Page 26 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 15

Protocol 1.11 Expression of the recombinant protein in the Baculovirus system
in suspension cultures

Materials 6. Add the required volume of the recombinant virus.
◦
Sf 9 cell culture 7. Incubate the spinner flask at 27 C spinning at
EX-CEL 405™serum-free medium for insect cells containing 30–70 rpm for 1 h.
◦
gentamycin sulphate, amphotericin B, and fetal calf 8. Equilibrate fresh medium at 27 C.
9. Transfer the spinner flask to the hood and adjust cell
serum
6
density to 1 × 10 cells/ml by adding the medium prepared
60-mm tissue culture plates
in Step 8.
Baculovirus high-titre virus stock
◦
10. Incubate the spinner flask at 27 C, spinning at
Microcentrifuge tubes
30–70 rpm, for 2–4 days.
50 ml sterile centrifuge tubes 11. Follow the progress of the infection by removing
Light microscope aliquots of the culture and examining under a microscope.
Spinner flask and spinner apparatus 12. Transfer 2 × 1.5 ml of the culture to microcentrifuge
Haemocytometer tubes and separate cells from the medium by centrifugation
27 C incubator at 2500 g for 5 min for analysis by SDS-PAGE.
◦
SDS-polyacrylamide gel electrophoresis system 13. Transfer the rest of the culture to sterile centrifuge
tubes and harvest the cells by centrifugation at 2500 g for
Method 5 min at room temperature.
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1. Pipette Sf9 cell culture containing 2 × 10 cells/ml 14. For non-secreted protein store the cell pellets at
into a spinner flask. –80 C; for secreted protein, keep the supernatant in sterile
◦
2. Pipette 1 ml of the culture into a 60-mm tissue tubes at –80 C.
◦
culture plate. 15. Lyse the cell pellet from Step 12 in an appropriate cell
3. Place the plate on a level surface for 30–40 min. lysis buffer and analyse the lysate and also the supernatant
4. Examine the plate to see if the cells are healthy. from Step 12 by SDS-PAGE for the presence of the
5. Calculate the volume of recombinant virus required to recombinant protein.
infect at a multiplicity of infection of 3–10 by the equation:
ml of virus required = multiplicity of infection
× number of cells/titre of
recombinant virus per ml

1.2.5 Recombinant protein expression properties of the host, chromosomal site of
in mammalian systems integration of the gene of interest, and potential tox-
icity of recombinant proteins to host cells. Delivery
1.2.5.1 Transient expression
of vector DNA can be achieved by either infection of
Expression by transient transfection methods using
the host cell line with a virus containing the recom-
mammalian cell lines is a convenient and rapid
binant gene of interest or by direct transfection of
method of producing recombinant proteins when
vector DNA.
E. coli systems fail to produce correctly folded,
Introduction of a gene of interest into the host cell
structurally homogeneous protein. Moreover, it is
line by viral infection is a convenient method since a
a method that is routinely used to produce proteins
large number of cells can be infected simultaneously.
for crystallization.
Systems employing Semliki Forest Virus, Vaccinia
Successful expression depends on several factors,
Virus, and Retoviral vectors are used. However,
such as efficiency of delivery of vector DNA to the
drawbacks include the requirement for special pre-
host cell, transcriptional and translational control
cautions when engineering and preparing the viral
elements on the vector, mRNA stability, genetic