Part:BBa_K608408

GST tag

Results

The non-purified, sonificated supernatant of a 50ml E.coli culture pellet was resuspended in 7.5ml PBS. This sample delivered 319,4 µg/µl of protein detected with a Bradford assay. A second identical sample was GST-purified with a Glutathion-Sepharose pull down assay and resulted in 1ml of sample with a protein concentration of 1,6 µg/µl. However both concentrations were a little above the straight calibration line values. Therefore the results are not perfectly quantified, but the numbers are still significant enough to provide a good proof of the functionality of the assay.

Then the GFP fluorescence of 100µl of supernatant of both samples was measured with a plate reader:

Non-purified: 180630

GST-purified: 243011

Thus the ratio of GFP absorbance / 1µg protein is:

Non-purified: 5642

GST-purified: 1432257

If the values of the Bradford assay are assumed as correct, the purified sample has a 254x higher GFP concentration than the non-purified sample. Due to 3 washing steps it can be assumed that there is barely any other protein left except GFP. Furthermore it can be estimated that only 17% of the total GFP amount present in the 50ml cell culture, could be captured using this assay.

Methods

Plate reader:

The fluorescence intensity and protein concentration were measured with the FLUOstar Omega, which is a multi-mode microplate reader.

Samples were pipetted into the microplate and analyzed via the plate reader. In this experiment we focused on the protein concentration and the fluorescence intensity of GFP.

Bradford-assay: A method to determine the total protein concentration.

To analyze the protein concentration of the samples, Coomassie Brillant Blue was pippeted to each sample. With the binding of the dye to the proteins the color changes from dark red to blue. The more protein in the solution the more Coomassie dye can bind to proteins and the more the color changes into blue. The absorption of bound Coomassie dye is 595nm. The absorbance is proportional with the amount of bound dye. With a series of Bovine Serum Albumin (BSA) measurements the exact protein concentration of the samples can be determined. BSA acts like a “marker” because the concentration of BSA is known and with a linear calibration line the exact protein concentration can be detected.

Usage and Biology

Prepare construct

• Cloning: Clone the GST Biobrick part BBa_K608408 beforte the N-terminus of the protein you wish to purify. You may want to put a Glycine linker inbetween the two parts, for better expression and folding proterties of the fusion protein. DO NOT USE THE RFC10 STANDARD, but another, which allows fusion proteins. We recommend the Gibson Assembly, it worked conviently for us.

• Vector: You may want to use a vector especially designed for protein expression. The iGEM vectors are all high copy plasmids, which can lead to an overload of protein in your E. coli cells. This can result in toxic effects or inclusion bodies.

• Transformation: you may want to chose a E. coli strain especially designed for protein expression. It is helpful for a successfull protein purification to use a protease deficient strain such as BL21.

Cell culture and Lysis

• Grow cells overnight after transformation in 2ml medium.

• Inoculate 250mL LB in a 1l flask

• Optional: induce with IPTG (500 µL)

• centrifuging cells in 50ml falcon tubes

• resuspend pellets in 7,5µl ice cold PBS

• sonificatie cells (4x 1 min with pause, maximal power, 1 pulse per second)

• Add 1,5X Protease inhibitor and 25µl of 10mM Lysozyme and let rotate at room temperature for 30min

• Centrifuge lysate for 10min at 500xg

• Preparation of glutathione sepharose beads. You may add 1X protease inhibitor to all used PBS.

• Gently shake the bottle of sepharose to resuspend the matrix.

• Use a pipet to remove sufficient slurry for use and transfer to an 15 ml falcon tube. (Dispense 1.33 ml of original sepharose slurry per ml of final volume required.)

• Sendiment the matrix by centrifugation at 500xg for 5 min. Carefully decant the supernatant.

• Wash the sepharose by adding 10 ml of cold 1xPBS per 1.33 ml of the original slurry of glutathione sepharose dispensed. Invert to mix. (Sepharose must be thoroughly washed with PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures).

• Sediment the matrix by centrifugation at 500xg for 5 minutes. Decant the supernatant.

• For each 1.33 ml of the original slurry, add 1 ml of 1xPBS. This produces a 50% slurry. Mix well prior to the subsequent pipetting steps. (Sepharose 4B equilibrated with PBS may be stored at 4°C for up to a month.

