Page 49 - Macromolecular Crystallography

38 MACROMOLECULAR CRYS TALLOGRAPHY

desalting) in series, or up to four affinity purifica- media involved and components of the media which
tions followed by two further chromatography steps are incompatible with affinity chromatography.
(e.g. desalt and ion exchange) in series. The eluates If the media are compatible with an affinity chro-
from the columns are followed by a UV detector matography step, then simple programmes similar
measuring protein absorbance at 280 nm. Such pro- to those described in Section 2.4.4.1 can be used.
cedures have been implemented in many structural However, it is recommended that wash steps are
genomic laboratories and have been reported on by interspersed with sample loading to maximize bind-
Kim et al. and Sigrell et al. (Kim et al., 2004; Sigrell ing and reduce any risk of the column becoming
et al., 2003). The instrument can be used to purify blocked. A standard protocol for affinity purifica-
up to six affinity-tagged proteins per day, resulting tion of secreted proteins is given in Protocol 2.10.
in 1–50 mg of protein per run at over 90% purity This protocol has been automated and combined
(Sigrell et al., 2003). Standard protocols supplied with further chromatographic steps (desalt or size
with the Äkta Explorer 3D are listed in Table 2.2. exclusion) using the Äkta Xpress system within the
The Äkta Xpress consists of multiple modules OPPF.
(systems with either two or four modules are cur- Some media are incompatible with affinity chro-
rently available) each with five column positions matography, for example many media used for
and five loop positions (Fig. 2.3). Each unit func- baculovirus growth contain EDTA which strips the
tions in a similar way to the Äkta Explorer 3D metals from IMAC columns. To avoid this problem,
system, allowing parallelization of up to four affin- the media can be buffer exchanged using dialysis,
ity purifications with one further chromatographic stirred-cell, or cross-flow filtration. Alternatively,
step or up to three affinity purifications with two fur- an additional chromatography step can be intro-
ther chromatography steps. This system has many duced in which interfering agents are removed, for
advantages over the Äkta Explorer 3D, as each unit example using lentil lectin affinity column to select
can be run independently, allowing different purifi- glycosylated proteins.
cation strategies to be run in parallel. In addition,
the increased number of inlets, outlets, and loops
allow for greater flexibility in protocols. In the OPPF, 2.4.5 Quality assessment
an Äkta Xpress consisting of four modules has been
For high-throughput purification pipelines, qual-
used to purify 16 His-tagged proteins using an
ity assessment (QA) procedures are required which
IMAC–gel filtration strategy in one overnight run
can be carried out in parallel in relatively high
(∼11 h purification time).
throughput. The QA steps routinely carried out in
the OPPF are summarized in Table 2.3. Other meth-
2.4.4.3 Secreted proteins
ods that have been used in HTP projects include one
Secreted proteins from eukaryotic cell lines present
and two-dimensional NMR screening and differen-
problems in purification due to both the volume of
tial scanning calorimetry.
Table 2.2 Preprogrammed Äkta protocols
2.4.5.1 Mass spectrometry
Protocol ÄKTA 3D ÄKTA Xpress Mass spectrometry (MS) is widely used to ascer-
tain the purity, total mass of the protein produced,
1. Affinity – desalt and detect any covalent modifications (Cohen and
2. Affinity – gel filtration Chait, 2001). Both electrospray ionization (ESI) and
3. Affinity – desalt – ion exchange
MALDI may be used although for intact proteins;
4. Affinity – desalt – ion exchange –
ESI has the advantage of being accurate to ∼1 Da.
desalt
Using the simple protocol described in Protocol 2.11,
5. Affinity – desalt – ion exchange – gel
the MS of whole protein samples can be readily
filtration
automated without the need for sample prepara-
6. Affinity – on-column cleavage – desalt
tion. This method has proved successful for the