Page 13 - Macromolecular Crystallography

2 MACROMOLECULAR CRYS TALLOGRAPHY

Table 1.1 Selection of host cells for protein amplification

Host Advantages Disadvantages
Prokaryotic expression: E. coli Rapid growth with high yields (up to 50% total Lack of post-translational processing
cell protein) Product may prove toxic to host
Extensive range of vectors Product incorrectly folded and inactive
Simple, well-defined growth media High endotoxin content
Eukaryotic expression: yeast Pichia pastoris; Rapid growth with ease of scale-up and Glycosylated product differs from
Saccharomyces cerevisiae processing mammalian systems
High yields (g/l in Pichia pastoris)
Inexpensive
Formation of disulphides
Glycosylation
Insect cell expression: Baculovirus High-level expression Glycosylated product may differ from
Formation of disulphides mammalian systems
Glycosylation Not necessarily fully functional
Mammalian expression Fully-functional product Expensive media
Slow growth rates
Filamentous fungi: Aspergillus nidulans; Secretion of large quantities into growth media Genetics not well characterized
Aspergillus niger
Cell-free systems Only protein of interest expressed Expensive reagents
Simple purification

1.2 Cloning and expression their melting temperatures (Tm) which should not
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be lower than 60 C; and the GC content, which
PCR cloning is usually the preferred route to con-

should range between 40% and 60%. The 5 -end
structing an expression vector containing the gene

primer which overlaps with the 5 end of the coding
of interest. The choice of vector will inevitably
sequence is designed to contain: a suitable restric-
depend on the source and characteristics of the gene
tion endonuclease recognition site for cloning into
product, the quantity of product required, and its

the expression vector; a 5 extension to the restriction
purification strategy. Detection and purification can
site; a start codon; and an overlapping sequence. The
be simplified by using a fusion partner, such as

3 -end primer overlaps the complementary DNA
glutathione-S-transferase (GST), a histidine tag, or a
strand and should supply: a second restriction site;
recognition motif sequence such as the c-myc epitope

a5 extension; a stop codon; and an overlapping
(see Section 1.2.5).
sequence. If tags or fusion partners are appended,
1.2.1 Construction of a recombinant E. coli additional bases may be required in the antisense
primertoensuresequencesareinframe. Theprimers
expression vector by PCR
are normally synthesized using a commercial syn-
Once a suitable vector has been selected the cod- thesizer. It is not usually necessary for oligonu-
ing sequence of the target protein to be cloned must cleotides to be purified for routine PCR. A typical
first be amplified from either genomic or a cDNA amplification protocol consists of 25 cycles each of a
template by PCR, for which suitable forward and denaturing step at 95 C for 1 min, an annealing step
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reverse oligonucleotide primers are needed. Arange to be calculated from the melting temperatures of
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of web-based software is available for designing the primers used, and an extension at 72 C for 1 min
primers (www.clcbio.com). Important considera- followed by a final 10 min extension step at 72 C.
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tions in primer design include: the primer length, The reaction conditions can be optimized, with the
which for most applications is between 18 and 30 number of cycles being increased for mammalian
bases; the chosen 5 and 3 -end primer sequences; genomic DNA. Purification of the fragment is not