Purification of fusion proteins

• Transfer supernatant from step 2)h) on top of slurry from step 4)f)

• Rotate falcon for 30 min

• Centrifuge at 500xg for 5min

• Collect supernatant in a separate 50ml falcon ( in case the protein does not attach well to the beads, this supernatant can be used for a second purification)

• Add 10ml of cold PBS with protease inhibitor to flask with the beads

• Rotate falcon for 10 min

• Repeat steps c) - f)

• Centrifuge at 500xg for 5min

• Collect supernatant in a separate 50ml falcon (in case the protein does not attach well to the beads, this supernatant can be used for a second purification)

• Eluate beads with 1ml elution buffer (50mM Tris pH 8, 10mM Glutathione) – transfer slurry into a 2ml eppi

• Centrifuge eppi at 13000rpm to make a stable pellet

• Transfer supernatant to a new eppi. Take care not to take up beads from the pellet.

• Optional: repeat j) – l) to wash the rest off the beads

Further reading

Sandra Harper, David W. Speicher (2008); „Expression and Purification of GST Fusion Proteins“; John Wiley and Sons, Inc.; DOI: 10.1002/0471140864.ps0606s52

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 85
  

Characterization by 2021iGEM_Shanghai_Metropolis

Improvement of an existing part

Compared to the old part BBa_K608408, we design a new part BBa_K4004007, which contains the GST and VP1-LTB fusion protein. The VP1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus.

The Group iGEM11_Freiburg aimed to transform protein expression in bacterial systems into an elegant, fast and affordable process. In this process, they used GST tag.

Based on the group’ contribution, our team design the new composite part BBa_K4004007 to express VP1-LTB fusion protein. After the composite part was inserted in a particular plasmid vector and entered an industrial bacteria.

Figure 13. The blast results about the DNA sequence of our new part BBa_K4004007 and the old part BBa_K608408..

First of all, we constructed a composite part BBa_K4004007 and transformed it into E.coli.

Furthermore, VP1 and VP1-LTB were successfully expression in E. coli predominantly as inclusion bodies. As a mucosal immune adjuvant, LTB enhances the antiviral ability of vp1.

In addition, we are trying to develop a new oral vaccine for Hand-foot-mouth disease. our project has a huge potential commercial market.

Usage and Biology

Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus 71 (EV71). The virus is an important pathogenic factor of hand, foot and mouth disease. Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus. Generally, the vaccinated population, especially infants and young children, are more compliant with oral vaccines, so we are trying to develop oral HFMD vaccines. Probiotics bacteria Bifidobacteria, as the natural host of the intestinal tract, can adhere to intestinal epithelial cells and are ideal oral live vaccine expression vectors, and related studies have found that their preventive effects on gastrointestinal pathogens are more significant. Therefore, we can use the bifidobacterium in lactic acid bacteria as an expression system to express EV71 vp1.

Figure 1. Concept map of the EV71 oral vaccine...

Construct design

We link vp1 and LTB with a linker. The vp1-LTB is inserted into plasmid pGEX, respectively (Figure 2 and 3).

Figure 2. The expression system of EV71 vp1...

Figure 3. Schematic map of expression system of pGEX-vp1-LTB plasmid...

The profiles of every basic part are as follows:

Usage and Biology

BBa_K4004001 is a coding sequence of from E. coli . Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus.

Usage and Biology

BBa_K4004005 is a coding sequence of E. coli, which has strong immunogenicity and adjuvant activity, and will not cause harm to the human body.

Usage and Biology

BBa_K4004003 is a plasmid backbone. pGEX plasmid allows cloning of gene of interest into bacterial expression vector with PreScission Protease cleavable N-terminal GST tag.

Experimental approach

PCR for VP1, VP1-linker and LTB fragments

Firstly, to amplify VP1 fragments and VP1-linker fragments from pUC57-VP1 and LTB fragments from pUC57-LTB, we added VP1-FP and VP1-RP into two tubes to amplify VP1 fragments, VP1-FP and VP1-linker-RP into another two tubes to amplify VP1-linker fragments, and LTB-FP and LTB-RP into another two tubes to amplify LTB fragments.

