Introduction -galactosidase fusion proteins;
We use the pUR series of plasmid vectors (Ruther and Müller-Hill, EMBO J.
(1983)). These plasmids carry polylinkers in the C-terminal portion of the
-galactosidase gene beyond the Eco RI site used for insertion in lgt11. Therefore, the
fusion proteins will generally retain -galactosidase enzymatic activity. This could be
useful for following purification or for affinity chromatography. In any case, select the
appropriate vector and clone your open reading frame segment (generally 200-500 bp)
into the polylinker using E. coli strain 71-18. Analyze the clone by restriction mapping
and/or DNA sequencing to confirm insert orientation and maintenance of the open
reading frame.
Analytical analysis of fusion proteins:
The idea here is to examine the size of your fusion protein; and to determine its
abundance and stability.
1) Grow 1 ml overnight culture in L-broth with 100 µg/ml ampicillin (no glucose).
2) Inoculate 2 fresh 1 ml cultures with 50 µl of o/n. Grow until A590 ~0.2-0.4 at 37°C (~2 h).
3) To one culture add IPTG to 1 mM (1:100 of 25 mg/ml). Incubate at 37° for 2 h.
4) Spin down cultures in 1.5 ml Eppendorf tube and drain liquid.
5) Resuspend pellet in 100 µl 2X SDS gel sample buffer (use Eppendorf mixer and resuspend completely: 20-30 minutes).
6) Boil 2-4 min in boiling H2O broth.
7) Spin 5 min Epp centrifuge.
8) Load 10 µl on 7.5% SDS polyacrylamide gel.
9) Stain gel with Coomassie blue.
10) Destain gel and visualize bands.
Preparative fusion protein purification
Plan A: Preparative SDS-gel electrophoresis (this is suitable for unstable fusion
proteins).
1) Grow 100 ml IPTG-induced culture, harvest by centrifugation and resuspend in
3-5 ml 2X SDS-gel sample buffer.
2) Boil 5 min and spin at 12,000 x g for 10 min.
3) Remove supernatant and load ~1 ml onto 1.5 mm thick 7.5% preparative SDS gel.
4 Stain gel with 0.25 M cold KCl. The fusion protein band appears as a white band where K+-SDS complexes precipitate. Alternatively, the gel can be stained with Coomassie blue without acetic acid and then detained in H2O.
5) Excise band and either pulverize it for direct injection or electroelute the protein and concentrate it by acetone precipitation.
Plan B: Insoluble aggregate purification. (This is suitable for abundant fusion proteins and when you want a lot of fusion protein, e.g. for coupling to CNBr-Sepharose for affinity purification).
1) Grow 1 L IPTG-induced culture; harvest by centrifugation and freeze at -70°C.
2) Thaw cell pellet on ice in 50 ml Buffer A (50 mM Tris-HCl pH 7.9, 200 mM NaCl, 2 mM EDTA, 2 mM b-mercaptoethanol, 100 µg/ml PMSF, 100 µg/ml PMSF, 1 µM pepstatin and 1 µM leupeptin).
3) Add solid lysozyme to 0.2 mg/ml - incubate on ice for 20 min.
4) Add triton X-100 to 1% - incubate on ice for 10 min.
5) Add Zwittergent 3-14 (CalBiochem) to 0.5% . Incubate on ice for 10 min.
6) Sonicate 3 x 15 seconds on ice using a Branson sonicator with a microtip.
7) Layer the lysate onto 10 ml of 40% sucrose in 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.2 M NaCl.
8) Centrifuge 30 min at 13K rpm in a Sorvall HB-4 swinging bucket rotor.
9) Resuspend aggregate pellet in 2 ml PBS.
10) Add 20 ml urea extraction buffer (8M urea, 0.5 M NaCl, 0.5 M Tris-HCl, pH 7.9, 1 mM EDTA, 30 mM b-mercaptoethanol, 1 µM pepstatin and 1 µM leupeptin).
11) Vortex vigorously until pellet is dissolved.
12) Dialyze at room temperature for 2 h against 2 L of 50 mM Tris-HCl pH 7.9, 0.5 M NaCl, 10% glycerol, 200 µg/ml PMSF with 1 change.
13) Remove insoluble material by centrifugation at 10,000 x g for 10 min.
14) The fusion protein is present in the supernatant at ~1-4 mg/ml and is ~80% pure. It can be used directly for immunization and should be stored at -20°C.
Immunization:
1) We use 3-5 lb. female New Zealand white rabbits.
2) Inject 50-200 µg fusion protein in 0.5 ml PBS + 0.5 ml complete Freund's adjuvant (Gibco) at multiple intradermal sites or subcutaneously.
3) At 3-week intervals - a boost of the same amount of antigen with incomplete Freund's adjuvant. After 2 boosts, the serum is usually high titer. The rabbits are bled and serum prepared.
4) I have kept boosting for 3-4 months and can still get usable (high titer ) serum.
Preparation of Serum from Blood:
1) Bleed rabbit from central ear artery into glass Corex tube. Allow blood to clot
at room temp for 1-2 h.
2) Use pipette to dissociate clot from sides of glass tube and leave o/n at 4°C for clot to contract.
3) Use pipette to remove serum; (should be approx. 1/3 of the total blood
volume). Spin at 1000 rpm in benchtop clinical centrifuge or 5K in Sorvall SS34
for 5 min to pellet RBC. Store at -20°C in conveniently sized aliquots.