Page 15 - Macromolecular Crystallography

4 MACROMOLECULAR CRYS TALLOGRAPHY

details of materials, including preparations of buffer result in low levels of expression or even prema-
reagents, may be found in standard laboratory ture termination. Not all of the 61 mRNA codons
manuals and on manufacturers’ web sites. are used equally (Kane, 1995). Rare codons tend to
For high-throughput (HTP) the gene of interest occur in genes expressed at low level and their usage
can be cloned in parallel into a variety of expres- depends on the organism. (The codon usage per
sion vectors containing different tags and/or fusion organism can be found in the Codon Usage Database
partners, and into vectors for a variety of expression (www.kazusa.or.jp/codon). To overcome this, site-
systems. Gateway™ (www.invitrogen.com) cloning directed mutagenesis may be carried out to replace
technology is discussed in Chapter 2. the rare codons by more commonly-occurring ones,
or alternatively by coexpression of the genes encod-
ing rare tRNAs. E. coli strains that encode for a
1.2.2 Prokaryotic expression systems number of rare codon genes are now commercially
available (see Section 1.2.2.1). There is also a possi-
The most effective way to maximize transcription bility that expression of high levels of foreign protein
is to clone the gene of interest downstream from may prove toxic to the E. coli host inducing cell
a strong, regulatable promoter. In E. coli the pro- fragility, therefore placing the recombinant cell at
moter providing the transcription signal consists a disadvantage. Specific post-translational modifi-
of two consensus sequences situated –1 and –35 cations, such as N- and O-glycosylation, phospho-
basesupstreamfromtheinitiationcodon. High-level rylation and specific cleavage (e.g. removal of the
expression vectors contain promoter regions situ- N-terminal methionine residue) required for full
ated before unique restriction sites where the desired functionality of the recombinant protein, will not be
gene is to be inserted, placing the gene under the carried out in bacteria.
direct control of the promoter. Differences between Probably the best example of regulatory gene
consensus promoter sequences influence transcrip- expression in bacteria is the lac operon, which is
tion levels, which depend on the frequency with extensively used in the construction of expression
which RNA polymerase initiates transcription. In vectors (Jacob and Monod, 1961). The lac pro-
addition to these regulatory elements, expression moter contains the sequence controlling transcrip-
vectors possess a selectable marker – invariably an tion of the lacZ gene coding for β-galactosidase,
antibiotic resistance gene. one of the enzymes that converts lactose to glu-
When choosing an E. coli expression system for cose and galactose. It also controls transcription
production of eukaryotic cDNA, the differences of lacZ , which encodes a peptide fragment of

between prokaryotic and eukaryotic gene control β-galactosidase. Strains of E. coli lacking this frag-
mechanisms must be addressed. In E. coli, the ribo- ment are only able to synthesize the complete and
some binding site (RBS) consists (in most cases) of functional enzyme when harbouring vectors car-

the initiation codonAUG and the purine-rich Shine– rying the lacZ sequence, for example pUC and
Dalgano sequences located several bases upstream. M13. This is useful for screening recombinants. The
Vectors have been constructed which provide all lac promoter is induced by allolactose, an isomeric
the necessary signals for gene expression including form of lactose, or more commonly, isopropyl β-d-
ribosome binding sites, strong regulatable promot- thiogalactoside (IPTG), a non-degradable substrate,
ers and termination sequences, derived from E. coli at a concentration of up to 1 mM in the growth
genes with the reading frame removed. Multiple medium. Basalexpression(expressionintheabsence
cloning sites (MCS) are provided in these vectors to of inducer) may be reduced by addition of glucose
facilitate insertion of the target gene. to the media. The lacUV5, tac, and trc promoters are
Eukaryotic DNA contains sequences recognized all repressed by the lac repressor.
as termination signals in E. coli, resulting in pre- The trp promoter is located upstream of a group of
mature termination of transcription and a truncated genesresponsibleforthebiosynthesisoftryptophan.
protein. Also, there are differences in codon pref- It is repressed in the presence of tryptophan but
erence affecting translation, which may ultimately induced by either 3-indolyacetic acid or the absence