To confirm whether we successfully amplified the fragments we wanted from the plasmids, we ran the electrophoresis of the fragments in the six tubes. We then scanned the gel, compared the strong bands with the markers, and identified VP1, VP1-linker and LTB fragments on the gel. If we got the expected results, we can extract the three types of fragments from the gel and continue our experiments: digestion of VP1 fragments and OE PCR of VP1-LTB fragments.

Figure 4. Gel electrophoresis of VP1, VP1-linker and LTB fragments after PCR...

Conclusion: Theoretically, VP1 fragment is 891bp in length; VP1-linker fragment is 948bp in length; LTB fragment is 604bp in length. Compared with the markers, the strong bands in the six tubes all fit in the right range, so it proved that our PCR for the three types of fragments was successful, and we could continue our experiments.

·OE PCR for VP1-LTB fragments

After obtaining VP1-linker and LTB fragments from PCR, we overlapped them through OE PCR. We added the two types of fragments, VP1-FP, and LTB-RP into one tube and waited for them to overlap. Then we conducted double digestion on the newly ligated fragments.

To confirm whether we successfully overlapped the two fragments, we ran the electrophoresis of the fragments in the tubes. We then scanned the gel, compared the strong bands with the markers and identified VP1-LTB fragments on the gel. If we got the expected results, we can extract the fragments from the gel and insert them into the vectors.

Figure 5. Gel electrophoresis of VP1-LTB fragments after OE PCR and enzyme digestion...

Conclusion: Theoretically, digested VP1-LTB fragment is 1552bp in length. Compared with the markers, the strong band fit in the right range, so it proved that our OE PCR for VP1-LTB was successful, and we could continue our experiments.

·Clonexpress Ligation reaction for pGEX-VP1-LTB

We had already obtained digested VP1-LTB fragment after PCR and OE PCR. In order to insert them into the vectors pGEX-6P-1 respectively, we first needed to use the same restriction enzymes, SalⅠ and BamHⅠ, to digest pGEX-6P-1 and make the plasmids available for ligation. We then run the gel electrophoresis of digested pGEX-6P-1, identified the fragments we wanted, and extracted them from the gel. After that, we conducted ClonExpress ligation reaction to ligate VP1-LTB fragment with pGEX-6P-1.

Figure 6. gel electrophoresis of pGEX-6P-1 after double digestion...

Conclusion: Theoretically, pGEX-6P-1 after double digestion of SalI and BamHI is 4975bp in length. Compared with the markers, the strong band fit in the right range, so we can continue to conduct ClonExpress ligation reaction for pGEX-6P-1-VP1-LTB.

Simultaneously, we used Snapgene to construct the profiles of the plasmids we constructed, pGEX-6P-1-VP1-LTB. We demonstrated and understood the parts of the plasmids: restriction gene, target gene, and multiple cloning site.

Plasmid transformation

In this part, we transformed the plasmids we constructed into E.coli to replicate them, then extracted and verified the plasmids, and transformed them into E.coli and L.casei again. Finally, we verified the expression of VP1-LTB proteins in the two types of bacteria.

Verification of transformed plasmids

·Electrophoresis of pGEX-VP1-LTB

After transforming the plasmids into E.coli DH5α to replicate them, we picked 6 individual colonies from each petri dish and extracted the plasmids from them. To confirm whether the extracted plasmids were the ones we required, we ran a DNA gel electrophoresis of the transformed plasmids; we also run gel electrophoresis of pGEX-6P-1 after single digestion on the same gel as a negative group. We then scanned the gel, compared the brightest DNA bands with the markers, and identifies pGEX-6P-1-VP1-LTB on the gel.

Figure 7. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB...

Conclusion: Theoretically, pGEX-6P-1 is 4984 bp long in linear shape; pGEX-6P-1-VP1-LTB is 6511 bp long in linear shape. pGEX-6P-1, which became linear-shaped after single digestion, should be around 5000bp in length, Compared with the markers, the plasmids in the twelve groups that we extracted from E.coli DH5α all fit in the right range. Therefore, we reached a preliminary conclusion that we succeeded in the experiments of plasmid transformation and obtained the plasmids we wanted.

