Page 17 - Macromolecular Crystallography

6 MACROMOLECULAR CRYS TALLOGRAPHY

levels (Miroux and Walker, 1996). For proteins with precaution it is essential to maintain antibiotic resis-
disulphide bonds, host strains have been produced tance. As ampicillin is inactivated by β-lactamases
which have a more oxidizing cytoplasmic environ- secreted by E. coli into the medium one may spin
ment. For exampleAD494 (Novagen) has a mutation overnight cultures and resuspend the pellet in
in thioredoxin (trxB) and Origami (Novagen) carries fresh media.
a double mutation (trxB, gor) in the thioredoxin and
glutathione reductase genes.
1.2.2.3 Engineering proteins for purification
Fusion proteins containing a tag of known size
1.2.2.2 Expression method and plasmid and function may be engineered specifically for
stability overexpression and detection, as well as for facil-
A single colony from a freshly-streaked plate of the itating purification by rapid two-step affinity pro-
transformed host is used to inoculate a 30–50 ml cedures directly from crude cell lysates (Table 1.2).
starter culture of Luria broth (LB) medium contain- Customized fusions may be constructed tailoring
ing the appropriate antibiotic. It is important not to to the specific needs of the protein. For exam-
allow starter cultures to grow above OD 600nm > 1. ple an N-terminal signal sequence can be used to
Cells should then be centrifuged and resuspended direct the recombinant product into the periplasm.
in fresh medium for inoculation of the main cul- Using an appropriate leader sequence, antibody
ture. Growth and induction conditions vary with fragments can be secreted into the periplasm
the vector–host expression system. Usually, cells and through the outer E. coli membrane into the
are grown to midlog phase before induction, either culture medium where they can be effectively
by a rapid shift in temperature or addition of an reconstituted. The oxidizing environment of the
inducer to the medium (Protocol 1.2). It is impor- periplasm allows disulphide-bond formation and
tant to maintain good aeration in the fermentation minimizes degradation. Expression vectors incorpo-
vessel. rating the ompT and pelB leader sequences upstream

Plasmid instability can arise when the foreign pro- of the 5 cloning sites are commercially available
tein is toxic to the host cell. During rapid growth (Stader and Silhavy, 1990; Nilsson et al., 1985).
plasmids may be lost or the copy number reduced, Proteins expressed as fusions with Staphylococcal
allowing the non-recombinant cells to take over. As a protein A can be purified to near-homogeneity

Protocol 1.2 Growth and induction of expression of a heterologous sequence from vector
P L promoter
◦
This is an analytical scale induction to check for expression 5. Move cultures to a water bath set at 40 C and continue
levels. growing the cultures at this temperature for 2 h.
E. coli host strain AR58 containing defective phage 6. Remove 1 ml aliquot from each for analysis.
lambda lysogen transformed with a recombinant vector 7. Record the OD 650nm ; typically it will be 1.3 or higher if
which carries antibiotic resistance for kanamycin (Kan R). the gene product is not toxic to the cell.
8. Harvest remaining cells by centrifugation and freeze
◦
1. Grow recombinant and control E. coli strains overnight at –70 C.
◦
at 32 C in LB containing kanamycin antibiotic. 9. For large-scale cultures, use 2-litre flasks (with baffles).
◦
2. Dilute the overnight culture 1 in 60–100 into fresh LB Induce by adding 1/3 volume prewarmed LB at 65 Ctothe
containing kanamycin. culture.
◦
3. Grow cultures at 32 C in a shaker until the OD 650nm
reaches 0.6–0.8. This protocol is adapted from Appelbaum, E. and Shatzman,
4. Remove a 1-ml sample for analysis. Pellet samples for A. R. (1999). Prokaryotes in vivo expression systems. In:
30 sec at 16,000 g in a microcentrifuge, decant the medium, Protein Expression Practical Approach, Higgins, S. J. and
and place tubes on dry ice. Hames, B. D., eds. Oxford University Press.