Page 12 - Macromolecular Crystallography

CHAPTER 1

Classical cloning, expression, and

purification

Jane Skelly, Maninder K. Sohi, and Thil Batuwangala

1.1 Introduction cytoplasmic, secretory, or cell surface proteins can
be achieved in cultured insect cells using recombi-
The ideal protein-expression strategy for X-ray
nant baculovirus vectors. Furthermore, in insects the
structural analysis should provide correctly folded,
post-translational modifications are similar to those
soluble, and active protein in sufficient quantities
of eukaryotes. For some very large molecules,
for successful crystallization. Subsequent isolation
the only feasible way of obtaining correctly-folded,
and purification must be designed to achieve a
active protein is by expression in mammalian cells.
polished product as rapidly as possible, involv-
Mammalian expression vectors are usually hybrids,
ing a minimum number of steps. The simplest and
containing elements derived from prokaryotic plas-
least expensive methods employ bacterial hosts such
mids and controlling sequences from eukaryotes
as Escherichia coli, Bacillus, and Staphylococcus but
such as promoters and transcription enhancers
if the target protein is from an eukaryotic source
required for the expression of foreign DNA. Alter-
requiring post-translational processing for full func-
natively, in vitro protein expression in cell-free
tionality, an eukaryotic vector–host system would
systems is being developed specifically for struc-
be appropriate – although it should be noted that
tural proteomics, where only the protein of interest
in many instances the lack of processing can prove
is expressed, improving the yield of stably-active
an advantage in crystallization (Table 1.1). Micro-
eukaryotic proteins as well as simplifying their
bial eukaryotes, such as yeast and filamentous
purification. Product size, stability, the presence of
fungi, process their gene products in a way that
disulphide bonds, and whether the product is likely
more closely resembles higher organisms. Yeast
to be toxic to the host are all important considera-
is non-pathogenic and its fermentation character-
tions when choosing a suitable expression system.
istics are well known. Both Saccharomyces cere-
Levels of expressed gene product are measured as
visiae and Pichia pastoris strains are used extensively
a percentage of the total soluble cell protein which
for large-scale expression of heterologous proteins.
can vary from <1% to >50% depending on several
Whereas yeast, unless supplied with an appropriate
factors:
leader sequence, export protein to the cell vac-
uole the filamentous fungi, Aspergillus nidulans and 1. the vector-host system;
Aspergillus niger, secrete their gene products directly 2. gene copy number;
into the growth medium. Secretion is often pre- 3. transcription and translation efficiency;
ferred because it facilitates recovery of the product. 4. mRNA stability;
DNA can also be inserted into the fungal genome 5. stability and solubility of gene product;
at a high copy number, although the genetics are 6. the conditions of fermentation and induction, as
less well characterized. High-level expression of detailed for each vector.
1