Difference between revisions of "Part:BBa K2549010"
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This part is the C-terminal fragment of Cfa. Cfa is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. Cfa demonstrates both rapid protein splicing and unprecedented thermal and chaotropic durability. The 122-124 residues of Cfa is mutated from EKD to GEP, which has been proved to imbue ultrafast DnaE split inteins with minimal extein dependence, thus improving split Intein-mediated protein cyclization. Cfa’C is used in our amplifier to accomplish some complex logic functions. It can also be used by other iGEM teams to assembly their intein-based protein libraries.
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This part is the C-terminal fragment of Cfa. Split intein is a useful protein engineering approach to combine signals<ref>Protein trans-splicing and its use in structural biology: opportunities and limitations. Volkmann G, Iwaï H. Mol Biosyst, 2010 Nov;6(11):2110-21  PMID: 20820635; DOI: 10.1039/c0mb00034e</ref><ref>Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules. Jeon H, Lee E, Kim D, ..., Kim S, Kwon Y. Anal Chem, 2018 Aug;90(16):9779-9786  PMID: 30028129; DOI: 10.1021/acs.analchem.8b01481</ref>. Cfa is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. Cfa demonstrates both rapid protein splicing and unprecedented thermal and chaotropic durability<ref>Improved protein splicing using embedded split inteins. Gramespacher JA, Stevens AJ, Thompson RE, Muir TW. Protein Sci, 2018 Mar;27(3):614-619  PMID: 29226478; DOI: 10.1002/pro.3357</ref><ref>Design of a Split Intein with Exceptional Protein Splicing Activity. Stevens AJ, Brown ZZ, Shah NH, ..., Cowburn D, Muir TW. J Am Chem Soc, 2016 Feb;138(7):2162-5  PMID: 26854538; DOI: 10.1021/jacs.5b13528</ref><ref>A promiscuous split intein with expanded protein engineering applications. Stevens AJ, Sekar G, Shah NH, ..., Cowburn D, Muir TW. Proc Natl Acad Sci U S A, 2017 Aug;114(32):8538-8543  PMID: 28739907; DOI: 10.1073/pnas.1701083114</ref>. The 122-124 residues of Cfa is mutated from '''EKD''' to '''GEP''', which has been proved to imbue ultrafast DnaE split inteins with minimal extein dependence, thus improving split Intein-mediated protein cyclization<ref>A promiscuous split intein with expanded protein engineering applications. Stevens AJ, Sekar G, Shah NH, ..., Cowburn D, Muir TW. Proc Natl Acad Sci U S A, 2017 Aug;114(32):8538-8543 PMID: 28739907; DOI: 10.1073/pnas.1701083114</ref>. CfaC is used in our amplifier to accomplish some complex logic functions. It can also be used by other iGEM teams to assembly their intein-based protein libraries.
  
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
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===Biology===
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=====Protein trans-splicing=====
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Protein trans-splicing is a remarkable biological process, whereby a full-length protein is reconstituted from two fragments through the formation of a peptide bond. It is a post-translational autoprocessing event.
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[[File:Intein.png|none|400px|thumb|Volkmann G at al stated: ''Canonical protein splicing mechanism utilized by split inteins. The N-precursor protein (comprising exteinN and inteinN) and C-precursor protein (containing inteinC and exteinC) are expressed from separate genes, followed by association of the inteinN and inteinC parts of the split intein (step 1). The assembled split intein is now active to catalyze the first step in the protein splicing reaction, an N–S acyl shift involving the first residue of inteinN (step 2). The thioester intermediate is then attacked by the first residue of exteinC in a trans-thioesterification reaction (step 3), which also leads to physical separation of inteinN from exteinN. Next, the last residue of inteinC (Asn) forms a succinimide ring, effectively cleaving inteinC from the esterified exteins (branched intermediate) (step 4). The split intein likely remains assembled after the trans-splicing reaction. The final reaction is a spontaneous S–N acyl shift between the esterified exteins, leading to peptide bond formation between exteinN and exteinC (step 5). Although the reactions shown in this scheme involve Cys residues at position 1 of both inteinN and exteinC, other residues (Ser and Thr) at these positions are possible.'']]
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=====CfaN and CfaC reported in Stevens AJ et al 2016=====
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[[File:CfaNC1.png|none|400px|thumb|Stevens AJ at al stated: ''Design of the Cfa split intein. (a) Sequence alignment of Npu DnaE and Cfa DnaE. The sequences share 82% identity with the differences (cyan) evenly distributed through the primary sequence. Catalytic residues and second shell “accelerator” residues are shown in orange and green, respectively. (b) The same residues highlighted in panel a mapped on to the Npu structure (PDB ID 4Kl5).'']]
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[[File:CfaNC2.png|none|400px|thumb|Stevens AJ at al stated: ''Characterization of the Cfa intein. (a) Splicing rates for Cfa and Npu as a function of temperature. Npu is inactive at 80 °C. Error = SD (n = 3). (b, c) Splicing rates for Cfa and Npu as a function of added chaotrope. Npu is inactive in 3 M GuHCl or 8 M Urea. Note: Cfa has residual activity in 4 M GuHCl (k = 7E-5). Error = SD (n = 3).'']]
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[[File:CfaNC3.png|none|400px|thumb|Stevens AJ at al stated: ''(a) Test expression in HEK293T cells of various IntN homologues (Npu, Mcht, Ava, and Cfa) fused to the C- terminus of the heavy chain of a mouse &alpha;Dec205 monoclonal antibody. Top: Western blot analysis (&alpha;Mouse IgG) of antibody levels present in the medium following the 96 h expression. Bottom: &alpha;-actin Western blot of cell lysate as a loading control. (b) Quantification of normalized expression yield by densitometry of &alpha;DEC205 HC-IntN signal in panel a. Error = SD (n = 4). '']]
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Note: [[Part:BBa_K2549009]] and [[Part:BBa_K2549010]] have the same Biology section.
  
