Page 38 - Macromolecular Crystallography

HIGH-THROUGHPUT CLONING, EXPRESSION, AND PURIFICATION 27

Protocol 2.1 LIC-PCR
1. Amplify the desired insert sequence using appropriately plates supplemented with the antibiotic appropriate for the
designed PCR primers. The 5 -end of the primers must plasmid used.

incorporate the following sequences: 6. Pick colonies and prepare expression-ready
plasmids.
sense primer: 5 GAC GAC GAC AAG ATX – insert specific 7. PCR screen colonies or plasmid minipreps as usual

sequence 3 with a vector-specific (e.g. T7) forward primer and your
antisense primer: 5 GAG GAG AAG CCC GGT* – insert gene-specific reverse primer.

specific sequence 3
Notes
2. Gel purify the PCR product and resuspend in TlowE The primer sequences described here are specific to the
buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Treat the Novagen Ek/LIC system and its related suite of vectors,
PCR product with T4 polymerase in the presence of dATP, they cannot be used with other, unrelated vectors in the
the reaction may be performed in either a sterile PCR plate LIC protocols.
or microcentrifuge tubes (this generates single-stranded 5 X- The first nucleotide of the insert-specific sequence must
overhangs at both ends of the PCR product as far as the complete the codon ATX.
first ‘T’ in the primer extensions using the examples above). *If C-terminal tag sequences are desired, additional bases
◦
Incubate at 22 C for 30 min (higher temperatures are may be required in the antisense primer to ensure the
unsuitable for this reaction). C-terminal sequences are in frame. If a C-terminal tag is
3. Inactivate the T4 polymerase enzyme by incubating at not desired a stop codon could be included in the
75 C for 20 min and store the prepared Ek/LIC insert at insert-specific sequence
◦
◦
−20 C if not used immediately. This prepared insert can **Assuming that this is a commercially prelinearized and
be annealed to any of the Ek/LIC vectors. T4 polymerase-treated vector, if not, the vector must be
4. Anneal approximately 100 ng of each insert with linearized, gel purified, and T4 polymerase treated in
◦
50 ng of vector** at 22 C for 5 min. Add EDTA to a final the presence of dTTP (this generates single-stranded 5
◦
concentration of approximately 6 mM, incubate at 22 C overhangs at both ends of the PCR product as far as
for 5 min. the first ‘A’ in the ends of the linear vector that will
5. Transform competent E. coli (with a competency greater complement the overhangs generated on the T4
8
than 10 c.f.u./µg DNA) with 1 µl of the annealing polymerase-treated PCR products).
reaction. Select for recombinants by plating on LB agar

genes (Studier et al., 1990). Regulated expression is For other production hosts (yeast, insect, and
achieved by using strains in which expression of a mammalian cells), standard promoter formats have
chromosomal copy of T7 polymerase is under the been used in combination with HTP cloning meth-
control of the lacUV5 promoter and hence inducible ods to produce vectors for expression screening
by the addition of IPTG (DE3 strains). In addi- (see Section 2.3.2). A particularly interesting devel-
tion, by incorporating the lac operator sequence opment is the use of multipromoter plasmids for
just downstream of the start of the T7 promoter, expression in two or more hosts from a single vec-
repression of this T7/lac promoter is achieved by tor. The construction of a dual E.coli (T7 promoter)
expression of the lac repressor (either in cis or trans). and baculovirus transfer vector (polH promoter)
Further levels of control can be obtained by coex- for expression in insect cells has been described
pressing T7 lysozyme (pLysS) at a low level in the (Chambers et al., 2004). A three-promoter vector
expression strain. T7 lysozyme inactivates any T7 (T7, p10, and hCMV or CAG promoter) is available
polymerase produced under non-inducing condi- from Novagen (pTriEX™) and its use reported for
tions, ensuring tight control of the T7 promoter comparing protein expression in E. coli and insect
vector. cells (Xu and Jones, 2004).