Page 20 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 9

Polyhistidine tags of other lengths (e.g. His4 or 1.2.3 Yeast expression systems
His10) may provide useful alternatives. The amount
Being eukaryotes, yeast cells carry out some of the
of enzyme, temperature, and length of incubation
post-translational processes found in mammalian
required for complete digestion varies according to
cell lines and rarely give rise to inclusion bodies.
the specific fusion protein. Thrombin, factor Xa, and
Pichia pastoris and Saccharomyces cerevisiae are both
enterokinase are the most commonly used proteases.
well characterized, easy to handle, and grow rela-
Thrombin in particular tends to cleave promiscu-
tively quickly to high densities in defined medium.
ously. Another disadvantage of fusions is the alter-
Pichia is particularly suited for large-scale produc-
ation in the sequence of the tagged protein that may
tion, being capable of yielding tens of milligrams
be necessary in order to supply the cleavage site.
per litre of fully functional recombinant material
For GST fusions it is advisable to use the PreScission
without loss of yield. P. pastoris is widely chosen
Protease cleavage site (www.gehealthcare.com). The
for analytical and structural studies. Pichia expres-
GST tag then can be removed and the protein puri-
sion vectors are commercially available for inducible
fied in a single step on the column (Protocol 1.4).
and constitutive expression, as well as the pro-
The PreScission Protease also has the useful prop-
duction of secreted proteins coupled with a fusion
◦
erty of being maximally active at +4 C thus allowing
partner for rapid purification and immunological
cleavage to be performed at low temperatures and so
detection. Pichia, being a methyltrophic yeast, can
improving the stability of the target protein. The pro-
utilize methanol as its sole carbon source in the
tease can be removed after cleavage using a HiTrap
absence of glucose. Expression is regulated by the
Benzamidine column. The GST 96 well Detection
AOX1 promoter, which controls the expression of
Module provides a convenient ELISA assay for test-
alcohol oxidase, the enzyme involved in the first
ing lysates. Cloning procedures are specific for each
stage of methanol production (Cregg et al., 1993).
vector and manufacturers’ instructions should be
Another yeast species that has been successfully
closely followed.
utilized for high-level expression of heterologous
Fusions may also be designed against which anti-
proteins is Candida utilis (Kondo et al., 1997).
bodies may be raised that can be used for detection.
One potential problem is that yeast cells can
An example is the tripeptide Glu-Glu-Phe motif
acidify the culture medium and may also contain
for the immunoaffinity of HIV enzymes which is
compounds that affect binding of His-tags to the
recognized by the YL1/2 monoclonal antibody to
resin. Detailed protocols for culturing and handling
α-tubulin (Stammers et al., 1991).
yeast cells are available from Clontech laboratories
To determine the optimum conditions, pro-
(www.clontech.com).
tein amplification should be monitored at various
stages during pilot experiments before scaling-up.
It should be emphasized that not all proteins are 1.2.4 Baculovirus expression system
amenable to amplification in E. coli. Considerable
time, effort, and hours of frustration can be spent in Expression in insect cells is a common method
constructing a suitable expression system and opti- of production of recombinant proteins for struc-
mizing yields. In particular, growth media, antibi- tural studies. The advantages of using insect cells
otics, and chemical inducers can be prohibitively include relatively high expression levels compared
expensive. This is a major consideration when scal- to other eukaryotic expression systems, expres-
ing up as large-scale fermentation involving high sion of multiple genes, capacity for expressing
cell densities may simply result in the loss of vec- unspliced genes, ease of scale up, simplified cell
tor through selection or, as mentioned above, the growth, and the possibility of protein production in
product may prove toxic to the host. Should the high density suspension cultures. In addition, post-
above-mentioned expression strategies fail to pro- translational processing modifications to eukary-
vide adequate levels of product in E. coli it is otic proteins expressed in insect cells are similar
advisable to switch to yeast or insect cells. to those of mammalian cells and this facilitates