Difference between revisions of "Part:BBa K1582004"

Latest revision as of 16:58, 23 September 2015

GFP+sJanus Fusion Protein

Sequence and Features

Usage

Admittedly the separation of proteins is difficult in biological experiments. More often than not, we use chromatography, but it is expensive. Extraction turns to be cheap, however, its low separation rate is really an obstacle.
Here comes the Janus, a kind of amphipathic protein, which is made into a fusion protein with the target and carry it to leave the bulk protein phase. We could use aqueous two-phase systems to make them separated from mixture, according to the property that Janus will direct to the phase of detergent in the system of detergent/polymer. GFP-sJanus fusion protein could be used to verify this purification system.
This part provide a new way to express purified GFP, and this method could also be used for other protein, which has been verified through our modeling. At the same time, we use GFP because the phenomenon of protein exraction could be very obvious.

Biology

sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. They shows thread-like structure, can be expressed in prokaryotic cells like E.coli, and are involved in transferring themselves to detergent phase spontaneously.

Reference

[1]Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands. Hydrophobins: multipurpose proteins.Annu Rev Microbiol. 2001;55:625-46.

Protein Expression

Figure 1. The result of protein GFP-sJanus expression. In this picture of gel, we can see that when induced by IPTG, the gene of GFP-sJanus expressed. The correspond place has been underlined by red line.

Figure 2. The result of protein BL21_GFP-sJanus expression. H is Protein marker. G is sample of liquid after filtration by Ni column. F is sample of media after removing impurity with 20mM MCAC.E is sample of liquid after removing impurity with 20mM MCAC. D is sample of media after removing impurity with 50mM MCAC. C is sample of liquid after removing impurity with 50mM MCAC. B is sample of media after removing impurity with 100mM MCAC.A is sample of liquid after removing impurity with 100mM MCAC.

Figure 3. The result of protein BL21_GFP-sJanus expression. A is Protein marker. B is sample of media after washing with 100mM MCAC. C is sample of liquid after washing with 100mM MCAC. D is sample of media after washing with 200mM MCAC. E is sample of target protein after washing with 200mM MCAC. F is sample of media after washing with 500mM MCAC. G is sample of target protein after washing with 500mM MCAC. H is sample of target protein after washing with 1000mM MCAC.

After that we use the target protein concentrating, we got our target protein GFP-sJanus, whose concentration is 50.1mg/ml.

Protein Extraction Kit

Background

ATPS (aqueous two-phase systems) is a novel technology to purify proteins. The mechanism of this system lies in the partition between two different phases.

Aims

Construct a brand new and standard way to purify proteins based on aqueous two-phase system.

Results

1. Confirm the strengths using aqueous two-phase systems
2. Successfully separate the target proteins from bulk protein phase based on aqueous two-phase systems at a high partition rate.
3. Construct a standard protocol to separate different kinds of proteins.

Pre-experiment

Process of this experiment

In the pre-experiment, the original concentration of our protein is about 50ug/ml, which volume is 200uL. We designed the pre-experiment just in order to make preparations for our next experiment. We added 5% (v/v) Berol 532 to protein solution. And then, we used shaker to make them mixed at the speed of 250r/min and 20 centigrade working about one hour. Centrifuge was used to make them separated and came into being two phases at the speed of 8000g for about 25min. In these two phases, the upper phase is rich-detergent and the lower phase is depleted-detergent phase. Because of the property of hydrophobin, fusion protein will stay in the detergent phase, and bulk protein stay in the water phase. We put the rich-detergent phase in another centrifuge cubes and added butanol which is 5 times volume of detergent. Centrifuge was used to make them separated and finally in the lower phase (water phase), we got pure target protein. The upper phase contains detergent (Berol 532), which can be recycled.

Reagents used in this experiment

Results of this experiment

Figure 4. In this picture of gel, the third lane is the original protein solution and the forth is the protein solution which used ATPS to purify. We can see that some bulk proteins have been removed. This fusion protein is GFP-sJanus. The first and second lane are GFP-sJanus, because we didn’t dilute the protein’s solution, the concentration of protein is so high (about 3mg/mL) that they overflowed. The next is 100ug/mL and 50ug/mL.

Figure 5. We used Bandscan5.0 to analysis the gel. We can see that other proteins have been removed all in the third and fourth lane. Meanwhile, we can see that other proteins have been removed to some degree in the fifth and sixth lane.

We calculated the efficiency of this experiment. It is 18.93% in the third and fourth lane and 46.17% in the fifth and sixth lane. The efficiency is a bit low and we analyzed the reasons. We added 10uL of Berol 532 in this system to extract the target protein. However, the total amount of protein is so large that our detergent can’t extract them all.

Formal experiment using Berol 532 as detergent and MCAC0 as buffer

Process of this experiment

In our first formal experiment, we changed some conditions. We increased the speed of shaker to 300r/min in order to make them mixed totally. Meanwhile, in the pre-experiment, we found that we didn’t need to use centrifuge to make them separated because they can divided into two phases automatically in few seconds at the room temperature(Berol 532, article says that its low solubility does not allow cloud point measurement). We used 20% (v/v) Berol to extract our fusion proteins. If less detergent used in this system, it will increase the final concentration of proteins, but it will decrease the efficiency of extraction because detergent is lacking in combing the fusion protein.
The volume of our system is about 5mL (We used original solution which got by centrifuge and high pressure after suspending by MCAC0). We added 1mL Berol 532(20% w/w) in our original solution. And then, we used shaker to make them mixed at the speed of 300r/min and 20 centigrade working about one hour. We put the tube at the room temperature and the mixture was separated into two phases in few seconds. We got the upper phase (2mL) and added water saturated butanol (5mL). Then we used centrifuge to make them separated at 4 centigrade (In this temperature, we can prevent protein from denaturation for a little longer time) at the speed of 3500rpm (8000g and 3500rpm both worked) working for 10 minutes. We got the lower phase, and it contains our target protein. We did this twice in order to increase the purity of our target proteins. We used technology of SDS-PAGE to test if this system works.

