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RFP+sJanus Fusion Protein

Sequence and Features

Protein Expression

Transforming:
Transformed the expression vectors(concentration:121.7ng/mL) we had already built before (pET-28a) into the Escherichia coli BL21, then incubated the bacteria for 12h (37℃).
Pre-expression:
Picked up the single bacteria colony on the culture dish by a tip and put it into a 5mL LB liquid medium, incubated in the shaking table for 6h (37℃), took 400μL from it for Glycerol Stocking (-80℃) and took 200μL centrifuge and the supernatant was discarded, 20μL ddH20 re-suspended and then made sample for glue leaking, added 5μL IPTG into the rest and incubated for 4h, took 200μL for centrifuging and the supernatant was discarded, 20μL ddH20 re-suspended and then made sample for glue leaking. The leaking result is as followed.

Figure 1. The result shows that after induction the bacteria expressed obviously large amount of the target protein (length:30~40) Expression

Added 5μL Glycerin bacteria into a 5mL LB liquid medium and incubated about 15h, then added them into the 1L LB liquid medium, incubated under 37℃ until the OD was 0.6~0.8(about 4h), then cooling the atmosphere to 4℃ and incubated 0.5h, added 1mL IPTG into the flask and incubated for 16h under 16℃.

Figure 1. The expression of RFP fusion protein. The color is very obvious

Purification:
Centrifuged the mixture 4000rpm for 30min, then re-suspended the sediment by 15mL McAc. Used the ultrasonication to destroy the bacteria, then centrifuged 18000rpm for 30min and purred the supernatant by Ni-NTA, took the sample of the sediment; mixture, McAc 20, 30, 50, 100, 200, 500 which was combined with medium; misture, McAc 20, 30, 50, 100, 200 which leaked out of the Ni-NTA. Then made sample for glue leaking. The leaking result is as follow.

Figure 1. According to the result, the best removing impure concentration is 30, the best elution concentration is 200, and we got the pure protein after McAc 200 elution.

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

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Figure 1. 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-inJanus. The first and second lane are GFP-inJanus, 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 1. 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 1. 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 1. 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 1. 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-inJanus and BFP-inJanus. We added 2%, 5% of Berol 532 in GFP-inJanus and 2%, 5% (v/v) of Berol 532 in BFP-inJanus. 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 1. In this picture of gel, the first lane is marker. The second lane is the original solution of GFP-inJanus. 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-inJanus in this gel.

Figure 1. 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 1. 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 1. 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 1. 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-inJanusm. The conditions doing this experiment is same as the second group.

Reagents used in this experiment

Results of this experiment

Figure 1. In this picture of gel, the first lane is original protein solution of RFP-inJanusm. 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 1. 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-inJanusm does work.

We did the fourth group of experiment. In this experiment, we used GFP-inJanus, BFP-inJanus, RFP-inJanus and RFP-inJanusm 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 1. We did many groups of experiment.

Reagents used in this experiment

Results of this experiment

Figure 1. The experiment used BFP-inJanus 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).