 
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===Functional Parameters===
 
===Functional Parameters===
 
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===References===

Latest revision as of 09:45, 7 October 2018


split intein Cfa C

This part is the C-terminal fragment of Cfa. Split intein is a useful protein engineering approach to combine signals[1][2]. Cfa is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. Cfa demonstrates both rapid protein splicing and unprecedented thermal and chaotropic durability[3][4][5]. The 122-124 residues of Cfa is mutated from EKD to GEP, which has been proved to imbue ultrafast DnaE split inteins with minimal extein dependence, thus improving split Intein-mediated protein cyclization[6]. CfaC is used in our amplifier to accomplish some complex logic functions. It can also be used by other iGEM teams to assembly their intein-based protein libraries.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 91
    Illegal SapI.rc site found at 21


Biology

Protein trans-splicing

Protein trans-splicing is a remarkable biological process, whereby a full-length protein is reconstituted from two fragments through the formation of a peptide bond. It is a post-translational autoprocessing event.

Volkmann G at al stated: Canonical protein splicing mechanism utilized by split inteins. The N-precursor protein (comprising exteinN and inteinN) and C-precursor protein (containing inteinC and exteinC) are expressed from separate genes, followed by association of the inteinN and inteinC parts of the split intein (step 1). The assembled split intein is now active to catalyze the first step in the protein splicing reaction, an N–S acyl shift involving the first residue of inteinN (step 2). The thioester intermediate is then attacked by the first residue of exteinC in a trans-thioesterification reaction (step 3), which also leads to physical separation of inteinN from exteinN. Next, the last residue of inteinC (Asn) forms a succinimide ring, effectively cleaving inteinC from the esterified exteins (branched intermediate) (step 4). The split intein likely remains assembled after the trans-splicing reaction. The final reaction is a spontaneous S–N acyl shift between the esterified exteins, leading to peptide bond formation between exteinN and exteinC (step 5). Although the reactions shown in this scheme involve Cys residues at position 1 of both inteinN and exteinC, other residues (Ser and Thr) at these positions are possible.


CfaN and CfaC reported in Stevens AJ et al 2016
Stevens AJ at al stated: Design of the Cfa split intein. (a) Sequence alignment of Npu DnaE and Cfa DnaE. The sequences share 82% identity with the differences (cyan) evenly distributed through the primary sequence. Catalytic residues and second shell “accelerator” residues are shown in orange and green, respectively. (b) The same residues highlighted in panel a mapped on to the Npu structure (PDB ID 4Kl5).
Stevens AJ at al stated: Characterization of the Cfa intein. (a) Splicing rates for Cfa and Npu as a function of temperature. Npu is inactive at 80 °C. Error = SD (n = 3). (b, c) Splicing rates for Cfa and Npu as a function of added chaotrope. Npu is inactive in 3 M GuHCl or 8 M Urea. Note: Cfa has residual activity in 4 M GuHCl (k = 7E-5). Error = SD (n = 3).
Stevens AJ at al stated: (a) Test expression in HEK293T cells of various IntN homologues (Npu, Mcht, Ava, and Cfa) fused to the C- terminus of the heavy chain of a mouse αDec205 monoclonal antibody. Top: Western blot analysis (αMouse IgG) of antibody levels present in the medium following the 96 h expression. Bottom: α-actin Western blot of cell lysate as a loading control. (b) Quantification of normalized expression yield by densitometry of αDEC205 HC-IntN signal in panel a. Error = SD (n = 4).


Note: Part:BBa_K2549009 and Part:BBa_K2549010 have the same Biology section.


References

  1. ↑ Protein trans-splicing and its use in structural biology: opportunities and limitations. Volkmann G, Iwaï H. Mol Biosyst, 2010 Nov;6(11):2110-21 PMID: 20820635; DOI: 10.1039/c0mb00034e
  2. ↑ Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules. Jeon H, Lee E, Kim D, ..., Kim S, Kwon Y. Anal Chem, 2018 Aug;90(16):9779-9786 PMID: 30028129; DOI: 10.1021/acs.analchem.8b01481
  3. ↑ Improved protein splicing using embedded split inteins. Gramespacher JA, Stevens AJ, Thompson RE, Muir TW. Protein Sci, 2018 Mar;27(3):614-619 PMID: 29226478; DOI: 10.1002/pro.3357
  4. ↑ Design of a Split Intein with Exceptional Protein Splicing Activity. Stevens AJ, Brown ZZ, Shah NH, ..., Cowburn D, Muir TW. J Am Chem Soc, 2016 Feb;138(7):2162-5 PMID: 26854538; DOI: 10.1021/jacs.5b13528
  5. ↑ A promiscuous split intein with expanded protein engineering applications. Stevens AJ, Sekar G, Shah NH, ..., Cowburn D, Muir TW. Proc Natl Acad Sci U S A, 2017 Aug;114(32):8538-8543 PMID: 28739907; DOI: 10.1073/pnas.1701083114
  6. ↑ A promiscuous split intein with expanded protein engineering applications. Stevens AJ, Sekar G, Shah NH, ..., Cowburn D, Muir TW. Proc Natl Acad Sci U S A, 2017 Aug;114(32):8538-8543 PMID: 28739907; DOI: 10.1073/pnas.1701083114