Page 29 - Macromolecular Crystallography

18 MACROMOLECULAR CRYS TALLOGRAPHY

folded, endoglycosidase sensitive glycoproteins for yeast cells which are difficult to break. Usu-
with defined glycosylation. The features of the CHO ally yeast cells are disrupted using a combination of
LecR cell line make it highly useful for preparing physical and chemical methods. The BeadBeater™
soluble glycoproteins that can be readily deglycosy- (www.biospec.com) disrupts micro-organisms with
lated prior to crystallization (Davis et al., 1993). better than 95% efficiency. Up to 80 g (wet weight)
of cells can be processed in a typical 3-min run.
Whereas chemical methods create contamination,
1.2.6 Cell-free systems the chief disadvantage of mechanical methods is the

Cell-free in vitro expression systems are currently generation of heat and aerosols. For this reason all
being developed at the Centre for Eukaryotic Struc- procedures should be carried out in an ice bath. Son-
tural Genomics at the University of Wisconsin. As ication should be carried out in batches of 100 ml in
these systems express only the protein of interest short bursts. Even if protease-deficient host strains
and require smaller volumes, lengthy concentra- such as lon − have been used it is still advisable
tion steps are avoided. The disadvantage is the to include protease inhibitors in the resuspension
expense of the reagents. Cell-free systems are avail- buffer. The serine protease inhibitors aprotinin and
able from QIAGEN (www.qiagen.com), Invtrogen 1 mM phenylmethanesulphonyl fluoride (PMSF) are
(www.invitrogen.com), and CellFreeSciences. commonly used but for proteins particularly suscep-
tible to proteolysis a cocktail of inhibitors to cope
with each class of protease may be required.
1.3 Protein extraction and isolation

1.3.1 Cell disruption 1.3.2 The removal of cell debris and
nucleic acids
The cells are harvested by low-speed centrifuga-
tion and resuspended in lysis buffer before disrup- Cell debris may be removed by centrifugation at
tion. Either chemical or mechanical methods may 10,000 g for 30 min. The nucleic acids being the
be used for disruption. The choice depends on major contaminant can be removed by precipita-
the source of the protein (that is bacterial, yeast, tion with a positively-charged polymer such as
insect, or mammalian, intracellular or extracellular) polyethyleneimine PEI (typically 0.5–1% of a 10%
and the physicochemical properties of the recom- solution). Addition of magnesium to the resus-
binant product, as well as the scale of the extrac- pension buffer will assist in the enzyme digestion
tion. For bacterial cells, enzymic digestion with of DNA by DNAse. Some loss of protein may
hen egg white lysozyme, which specifically catal- occur by copreciptation, which is especially the case
yses the hydrolysis of 1,4 glycosidic bonds in the with some DNA-binding proteins. This can usually
peptideoglycan cell wall of Gram-positive bacteria, be avoided by a 1:1 dilution of the crude extract
is a gentle procedure which minimizes denatura- with buffer.
tion of the product. For Gram-negative bacteria, for
example E. coli, metal chelators such as EDTA are
1.3.3 Refolding strategies
requiredtochelatecationsthatmaintaintheintegrity
of the outer lipopolysaccharides. Chemical disrup- High-levelexpressionoffull-lengthproteinsin E.coli
tion methods require a cocktail of anions, reduc- may result in the production of inclusion bodies.
ing agents, non-anionic detergents, and chaotropic These are insoluble, inactive aggregates result-
agents in order to avoid irreversible denaturation of ing from inappropriate folding and association via
the product. Detergent-based lysis reagents are com- hydrophobic interactions. The proteins are function-
mercially available, including BugBuster™ (Merck) ally inactive in their aggregated state. The formation
Fastbreak (Promega). of inclusions can be advantageous for purification,
Mechanical disruption methods include sonica- provided the protein can be successfully solubilized
tion, high-speed homogenization using a French and renatured into its active form. This involves
press, and bead milling, which is especially suitable isolation of inclusions and removal of unwanted