A-B Toxin

8.23-8.26

preliminary purification of chimeric fusion protein: GD5 and TEG

Materials:

Plasmid pSW55-GD5(chimeric fusion protein carries a yeast transcription factor Gal4,an antibody fragment specific for the tumor-associated ErbB2 antigen and an internal DT translocation domain)

Plasmid pWF47-TEG(chimeric fusion protein carries a yeast transcription factor Gal4, an EGF receptor ligand TGF-a, and an internal Pseudomonas exotoxin A translocation domain)

DH5-a,

BL21,

Amp LB plates,

100ug/ml ampicillin LB liquid media,

TIANprep Rapid Mini Plasmid Kit(TIANGEN○R),

100ug/ml ampicillin LB liquid media containing 0.6% glucose,

Lysis buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea

Binding buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 10 mM imidazole

Wash buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 50mM imidazole

Elution buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 250 mM imidazole

Cleansing buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 1 M imidazole

Ni2+ affinity chromatography

Methods

8.23

1.We got the plasmids of GD5 and TEG from Winfried Wels, Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Germany

2.Transform these two plasmids into DH5-a, and incubated at Amp LB plates over night at 37oC.

8.24

1.Pick up a single colony of TEG and of GD5

2.Transform these two colonies into 100 µg/ml ampicillin LB liquid media, grow 12h at 37ºC, 200rpm 3.Extract plasmids GD5 and TEG by TIANprep Rapid Mini Plasmid Kit(TIANGEN○R)

4.Transform plasmids GD5 and TEG to BL21, grow overnight at Amp LB plates at 37ºC

8.25

1. Pick up single colony of plasmids GD5 and TEG to 2 ml LB medium containing 100 µg/ml ampicillin and 0.6%glucose and grow 2.5h at 37ºC and 200rpm (11:15am)

2. Dilute the culture to 100ml l fresh in LB medium containing 100 µg/ml ampicillin and 0.6 % glucose , grow at 37ºC to an OD600 of 0.6 (2:00pm)

3. TEG OD600=0.30, GD5 OD600=0.38 (3:50pm)

4. TEG OD600=0.38, GD5 OD600=0.51 (5:00pm)

5. TEG OD600=0.55, GD5 OD600=0.64 (6:00pm)

6.Add IPTG to a final concentration of 0.5mM and expression is induced for 1.75h at 37ºC, 200rpm (6:10pm)

7. The cells were divided into 6 centrifuge tubes( 7:55pm)

8. Harvest the cell at 4ºC by centrifugation at 9000rpm for 15 min. (8:30pm)

9. Resuspend the cell in 27ml lysis buffer (1g cells = 25ml lysis buffer)

10. Thel cells are lysed by sonication for 3 min on ice (5s on, 3s off) (10:10pm)

11. The lysate is gently shaken for 1.5 h at room temperature (26ºC)

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12. Centrifuged the cells at 4ºC in for 40 min at 9200rpm and then collect the supernatant (8.27 0:40am)

13. Repeat step 12 (1:40am)

14. The supernatant is collected, 10 mM imidazole final concentration is added and stored at 4ºC. (2:00am)

15. Purify GD5 and TEG via Ni2+ affinity chromatography by the protocol Purification of the chimeric fusion protein via Ni2+ affinity chromatography

16. Proteins GD5 and TEG are determined by SDS-PAGE and Coomassie brilliant blue staining

Results:

Supposed Results:

We can see only one band in the elution samples of TEG and GD5, TEG is 38kDa, GD5 is 68kDa

Actual Results:

Schematic representation of the TEG fusion gene in the E. coli expression plasmid pWF47-TEG

E.coli which expressed plasmid pSW55-GD5