Page 19 - Macromolecular Crystallography

8 MACROMOLECULAR CRYS TALLOGRAPHY

provides lac Iq repressor and confers resistance to commercially in a variety of vectors with MCSs
ampicillin. Novagen’sPETsystemoffersawidevari- ensuring easy transfer of inserts.
ety of fusion tags including both N and C-terminal Invariably, for crystallization it is desirable to
polyhistidines (www.novagen.com). Other widely- remove the tag thus avoiding any possible inter-
used tags include a calmodulin-binding peptide ference with folding and tertiary structure. This
(www.stratagene.com), the maltose binding protein may prove problematic, particularly if the prote-
(www.westburg.nl), polyarginine, and cellulose- olytic cleavage site introduced into the vectors for
binding tag. These are all helpfully reviewed by this purpose, is not unique. Shorter fragments may
Terpe (2003). Another tag utilizes the stability char- result, leading to microheterogeneity. Histidine-
acteristics of E. coli thioredoxin, which when used tagged protein appears to be less of a problem in
as a fusion confers its heat tolerance and solubility this respect since successful crystallization and high-
properties upon the recombinant protein (Yasukawa resolution structure solution has been achieved with
et al., 1995). Providing the target sequence with a the protein-polyhistidine sequence (His6) remaining
C-terminal tag will ensure that only full-length pro- intact. Cleavage may be carried out either on the
tein is purified. All these fusion tags are available immobilized media or after elution of the product.

Protocol 1.4 Purification of soluble GST-tagged recombinant protein and cleavage
of the GST tag using thrombin and factor Xa

Materials 8. Remove the protease using a Hi-trap benzamidine
Binding buffer: 1 × PBS (140 mM NaCl, 2.7 mM KCl, column.
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.0–8.0)
Elution buffer: 50 mM Tris-HCl, 10 mM reduced GSH, Cleavage of the fusion protein on the column:
pH 7.0–8.0
Prepacked MicroSpin™GST or GSTrap FF columns 1. Add the cell lysate to a prepacked MicroSpin™GST
(GE Healthcare) column or GSTrap FF column equilibrated with the binding
buffer.
Hi-trap Benzamidine FF column
2. Wash with the binding buffer.
SDS-polyacrylamide gel electrophoresis system
3. If using a GSTrap FF, connect the column directly to a
◦
Thrombin: 500 units in 0.5 ml PBS (stored at –80 C)
Hi-trap benzamidine FF column.
Factor Xa: 400 units in water to give a final solution of
4. Cleave the fusion protein with a site-specific protease
1 Unit/µl stored at –80 C (thrombin, factor Xa or any other protease).
◦
5. Collect the flow through sample and analyse on a
SDS-PAGE or by mass spectroscopy.
Method
Cleavage of the fusion protein off the column:
For scale-up:
GSTrap FF (1 ml) column binds 10–12 mg fusion protein
1. Add the cell lysate to a prepacked MicroSpin™GST
or GSTrap FF column equilibrated with the binding buffer. GSTrap FF (5 ml) column binds 50–60 mg fusion protein
2. Wash the column with the binding buffer.
3. Elute the fusion protein with the elution buffer. 1. Equilibrate the column with 5 column volumes of the
4. Cleave the eluted fusion protein with site-specific binding buffer.
protease thrombin or Factor Xa. 2. Maintain loading flow rate 0.2–1 ml /min for 1 ml
5. Desalt the sample using a Hi-trap desalting column. column and 1–5 ml/min for 5 ml column.
6. Add the sample to a MicroSpin™GST or GSTrap FF 3. Wash with 5–10 column volumes of binding buffer.
column equilibrated with the binding buffer. 4. Elute with 5–10 volumes of elution buffer.
7. Collect the eluate and analyse it by SDS-PAGE or by
mass spectroscopy. Adapted from protocol supplied by GE Healthcare.