description

This records what we try to purify the A-B Toxin from Winfried Wels, Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Germany. And importantly, we have taken many improve to the protocol we got, and got many helps from the teachers and professors. Until today, we extract a large amount of proteins and tried in the hela cell. Figure 1. this is the coding sequence of pWF47-TEG, which has flag, DNA binding domain, translocation domain, receptor binding domain and ect. Figure 2. this is the coding sequence of pSW55-GD5, which has the similar domain as the TEG coding sequence.

practice(8.16~8.22)

2014.8.16

Because of our plasmid can only form inclusion body in the E.coli, we decide to use a plasmid which can produce inclusion body in the E.coli. Incubate the transformed bacteria(DH5α) in the incubator.(plasmid is a eukaryotic protein and will form inclusion body, size is about 69kDa)
Transformation: https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/Preparation_work#bacteria_transformation

2014.8.17

1. Select the single colony in the plate of DH5α, and add 100 µg/ml ampicillin 300ul LB medium.
2. After shake it in the 37 ºC shaker for 12hrs, use the TIANprep Rapid Mini Plasmid Kit(TIANGEN○R) to extract the plasmid.
3. Transform the plasmid into the BL21.
4. Incubate it in the 37 ºC incubator for 12hrs.

2014.8.18

1. select the single colony on the plate and use 250ml 100 ug/ml ampicillin LB medium to incubate it.
2. Make the protein extract buffer
   • https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/Purify#Materials:

2014.8.19

‘’Extract the protein from the broth.’’

1. Harvest the cell at 4C by centrifugation at 9000rpm for 15 min.
2. Resuspend the cell in 27ml lysis buffer (1g cells = 25ml lysis buffer)
3. Thel cells are lysed by sonication for 3 min on ice (5s on, 3s off)
4. The lysate is gently shaken for 1.5 h at room temperature (26ºC)

2014.8.20

‘’Use the Ni2+ column to purify the protein’’
https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/Ni2%2B_affinity_chromatography#Purification_of_the_chimeric_fusion_protein_via_Ni2.2B_affinity_chromatography

2014.8.21

1. make the 10%SDS
2. make the 10%APS(Ammonium Persulfate)
3. make the Coomassie Brilliant Blue

2014.8.22

Use the SDS-PAGE to run the protein sample
https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/SDS_PAGE#protocol

The primary extract of 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

2014.8.23

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 37 ºC.

2014.8.24

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 37 ºC, 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 37 ºC

2014.8.25

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 37 ºC 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 37 ºC 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 37 ºC, 200rpm (6:10pm)
7. The cells were divided into 6 centrifuge tubes( 7:55pm)
8. Harvest the cell at 4C 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 (26 ºC)

2014.8.26

12. Centrifuged the cells at 4 °C 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 4°C. (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:

Fig.3 Schematic representation of the TEG fusion gene in the ‘E.coli’ expression plasmid pWF47-TEG

Fig.4 ‘E.coli’ which expressed plasmid pSW55-GD5

protein concentration measurement

Both consider the accuracy and complication of the procedure, we did not use the western-blot or the nanodrop but the Bradford assay.

2014.9.2

Make the principle curve and compare the sample with the BSA data. https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/Bradford#Protocol

Primary Refolding(2014.9.4~9.6)

2014.9.4~9.5

1. Use the 0.3mM DDT to deal with the purified protein sample for 2hrs
   • for 2ml purified sample, add about 0.6ul 1M DDT to the sample.
2. Use the refolding buffer to deal with it for 2 days(the sample concentration must lower than 100mg/ml)

Composition

Refolding buffer: 100mM tris-HCl, 8mM oxidized glutathione, 0.5M L-arginine, 150mM NaCl

2014.9.6

Run the SDS-PAGE to test if the protein has activity or not.

Note:

In this procedure, we got a mistake that we thought this process of just reduced the disulfide bonding as a operation of dilution refolding. So we haven’t gotten any effective results in this primary try.

Second try(2014.9.8~2014.9.13)

Background

We concluded that in the primary trying, we had many wrong and bad operation, and in this day we did a second attempt.
We did some improvement to the procedure:

1. the time of centrifuge to harvest the cell, we decrease it to 4000 rpm, because it did not need 9000 rpm to harvest the cell(E.coli is easy to harvest), and the over high speed is harmful to the machine.
2. the Ni2+ column purify, we didn’t filter in the primary try, and the impurity will affect the column
3. the refold, we made a mistake as we said before, we changed it and after the reduction ,we did the dialysis refolding.

Results

Third try(2014.9.22~2014.9.30)

Background

Repeat experiment, due to the low concentration of protein, we enlarge the system of broth to 2L, which means that for each protein we make a 2L broth.

Results

Ask for prof’s help(2014.10.1~2014.10.2)

Without any ideal results, we visit professor Hongming Zhang, who is concentrate on the structure of protein analysis, and have rich experience of purify of protein. He gave us mainly two advices, we can just do not add urea to in the cell lysis system and just use DNase, RNase, Protease and lysozyme to deal with the lysis, and use some low salt buffer to wash and centrifuge it, remove the supernatant and remain the deposit in the every step. At last, when the deposit become white and pure, using buffer contain urea to dissolve it. And the concentration of protein can be control and largely improved.

Forth try(2014.10.4~2014.10.11)

Background

This experiment happen in the National holiday, and we can explore different methods to purify and refold the protein. We two persons divide into two direction to do the experiments. Long will do the repeat experiments and improve it. Su will do the new method that Prof.Zhang told us.

Protocol of Zhang’s

Results

The arrangement of results before(2014.10.12)

Fifth try(2014.10.13~2014.10.16)

Background

The more important thing changed to the how to refold the protein and make it active, Su will do the Zhang’s method and Long will do a new method that Prof.Yu told.

Protocol of Yu’s

Results