Difference between revisions of "Part:BBa K2933102"
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===Usage and Biology===
 
===Usage and Biology===
 
This composite part is made up with two basic parts, the His-Sumo tag and our target protein SPG-1. It encodes a protein which is SPG-1 fused with His-Sumo tag. The fusion protein is about 42.2kD. In order to gain the highly purified target protein, we add His-Sumo tag and there is a cutting site of Sumo Proteasein the first part so that the fusion protein can be cut off at the cutting site by Sumo Protease. It is convenient for us to purify our target protein.<br>
 
This composite part is made up with two basic parts, the His-Sumo tag and our target protein SPG-1. It encodes a protein which is SPG-1 fused with His-Sumo tag. The fusion protein is about 42.2kD. In order to gain the highly purified target protein, we add His-Sumo tag and there is a cutting site of Sumo Proteasein the first part so that the fusion protein can be cut off at the cutting site by Sumo Protease. It is convenient for us to purify our target protein.<br>
 +
===Molecular cloning===
 +
 +
 +
===Expression and purification===
 +
'''Pre-expression:'''<br>
 +
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 +
 +
'''Massive expressing:'''<br>
 +
After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.<br>           
 +
 +
'''Purification of GST fusion proteins:'''<br>
 +
 +
'''Anion exchange column:'''<br>
 +
According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein. Since the isoelectric point of our protein is around 9.5 in theory, we choose buffer pH of 7.4 and use anion exchange column for purification.
 +
The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.
 +
Balance the selected column with liquid A. Through the AKTApure protein purification system, the samples are loaded to the column at a flow rate of 0.5ml/min, and continue washing for 5min. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak. Use SDS-PAGE to check the result.<br>
 +
 +
'''Gel filtration chromatography:'''<br>
 +
The collected protein samples are concentrated in a 10 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 200 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.<br>
 +
<p style="text-align: center;">
 +
  [[File:--File-T--TJUSLS China--SPG gel jiaotu+fengtu jpg--.png]]<br>
 +
'''Figure 1.'''  (a) The result of gel filtration used the superdex75 column with the AKTA system, which shows that the target protein is monomeric. (b) The result of SDS-PAGE. And the target protein is about 30kD.<br>
 +
</p>

Revision as of 04:49, 8 September 2019


His+Linker a+Sumo+Linker b+SPG-1

This part encodes the fusion protein of His-Sumo tag and SPG-1 to promote the expression and purification of target protein(SPG-1).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with two basic parts, the His-Sumo tag and our target protein SPG-1. It encodes a protein which is SPG-1 fused with His-Sumo tag. The fusion protein is about 42.2kD. In order to gain the highly purified target protein, we add His-Sumo tag and there is a cutting site of Sumo Proteasein the first part so that the fusion protein can be cut off at the cutting site by Sumo Protease. It is convenient for us to purify our target protein.

Molecular cloning

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.

Massive expressing:
After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.

Purification of GST fusion proteins:

Anion exchange column:
According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein. Since the isoelectric point of our protein is around 9.5 in theory, we choose buffer pH of 7.4 and use anion exchange column for purification. The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles. Balance the selected column with liquid A. Through the AKTApure protein purification system, the samples are loaded to the column at a flow rate of 0.5ml/min, and continue washing for 5min. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak. Use SDS-PAGE to check the result.

Gel filtration chromatography:
The collected protein samples are concentrated in a 10 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 200 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

--File-T--TJUSLS China--SPG gel jiaotu+fengtu jpg--.png
Figure 1. (a) The result of gel filtration used the superdex75 column with the AKTA system, which shows that the target protein is monomeric. (b) The result of SDS-PAGE. And the target protein is about 30kD.