·Restriction enzyme double digestion of pGEX-VP1-LTB

We had tested the plasmids we extracted from E.coli DH5α in terms of their length. To further confirm whether the plasmids carried the fragments we wanted, we used SalⅠ and BamHⅠ to cut off the fragments between their cutting sites from pGEX-6P-1-VP1-LTB; simultaneously, we conducted single digestion on each group as the negative control group. Then we run the gel electrophoresis of the digested plasmids to separate and identify the fragments cut off from the plasmid.

Figure 8. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB after double digestion...

Conclusion: Theoretically, VP1 fragment is 891bp in length; LTB fragment is 604bp in length；pGEX-6P-1 after double digestion by SalⅠ and BamHⅠis 4975 in length. Compared with the markers, sample 3 of pGEX-6P-1-VP1-LTB was correct as well. However, in sample 4 B+S (pGEX-6P-1-VP1-LTB after double digestion by SalⅠ and BamHⅠ), the weaker bands, which should represent the VP1-LTB fragments, did not appear on the gel, so we recognized the plasmids transformation in sample 4 of pGEX-6P-1-VP1-LTB as a failure. We discarded this sample and sent the samples that were proved right after enzyme digestion to a company for sequencing.

Sequencing for VP1 and VP1-LTB fragments on transformed plasmids

After enzyme digestion and gel electrophoresis of pGEX-6P-1-VP1-LTB, we tentatively confirmed that the experiment of plasmid transformation into E.coli DH5α was successful, and we obtained the plasmids we wanted. To further verify whether there was any mutation in VP1-LTB fragments on the plasmids, we needed to obtain and compare the upstream and downstream sequences of the two fragments.

Proof of function

·SDS-PAGE and Coomassie Brilliant Blue staining for whole bacteria, supernate, and precipitation

We transformed pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB into E.coli BL21 respectively and incubated them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and precipitation of E.coli BL21 and then stained the gel through Coomassie Brilliant Blue Staining.

Figure 9. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining(W: whole bacteria; S: supernatant; P: precipitation)...

Conclusion: After Coomassie Brilliant Blue Staining, we found that the extent of the brightness of the band in the P group was comparable to that in the W group, while the band in the S group was nearly invisible. In other words, GST, GST-VP1 and GST-VP1-LTB had all been successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the form of inclusion body. Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E. coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigencity of VP1 and VP1-LTB proteins.

Figure 10. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins...

Expression optimization

In order to find the optimum condition under which the proteins were expressed the most, we selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted them with IPTG solution of different concentration (IPTG=1mM/10mM). Then we ran a PAGE gel of them and then marked the proteins with Coomassie Brilliant Blue Staining Solution. To visualize and compare the expression of proteins under different conditions, we used the software ImageJ to quantify specific bands on the gel, collected and arranged the data, and constructed a broken line graph with OD600 the x- axis and the gray value as the y-axis.

Figure 11. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions...

Figure 12.Model...

Conclusion: Via this graph of model, we determined the optimum expression conditions of VP1 and VP1-LTB respectively in E.coli BL21: VP1 was expressed the most under OD600=0.5, IPTG=10mM; VP1-LTB was expressed the most under OD600=0.8, IPTG=1mM. Besides, we could see that when OD600 was over 0.8, the expression of VP1-LTB proteins was significantly higher than that of VP1 proteins in E.coli BL21. This meant that LTB could promote the expression of VP1 in E.coli BL21 during its late growth period.

Future plan

We combined VP1 and LTB to construct the recombinant expression system and used E.coli to express the proteins. We detected the protein expression with IPTG solution of different concentration at different growth stage, constructed related models, and determined the optimum expression conditions. All these laid a solid foundation for the development of HFMD oral vaccine.

1.Currently, we just verified the viability of our concept and determined the optimized condition of protein expression in E.coli. However, because of the difficulty in protein expression in L.casei, we would need to optimize the incubation and electroporation of L.casei, or improve and reconstruct the plasmids, to realize the expression of GST-VP1-LTB in L.casei.

2.If L.casei did express the proteins after the improvement of our design, the next part of our project would be that we let the mice take the transformed L.casei via gavage, and then measured the level of anti-VP1-LTB IgG in the serum of the mice and the level of IgA in their feces. Finally, we could verify immunogenicity of our oral vaccine.