Page 48 - Macromolecular Crystallography

HIGH-THROUGHPUT CLONING, EXPRESSION, AND PURIFICATION 37

Protocol 2.9 IMAC-SEC purification

Charge a column using 1.5 × column volume 0.2 M nickel 6. Inject this peak directly onto an equilibrated gel filtration
sulphate. column (S200 or S75).
7. Elute the protein with 1.2 × gel filtration column
1. Wash with 1.5 × column volume water. volume of Gel Filtration Buffer (20 mM Tris pH 7.5, 200 mM
2. Equilibrate with 2 × column volume Binding/Wash NaCl, reducing agents as desired) collecting fractions.
Buffer (50m M Tris pH 7.5, 500 mM NaCl, 20 mM 8. Run an SDS-PAGE gel on the fractions and pool those
imidazole). containing the protein of interest.
3. Load the filtered lysate containing the protein of interest.
4. Wash the column with 5 × column volume Binding/ After affinity purification, the protein usually has high purity;
Wash Buffer or until the absorbance at 280 nm becomes however some non-specific binding to the resin can occur,
stable. along with binding of any truncations of the target protein.
5. Elute the protein with 5 × column volume of Elution
Buffer (50 mM Tris pH 7.5, 500 mM NaCl, 500 mM
imidazole) collecting the peak detected at 280 nm.

oligomeric state of the protein when the retention This process can be fully automated by perform-
volume is compared to that of standard proteins. ing the protease cleavage step within a Super-
In the event that a third chromatography step is loop™ connected to the purification instrument. For
necessary to achieve acceptable purity, then an ion on-column cleavage, either an untagged protease
exchange step is normally inserted between the or a protease with a different tag to the protein of
affinity and SEC columns. This usually consists of interest is used. Here, the target protein is bound to
affinity chromatography followed by ion exchange the affinity column, one column volume of protease
and then size exclusion chromatographies. As the solution applied, and the column incubated for the
affinity elution buffer is usually not compatible with amount of time required for cleavage to occur. The
the ion exchange chromatography step, a buffer column is then washed to remove both the protein
exchange is normally included between these stages. of interest and the protease. Further purification is
In many cases the recombinant protein expression then necessary to remove the protease from the sam-
construct has been designed to include a protease ple. On-column cleavage strategies are more readily
cleavage site (Section 2.2.2.2). Therefore tag cleav- automated than an in-solution strategy; however, in
age is included in the purification strategy. There some cases cleavage is less likely to go to completion.
are two basic tag removal strategies: either cleav- This is most likely due to the protein being tightly
age of the protein in solution or when bound to bound to the resin and therefore the protease is ster-
an affinity column. In general, tagged-proteases ically hindered from approaching the cleavage site.
are used in order to facilitate their removal from
the protein sample. In the first case, the strategy 2.4.4.2 Instrumentation
may include an automated affinity step followed Automation of the strategies described above can be
by buffer exchange into the conditions compatible achieved using instruments supplied by GE Health-
with the protease. Conveniently, both 3C and Tev care. Many of the procedures described above are
proteases cleave efficiently in the SEC buffer (Pro- preprogrammed to allow ease of implementation of
tocol 2.9). The protease is then added off-line and HTP automated purification techniques.
incubated in solution; for Tev and 3C this is typically The Äkta Explorer 3D is equipped with positions
◦
overnight at 4 C. Postcleavage, a second automated for up to seven column and has two loop posi-
affinity chromatography step followed by size exclu- tions. This instrument allows sequential automation
sion chromatography can be performed in order to of up to six affinity purifications followed by a fur-
gain pure cleaved protein (Kim et al., 2004). ther chromatography step (e.g. size exclusion or