Part:BBa_K3715013
T7 promoter+RBS+Linker a+His+Super6+Linker b+T7 terminator
This part encodes a protein called Super6, which is a mutation of PETase.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 863
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 863
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 878
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 863
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 863
Illegal NgoMIV site found at 138
Illegal NgoMIV site found at 192
Illegal NgoMIV site found at 219 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with five basic parts, the RBS, two cutting sites NdeI and XhoI (linker a,b) , the His tag,and our target protein Super6. It encodes a protein which is Super fused with His tag. The fusion protein is about 31.49kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Super and combine the two parts with the cutting site of protease. The fusion protein can be cut off at the cutting site by protease. It is convenient for us to purify our target protein.
Origin(organism)
Ideonella sakaiensis
Molecular cloning
First, we used the vector pET21a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(50μg/mL) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 900ml LB medium and added antibiotic to 50 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 5-6 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.
Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-His tag fused with target protein.
- First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
- Second, add the protein solution to the column, let it flow naturally and bind to the column.
- Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
- Forth,add Ni-Washing buffer several times. Check as above. Collect the eluted proteins for further operation.
Figure 2. The result of SDS-PAGE.
Gel filtration chromatography:
The collected protein samples are concentrated in a 30 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 75 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.
Figure 3. The result of gel filtration used the superdex75 column with the AKTA system.
Enzyme activity determination
We use HPLC equipment to measure the peak area of the product of PET(MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.
Figure 4. Enzyme activity determination, compared with wild type.
Conclusion
In conclusion,the enzyme activity and thermostability of Super6 has greatly improved ，compared with WT(wild type).
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