Page 31 - Macromolecular Crystallography

20 MACROMOLECULAR CRYS TALLOGRAPHY

(nucleic acids, proteins, and endotoxins), and a choice of media is governed by scale of produc-
final polishing step. The programmable AKTA pilot tion and resolution. Functional properties have
system (GE Healthcare) is ideal for method devel- been combined with matrices of various strengths
opment and small and medium scale preps. AKTA and porosities to optimize flow rates and selectiv-
Xpress is a dedicated, high-throughput system. ity. For intermediate purification Sepharose Fast
Flow™ is routinely used for general and large-
scale separations and Sepharose High Performance
1.3.4.1 Primary isolation and concentration
media is preferred for high-resolution applications.
Theinitialstageisthecapture, stabilization, andsub-
The small-scale High Trap columns are useful for
sequent enrichment of soluble product. If the pI is
method development. The Tricorn High Perfor-
known, ion exchange (IEX) on a rigid matrix can be
mance columns are a new generation of high-
used for concentration and as a preliminary clean-
resolution of columns which come prepacked with
up. Supernatant (see Section 1.7.2) can be applied
MonoQ, MonoS, Superdex 200, and Superdex 75
directly to Sepharose Fast Flow™. STREAMLINE™
from GE Healthcare. At the final polishing phase
(www. gehealthcare.com) expanded bed adsorption
there is frequently a trade off between recovery and
is particularly suitable for secreted proteins in large
resolution, as peak-cutting may be necessary.
volumes of crude supernatant as no preliminary
Affinity chrmomatography exploits the biological
clean-up is required. At this stage buffers should
properties of the molecule by reversible absorption
be relatively inexpensive and any additives required
to an immobilized ligand coupled to an insoluble
for maintaining stability should be easy to remove
support. A wide variety of media is available for
at a later stage if necessary. The volume of super-
affinity applications (Table 1.3). For immunoaffin-
natant can be reduced either by ultrafiltration or
ity, antibodies raised against the target protein are
selective precipitation with salts, organic solvents,
coupled to an activated adsorbant, for example
or long-chain polymers, for example polyethylene
cyanogen bromide activated Sepharose™. The high
glycol (PEG). Ammonium sulphate is commonly
binding capacities and specificities require harsh
used because of its high solubility and stabilizing
conditions for elution, often requiring denaturing
properties. Ethanol and acetone have proved suc-
conditions.
cessful in the fractionation of extracellular proteins
suchasplasmaproteins, polypeptidehormones, and
in the extraction of histones from non-recombinant 1.3.5 Product analysis
sources.
For crystallization at least 95% purity is desirable.
SDS PAGE stained with Coomassie™ Brilliant Blue
1.3.4.2 Chromatography R-250 provides a crude but reasonable primary indi-
To avoid loss of active material the number of cator of purity and expression. Most minor con-
chromatographic steps must be minimized. This taminants (<5%) can be detected by silver staining.
is best achieved by combining chromatographic For enzyme isomers and proteins which differ in
steps in a logical sequence to maximum effect. charge but not size, analytical isoelectric focus-
Buffer exchange, desalting, dialysis, and ultrafil- ing and/or two-dimensional PAGE is necessary.
tration should be avoided where possible between All these electrophoretic methods can be carried
chromatographic stages. Size-exclusion (GF), which out using the Phast System™ (GE Healthcare) or
desalts and dilutes the sample, should follow equivalent. Proteins which possess an optical chro-
a concentration step such as ion-exchange (IEX), mophore can also be assessed using the ratio of the
and ion-exchange would not be appropriate after absorption peak in visible spectrum to the absorp-
ammonium sulphate fractionation because of the tion at 280 nm. The Protein 200-HT2 assay of Agilent
extensive desalting required to allow the protein to Technologies (Palo Alto California) identifies, deter-
bind. Instead, hydrophobic interaction chromatog- mines size, and quantitates proteins from 14 kD
raphy (HIC), which binds proteins preferentially to 200 kD. Microheterogeneity caused by chemical
at high ionic strengths, could be substituted. The modification, partial denaturation, or incomplete