Difference between revisions of "Part:BBa K4241019"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4241019 short</partinfo>
 
<partinfo>BBa_K4241019 short</partinfo>
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<partinfo>BBa_K4241019 SequenceAndFeatures</partinfo>
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===Overview===
 +
This is composite part is RFP fused as a target protein of interest via inclusion expression. The expression is powered by a composite enhanced T7 promoter region with an enhanced translation initiation region, this results in a combined effect of up to a 40-fold increase in protein expression. This part also features the ability for DTT mediated cleavage for downstream purification purposes.
 +
It is noted that this is a mutant version.
 +
<br/>
 +
<br/>
 +
This is derived from the following publications: <br/>
 +
https://www.nature.com/articles/s42003-020-0939-8 <br/>
 +
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173929/ <br/>
  
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It is the intein fused RFP, which can be cleaved and purified with DTT.
 
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
The typical T7 consensus sequence used in the ubiquitous pET system, such as in popular plasmids such as pET28a has inherent flaws that inhibit the maximal expression and yield of proteins. Firstly, The nucleotide sequence of the T7 promoter which is derived from the consensus φ10 promoter in the T7 phage. The original consensus sequence is 23 nucleotides long spanning from -17 to +6 bases relative to the mRNA start site. However it is noted that in the pET system, the sequence is truncated at 19 nucleotides from -17 to +2 bases relative to the mRNA start site. By incorporating the full consensus sequence, the protein expression and yield is thus enhanced. Secondly, the Translation Initiation Region (TIR) is a stretch of nucleotudes that are recognised by the 30S ribosomal subunit during translation initiation. TIR-2 is an engineered translational initiation region. When combined with the enhanced T7pCONS promoter yields an increase of 121-fold over the standard pET28a in the synthesis of sfGFP.
 +
<br/>
 +
ELK16 is the basis of a self-assembling peptide-fused protein expression system, SA-ELK16 system. ELK16 indues the formation of active fusion protein aggregates in E. coli, these aggregates are easiily and cheaply purified by centrifugation. The protein of interest can then be seperated by cleaving intein via DTT mediated cleavage. The summation of this allows for high purity, cost-effective purification scheme.
 +
<br/>
 +
<br/>
 +
Purification scheme:<br/>
 +
1. Express fusion protein and sonicate cells <br/>
 +
2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom. <br/>
 +
3. Treat with DTT to cleave the protein of interest from the aggregates <br/>
 +
4. Centrifuge and remove supernatent. The pure, protein will be solubized whereas the uncleaved protein and insoluble aggregates will remain at the bottom.<br/>
 +
 +
===Results===
 +
To compare the relative expression of the enhanced T7 and the enhanced TIR: The pET system, pET system with cloned RBS and enhanced T7 were cloned into pET28(a)-FGF2 and the yield of the 21kDa product was compared. The constructs were then transformed into T7 Express lysY Competent E. coli.
 +
[[File:BBa K4241010_1.png|center]]
 +
Fig.1. The SDS PAGE result of expression of a 21kDa using standard pET system and TIR (control), pET system with cloned RBS (Normal T7+RBS) and enhanced T7 with TIR-2 (T7pCONS+TIR-2).
 +
After sonication, the protein is released from the cells. In cells expressing intein-ELK16, the RFP-tagged fusion protein remains in an aggregated and insoluble fraction.
 +
[[File:BBaK4241018-1.jpeg|center]]
 +
Fig. 2. Left: Sonicated bacteria RFP-Intein-ELK16 system. Right: Sonicated bacteria 6xHis-SUMO-RFP.
 +
<br/>
 +
[[File:BBaK4241018-2.jpeg|center]]
 +
Fig. 3. SDS PAGE results depicting cleaved RFP after overnight DTT cleavage of RFP-intein fusion.
 +
<br/>
 +
<br/>
 +
The RFP was precipitated in 2.5% acetic acid to release the RFP from the intein fusion. It was then resolubized in with urea and high salt solution. The ELK16 remains in the insoluble fraction.
 +
[[File:BBaK4241018-3.jpeg|center]]
 +
Fig. 4. RFP is denatured with acetic acid (as shown by the loss of color), but could be refolded in 8M Urea to regain RFP red colour rapidly to be back to natural folding.
 +
 +
 +
===Parts that comprise this composite component===
 +
T7pCONS-TIR-2 - Part:BBa_K4241015 <br/>
 +
highly engineered mutant of red fluorescent protein from Discosoma striata (coral) - Part:BBa_E1010 <br/>
 +
Mxe_GyrA_Intein with PT linker and ELK16 - Part:BBa_K4241018 <br/>
 +
Double stop codon - Part:BBa_K4241018 <br/>
 +
double terminator (B0010-B0012) - Part:BBa_B0015 <br/>
 +
 +
===References===
 +
Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., &amp; Wang, J. (2018). Rapid and efficient production of cecropin a antibacterial peptide in escherichia coli by fusion with a self-aggregating protein. BMC Biotechnology, 18(1). https://doi.org/10.1186/s12896-018-0473-7
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4241019 SequenceAndFeatures</partinfo>
 