Reagents used in this experiment

Results of this experiment

Figure 6. In this picture of gel, the first lane is the original solution of protein. We can see that it contains many other proteins. The second lane is the lower phase (water phase), we can see that the target protein have been removed and some bulk proteins stay in the water phase. The third and fourth lane are the water phase got in the reverse extraction. We can see that the target proteins have been concentrated and some other proteins have been removed.

Figure 7. This picture was shot in ultraviolet, we can see clearly that our target protein stay in the detergent phase because of the blue fluorescence.

Figure 8. We used Bandscan5.0 to analysis the gel. We can see that target proteins have been purified by ATPS. In this process, other proteins stay in the water phase and our target protein stay in the detergent phase. When extracted by saturated butanol, target protein was concentrated in the water phase. The picture of gel shows that when used it twice, the purity will increase again

We calculated the efficiency of this experiment. It is 67.54% in the twice and fourth lane and 75.06% in the fourth and fifth lane. The efficiency of it is high to some degree, but we want get a higher separation rate and increase the purity of our target protein. In our next experiment, we used many buffer and different detergent to achieve it.

Formal experiment using Berol 532 as detergent and HAc/NaAc as buffer

Process of this experiment

In this experiment, the fusion proteins we used are GFP-sJanus and BFP-sJanus. We added 2%, 5% of Berol 532 in GFP-sJanus and 2%, 5% (v/v) of Berol 532 in BFP-sJanus. We added 200uL HAc/NaAc(pH=7.5) to 4800uL protein solution. And then, we used shaker to make them mixed at the speed of 300r/min and 20 centigrade working about one hour. We added butanol which is 5 times volume of detergent to do reverse extraction. Centrifuge was used to make them separated and finally in the lower phase (water phase), we got pure target protein.

Reagents used in this experiment

Results of this experiment

Figure 9. In this picture of gel, the first lane is marker. The second lane is the original solution of GFP-sJanus. The third lane is the system adding 2% and the fourth is that adding 5%. We can see clearly that other proteins have been removed. Unfortunately, because of some unknown reasons, there isn’t any line about BFP-sJanus in this gel.

Figure 10. We used Bandscan5.0 to analysis the gel. The percentage of our target protein in total proteins is 11.5% before separating. It increased to 59.1% in the third lane(2% Berol 532) and 88.7% in the fourth lane(5% Berol 532). Many other proteins have been removed.

Figure 11. We can see the green florescence in the upper phase. This picture was shot in ultraviolet.

We did the second group of experiment. In this experiment, the concentration of protein solution and volume of Berol 532 changed. The total volume of protein solution is 6mL. The conditions doing this experiment is same as above.

Reagents used in this experiment

Results of this experiment

Figure 12. In this picture of gel, the first lane is original protein solution. The second and third lane are proteins separated by ATPS. The former is 2% of Berol 532; the latter is 5% of Berol 532. We can see that the system added 5% of Berol 532 get the best result.

Figure 13. We used Bandscan5.0 to analysis the gel. The percentage of our target protein in total proteins is 26.4% before separating. It increased to 74.6% in the third lane(5% Berol 532). Many other proteins have been removed. However, there isn’t any line detected in the second one.

We did the third group of experiment. In this experiment, the protein we used is RFP-sJanus-m. The conditions doing this experiment is same as the second group.

Reagents used in this experiment

Results of this experiment

Figure 14. In this picture of gel, the first lane is original protein solution of RFP-sJanus-m. The second and third lane are proteins separated by ATPS. The former is 2% of Berol 532; the latter is 5% of Berol 532. We can see that the system added 5% of Berol 532 get the best result.

Figure 15. We used Bandscan5.0 to analysis the gel. The percentage of our target protein in total proteins is 19.5% before separating. It increased to 78.6% in the second lane(2% Berol 532) and 60.9% in the fourth lane(5% Berol 532). Many other proteins have been removed. This experiment confirmed that RFP-sJanus-m does work.

We did the fourth group of experiment. In this experiment, we used GFP-sJanus, BFP-sJanus, RFP-sJanus and RFP-sJanusm to test this system. The concentration of original protein solution was detected by BCA measurement. The conditions doing this experiment is same as the second group.

Figure 16. We did many groups of experiment.

Reagents used in this experiment

Results of this experiment

Figure 17. The experiment used BFP-sJanus failed but other groups succeeded. We can see that 5%(v/v)of Berol 532 is suitable for all groups. After calculating, we found that the efficiency of systems adding 5% of Berol 532 are the highest. It’s correspond to the results measured by BCA.

Formal experiment using Berol 532 as detergent and Reppal PES100 as polymer

It has been reported that polymer can increase the efficiency of extraction and the system can be divided into two phases automatically without above cloud point. We used Reppal PES100 as polymer to do this experiment. However, it doesn’t work.

Formal experiment using Triton X-100 as detergent and HAc/NaAc as buffer

The cloud point of Triton X-100 is 64-65 centigrade. We changed detergent and did this experiment again. We put the tubes in the heat block to make system separated. However, there aren’t any florescence in the detergent phase (the lower phase).

Formal experiment using Triton X-114 as detergent and HAc/NaAc as buffer

The cloud point of Triton X-114 is 22 centigrade. We changed detergent and did this experiment again. We made the system separated at the room temperature, and used centrifuge to get a better separation. However, there aren’t any florescence in the detergent phase (the lower phase).