. 6. The temperature is reduced to 25 C and the cultures
6. The temperature is reduced to 20 C. After 30 min at incubated for a further 18 h.
◦
◦
20 C, the cultures are induced with 0.5 mM IPTG and are 7. The culture is then centrifuged at 6000 g for 15 min to
incubated for a further 20 h at 20 C and 200 rpm. obtain the pellet.
◦
7. Product is harvested by centrifugation at 6000 g for
15 min and frozen for later processing.