Page 16 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 5

of tryptophan in the growth medium (i.e. a defined The T7 RNA polymerase recognizes the bacterio-
minimal medium such as M9CA). The tac promoter phage T7 gene 10 promoter, which is carried on
is a synthetic hybrid containing the –35 sequence the vector upstream of the gene of interest. Being
derived from the trp promoter and –10 from lac.It is more efficient than the E. coli RNApolymerase, very
several times stronger than either lac or trp (Amann high levels of expression are possible. Up to 50% of
et al., 1983). the total cell protein can be attained in a few hours
pBAD expression (www.invitrogen.com) utilizes after induction. First, the target gene is cloned using
the regulatory elements of the E. coli arabinose an E. coli host which does not contain the T7 poly-
operon (araBAD), which controls the arabinose merase gene. Once established, the plasmids are
metabolic pathway. It is both positively and neg- then transferred into the expression host harbouring
atively regulated by the product of the araC gene, the T7 polymerase under the control of an inducible
a transcriptional regulator which forms a complex promoter, usually lacUV5. Induction is by addition
with l-arabinose (Ogden et al., 1980). The tight reg- of IPTG. Besides high-level expression, the system
ulation provides a simple but very effective method offers the advantage of very tight control. Since the
for optimizing yields of soluble recombinant pro- host-cell RNApolymerase does not recognize the T7
tein at levels just below the threshold at which they promoter, it prevents basal expression which might
become insoluble. Induction is by the addition of prove harmful to the host. Control can be tightened
arabinose. Again, basal expression may be repressed even further by coexpressing T7 lysozyme from an
by the addition of 2% glucose to the growth medium, additional plasmid (pLysS/pLysE) in the expression
an important consideration if the protein of inter- strain which inactivates any spurious T7 polymerase
est is known to be toxic to the host. Currently there produced under non-inducing conditions. An exten-
are nine pBAD expression vectors with a variety of sive series of derivatives of the original pET vec-
vector-specific features. tors constructed by Studier and Moffatt (1986) are
Bacteriophage lambda P L is an extremely pow- commercially available (www.novagen.com).
erful promoter responsible for the transcription of
bacteriophage lambda DNA. P L expression systems 1.2.2.1 Bacterial hosts
offer tight control as well as high-level expres- Most E. coli host strains used for high-level expres-
sion of the gene of interest. The P L promoter is sion are descended from K12. E. coli strains should
under the control of the lambda cI repressor pro- ideally be protease deficient, otherwise some degree
tein, which represses the lambda promoter on an of proteolysis is more or less inevitable as evident
adjacent operator site. Selected E . coli host strains in multiple banding on SDS gels. For this reason,
synthesize a temperature-sensitive defective form of E. coli B strains deficient in the ATP-dependent lon
the cI repressor protein, which is inactive at temper- (cytoplasmic) and ompT (periplasmic) proteases are
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atures greater than 32 C. Expression is induced by normally used. As in the case of T7 polymerase,
a rapid temperature shift. The host cells are usually some vectors require host strains carrying addi-
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grown at 28 Cto32 C to midlog phase when the tional regulatory elements for which a variety of
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temperature is rapidly adjusted to 40 C as described derivatives of BL21 strains are commercially avail-
in Protocol 1.2. Alternatively, the cI repressor may able. However, BL21 does not transform well so an
be placed under the control of the tightly-regulated alternative strain for cloning and maintenance of
trp promoter, and expression is then induced by the vector should be used, for example JM105. E. coli
addition of tryptophan. With no tryptophan present, strains that encode for a number of rare codon genes
the cI repressor binds the operator of P L , preventing include: BL21 (DE3) CodonPlus-RIL AGG/AGA
expression. However, in the presence of trypto- (arginine), AUA (isoleucine), and CUA (leucine)
phan the tryptophan–trp repressor complex forms (www.stratagene.com); and Rosetta or Rosetta
and prevents transcription of the cI repressor gene, (DE3) AGG/AGA (arginine), AUA (isoleucine), and
allowing transcription of the cloned gene. Induc- CCC (proline) (www.novagen.com). For membrane-
tion can be achieved at lower temperatures although bound proteins, expression in mutant strains C41
basal expression can be a problem. (DE3) and C43 (DE3) could improve expression