Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 205)

and binds to the N-terminal co-expressed affinity tag in the fusion protein. Subsequent to the
column binding and washing steps, the eXact protease performs a specific, controlled cleavage
and removal of the tag from the fusion directly on the cartridge, resulting in the release of
highly purified recombinant protein with a native N-terminus. This cleavage reaction is
triggered with a sodium fluoride-containing buffer and can take between 30 min to 15 hrs
(overnight). During elution, the cleaved Profinity eXact tag remains tightly associated with
the resin’s immobilized protease, eliminating the need for additional steps to remove the
protease and the tag. The yield of Profinity eXact cartridges is approximately 3 mg/ml of
purified maltose binding protein (from Profinity eXact Control Protein Lysate). The total
capacity of Profinity eXact cartridges will also depend on several other factors, such as flow
rate during sample application, sample concentration, the temperature at which the purification
is carried out, and the composition of the buffers used for the separation. The Profinia eXact
methods are designed for the recommended buffer formulations provided in Table 2 (see the
eXact System Manual for additional information) and are optimized to provide fast, easy, and
reproducible chromatographic separations including integrated desalting and cartridge
regeneration.
The protease used in the Profinity eXact system generally binds most effectively at neutral
pH using a sodium phosphate or tris-based buffer that does not contain Cl
- ions or other
triggering anions (see Table 3). Elution is generally achieved by triggering anions such as
fluoride or azide. When possible, the sample should be prepared in the composition of the
binding/equilibration buffer. Refer to Section 5.3 of the Profinity eXact System Manual for
detailed sample preparation guidelines.
Note: increased recoveries of target protein can be achieved during purification by using sample
load volumes of 10 ml or greater. This is best accomplished by diluting the sample to a
minimum of 10 ml into binding buffer. In cases where the concentration of the sample is
low and the sample volume is large (i.e. > 25 ml for a 1 ml cartridge) it may not be practical
to dilute the sample further with binding buffer. When working with dilute samples, the flow-rate
of the sample loading step may be adjusted in the Profinia Programmed Method mode in order
to minimize the cleavage and loss of the sample that may occur under prolonged sample
loading conditions. Immediately prior to loading on the instrument, samples should be
centrifuged to remove cellular debris and filtered through a 0.2 or 0.45 µm filter to prevent
clogging of the Profinia system filter and cartridge. This is especially important when loading
large volumes of cell lysate. Samples should be keep on ice or cooled using the Profinia
Cooling Accessory until ready to load on the system.
2.2 eXact Method Cartridge Selection
The Profinia eXact methods are designed to use 1 ml or 5 ml Profinity eXact cartridges.
Since the target protein content of cell lysates varies between preparations and different
proteins, use a sample size such that the amount of target protein in the sample does not
grossly exceed the maximum cartridge binding capacity. The cartridge binding capacity for
a 1 ml Bio-Rad eXact cartridge is approximately 3–4 mg target protein. If the anticipated
amount of target protein in the starting sample exceeds the capacity of a 1 ml cartridge,
then it is recommended to either select the 5 ml cartridge purification option or to decrease
the sample volume applied to a 1 ml cartridge. To minimize the possibility of cross
contamination, it is recommended that individual Profinity eXact and desalting cartridges
are dedicated to the purification of a unique samples or target protein. The Profinia eXact
methods include regeneration steps for the Profinity eXact cartridge to strip the bound affinity
tag following elution. In the methods that include integrated desalting, use of the recommended
buffers in Table 2 will ensure that the Bio-Rad P6 Desalting cartridges are cleaned, stored,
and ready for re-use at the completion of the method.
198