  
  

Latest revision as of 05:56, 12 October 2022


RFP_Mxe_GyrA_Intein_PT_ELK16


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1482
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1305
    Illegal NgoMIV site found at 1320
    Illegal AgeI site found at 655
    Illegal AgeI site found at 767
  • 1000
    COMPATIBLE WITH RFC[1000]

Overview

This is composite part is RFP fused as a target protein of interest via inclusion expression. The expression is powered by a composite enhanced T7 promoter region with an enhanced translation initiation region, this results in a combined effect of up to a 40-fold increase in protein expression. This part also features the ability for DTT mediated cleavage for downstream purification purposes. It is noted that this is a mutant version.

This is derived from the following publications:
https://www.nature.com/articles/s42003-020-0939-8
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173929/


Usage and Biology

The typical T7 consensus sequence used in the ubiquitous pET system, such as in popular plasmids such as pET28a has inherent flaws that inhibit the maximal expression and yield of proteins. Firstly, The nucleotide sequence of the T7 promoter which is derived from the consensus φ10 promoter in the T7 phage. The original consensus sequence is 23 nucleotides long spanning from -17 to +6 bases relative to the mRNA start site. However it is noted that in the pET system, the sequence is truncated at 19 nucleotides from -17 to +2 bases relative to the mRNA start site. By incorporating the full consensus sequence, the protein expression and yield is thus enhanced. Secondly, the Translation Initiation Region (TIR) is a stretch of nucleotudes that are recognised by the 30S ribosomal subunit during translation initiation. TIR-2 is an engineered translational initiation region. When combined with the enhanced T7pCONS promoter yields an increase of 121-fold over the standard pET28a in the synthesis of sfGFP.
ELK16 is the basis of a self-assembling peptide-fused protein expression system, SA-ELK16 system. ELK16 indues the formation of active fusion protein aggregates in E. coli, these aggregates are easiily and cheaply purified by centrifugation. The protein of interest can then be seperated by cleaving intein via DTT mediated cleavage. The summation of this allows for high purity, cost-effective purification scheme.

Purification scheme:
1. Express fusion protein and sonicate cells
2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom.
3. Treat with DTT to cleave the protein of interest from the aggregates
4. Centrifuge and remove supernatent. The pure, protein will be solubized whereas the uncleaved protein and insoluble aggregates will remain at the bottom.

Results

To compare the relative expression of the enhanced T7 and the enhanced TIR: The pET system, pET system with cloned RBS and enhanced T7 were cloned into pET28(a)-FGF2 and the yield of the 21kDa product was compared. The constructs were then transformed into T7 Express lysY Competent E. coli.

BBa K4241010 1.png

Fig.1. The SDS PAGE result of expression of a 21kDa using standard pET system and TIR (control), pET system with cloned RBS (Normal T7+RBS) and enhanced T7 with TIR-2 (T7pCONS+TIR-2). After sonication, the protein is released from the cells. In cells expressing intein-ELK16, the RFP-tagged fusion protein remains in an aggregated and insoluble fraction.

BBaK4241018-1.jpeg

Fig. 2. Left: Sonicated bacteria RFP-Intein-ELK16 system. Right: Sonicated bacteria 6xHis-SUMO-RFP.

BBaK4241018-2.jpeg

Fig. 3. SDS PAGE results depicting cleaved RFP after overnight DTT cleavage of RFP-intein fusion.

The RFP was precipitated in 2.5% acetic acid to release the RFP from the intein fusion. It was then resolubized in with urea and high salt solution. The ELK16 remains in the insoluble fraction.

BBaK4241018-3.jpeg

Fig. 4. RFP is denatured with acetic acid (as shown by the loss of color), but could be refolded in 8M Urea to regain RFP red colour rapidly to be back to natural folding.


Parts that comprise this composite component

T7pCONS-TIR-2 - Part:BBa_K4241015
highly engineered mutant of red fluorescent protein from Discosoma striata (coral) - Part:BBa_E1010
Mxe_GyrA_Intein with PT linker and ELK16 - Part:BBa_K4241018
Double stop codon - Part:BBa_K4241018
double terminator (B0010-B0012) - Part:BBa_B0015

References

Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., & Wang, J. (2018). Rapid and efficient production of cecropin a antibacterial peptide in escherichia coli by fusion with a self-aggregating protein. BMC Biotechnology, 18(1). https://doi.org/10.1186/s12896-018-0473-7