Page 45 - Macromolecular Crystallography

34 MACROMOLECULAR CRYS TALLOGRAPHY

2.4.1.2 Insect and mammalian cells auxotroph is required to achieve high levels of incor-
Insect cells are conveniently grown to multilitre scale poration. One such auxotroph is the BL21-derived
in suspension using Erhlemeyer flasks (either glass strain, B834. In addition, the biomass of cultures
or plastic). Alternatively, disposable plastic Wave™ and the yield of protein are often depressed when
Bioreactors (Wave Biotech) offer an affordable and compared to non-SeMet containing medium due to
easy-to-use solution for growing 5–10-L volumes the toxic side-effects of SeMet labelling of all the
of cell culture (Weber et al., 2002). For HEK 293T proteins within the cell. SeMet labelling within the
cells we have developed a scale-up process based on OPPF is performed using the methods described
growth in attached culture in roller bottles (see Pro- in Protocol 2.6 for both IPTG induction and
tocol 2.4). Recently, there have been several reports autoinduction of protein expression. These meth-
of the use of suspension cultured HEK cells for large- ods routinely produce greater than 99% selenome-
scale transient expression of proteins (Pham et al., thinone incorporation as determined by mass
2003; Durocher et al., 2002; Baldi et al., 2005). spectrometry.
If a methionine auxotroph is not used, the methio-
nine within the culture can be replaced by SeMet
2.4.2 Selenomethionine labelling
using a methionine biosynthesis inhibition or ‘poi-
Labelling of proteins with selenomethionine (SeMet) soning’ method such as that outlined in Protocol 2.7.
represents a standard approach to determining This method has the advantage of using any E. coli
phases during protein structure solution by X-ray strain available and thus the initial growth of the
crystallography using multiwavelength anomalous culture is not reduced, however the method is more
dispersion methods (Hendrickson et al., 1990). laborious than using the methionine auxotroph.
Therefore, HTP structural genomics not only Stols et al. have reported the successful SeMet
requires a high yield of pure protein, but also a labelling of proteins (over 95% incorporation) with
high percentage incorporation of selenium. Sub- IPTG induction and a ‘poisoning’ pathway using
stitution of methionine for selenomethionine dur- the 2-L PET bottle methodology (Stols et al., 2004).
ing in vivo protein expression can present many This was taken a stage further by Sreenath et al.
problems. Many E. coli cell lines have their who utilized the bottles with autoinduction of the
own methionine sources, therefore a methionine methionine auxotroph B834 to give over 90% SeMet

Protocol 2.5 Scale-up of E. coli culture

IPTG induction protocol Auto-induction protocol using OnEx Solutions
1. A single colony is inoculated into 20 ml of GS96 medium from Merck Biosciences
with 1% w/v glucose and antibiotics in a 50 ml falcon tube. 1. As for IPTG induction, a single colony is inoculated into
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The culture is incubated at 37 C and 225 rpm overnight 20 ml of GS96 medium with 1% w/v glucose and antibiotics
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(∼16 hours). in a 50 ml tube. The culture is incubated at 37 C and
2. The overnight culture is diluted 1 in 100 into 1 L of 225 rpm overnight (∼16 hours).
GS96 medium supplemented with 1% w/v glucose and 2. The overnight culture is diluted 1 in 100 into 1 L of GS96
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antibiotics. The cultures are then incubated at 37 C medium supplemented with 20 ml OnEx Solution 1, 50 ml
and 225 rpm until OD 595 ≈ 0.6. OnEx Solution 2 and 1 ml OnEx Solution 3 along with the
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3. The temperature is reduced to 20 C. After 30 min relevant antibiotics. The cultures are incubated at 37 C and
at 20 C, the cultures are induced with 0.5 mM IPTG 225 rpm for 4 h.
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and are incubated for a further 20 h at 20 C 3. After this 4-h growth period, the temperature is reduced
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and 225 rpm. to 25 C and the cultures incubated for a further 20 h.
4. Product is harvested by centrifugation at 6000 g for 4. The cultures are then centrifuged at 6000 g for 15 min
15 min and then frozen. to obtain the pellet containing the protein, which can be
frozen.