Page 44 - Macromolecular Crystallography

HIGH-THROUGHPUT CLONING, EXPRESSION, AND PURIFICATION 33

Protocol 2.4 Transient transfection of HEK 293T cells
Materials in a 96-well plate then add 2 µl of PEI stock solution
Polyethylenimine (PEI) stock:The transfection reagent (1 mg/ml); mix well by pipetting and incubate for
(Aldrich, catalogue number 40,872–7 ‘25 kDa branched approx. 20 min at RT.
PEI’). Stock solution of PEI is prepared as follows: 3. Add 140 µl DMEM containing 2% FCS to the DNA/PEI
cocktail and mix.
prepare stock solution of 100 mg/ml PEI in water, mix
4. Remove media from the cells in the 24-well plate and
and further dilute to 1 mg/ml, neutralize with HCl and
add the DNA/PEI cocktail to each well (total vol. 200 µl).
filter sterilize; store 5 ml aliquots frozen.
5. After 3–4 h at 37 C, top up well with 860 µl of DMEM
◦
Preparation of DNA:The plasmid DNA quality is critical
containing 2% FCS.
for a successful transfection, e.g. prepare using the 6. Leave to express for 3–4 days, then collect medium, spin,
endotoxin-free mega kit from Qiagen. Acceptable OD filter 0.22 µm, then go on to protein purification (method
ratios should be >1.9. depends on the tags used etc.).
Cells: HEK-293T or 293S GnTI- are grown in DMEM
supplemented with l-Gln (Gibco cat. 25030–024) and For transfection in roller bottles the protocol is scaled up as
2
non-essential amino acids plus 10% FCS. Lower to 2% follows: For each roller bottle (2125 cm ) 0.5 mg DNA is
serum during expression. diluted with 50 ml serum-free DMEM and 1 ml PEI added.
Following incubation at room temperature for 20 min,
Transfection protocol in 24-well plates 100 ml DMEM containing 2% FCS is added to the
1. Seed cells to be 90% confluent on the day of transfection mixture and this added to the cells. After 4 h
transfection. the media in the roller bottle is topped up to 250 ml and the
2. For each well of a 24-well plate dilute 1 µg DNA bottle gassed with 5% CO 2 in air and incubated for
◦
(typically from 0.5 µg/µl) with 60 µl serum-free DMEM 3–4 days at 37 C with continuous rolling.

screening is necessary. The larger volumes required protocols for E. coli culture and induction using
present a challenge for high-throughput facilities as both IPTG induction and autoinduction are given
plate-based formats are no longer viable. In this in Protocol 2.5.
section, the technologies available for scaling-up Another simple culture system for recombinant
of cultures, parallel purification of proteins, and protein production from E. coli is the use of 2-L
quality assessment of the protein produced are polyethylene terephthalate (PET) beverage bottles
reviewed. which are cheap and disposable avoiding the cost
and time for sterilization (Millard et al., 2003).
These bottles are readily available, inexpensive and
2.4.1 Culture systems
can be used in conventional flask holders. Up to
2.4.1.1 E. coli 1-L cultures can be grown per bottle, although
The most commonly used culture system for scale- lower volumes (100–250 ml) are more common for
up of E. coli is the shake flask. This conven- high-density cultures. The system has proved suc-
tional methodology has been shown to produce the cessful for growth of E. coli using Luria broth
quantity and quality of protein required with good (LB), terrific broth (TB), and M9 media (Millard
scalability from plate-based expression screening. et al., 2003). In addition, Sreenath et al. (2005)
In general, cell-line, medium, growth conditions, have reported the use of PET bottles for pro-
and induction method are determined by the small- duction of proteins using autoinduction medium.
scale expression screen. Typically, the shake-flask Protocols for growth in conventional shake-
is filled to one-quarter of its capacity to allow for flasks can be readily converted to the PET bottle
adequate aeration of the culture. The generic OPPF system.