Difference between revisions of "Part:BBa K5184022"
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<partinfo>BBa_K5184022 short</partinfo>
 
<partinfo>BBa_K5184022 short</partinfo>
  
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G1M5 is the mutated, less hydrophobic version of the signal peptide of the G1 cyclomaltodextrin glucanotransferase (CGtase) of Bacillus sp., which allows the extracellular secretion of the bacterial enzyme. The signal peptide can be divided into three regions: an N-terminus region with several positively charged amino acids, a central hydrophobic region, and a hydrophilic cleavage region. The N-terminus region was found to act as intramolecular chaperones, and to be in cooperation with SecA and SecB to act as factors for protein targeting to the membrane.
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===Abstract===
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Since both SVPs and MVPs are both cysteine-rich proteins that are easily folded incorrectly while using <i>E. coli</i> as chassis, we aim to find out a method that can solve this problem. We discovered that through utilizing the G1M5-SUMO tag, solubility of the proteins expressed can be increased and successful expression is enabled. This investigation hence has reference value in <i>E. coli</i> tag selection for expression of cysteine-rich peptides for the iGEM community.  
  
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Constructs containing the signal peptide G1M5 allows extracellular secretion of the fusion protein. In context of our project this will allow export of spider venom peptide into extraceullar spaceafter translation in cytoplasm, of which can lower production cost and make production process more efficient. This exporting process can also provide a more oxidative environment that aids the formation of disulphide bridges that is essential for the correct folding of spider venom peptides."
 
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===Usage and Biology===
 
===Usage and Biology===
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G1M5 is the mutated, less hydrophobic version of the secretion signal peptide of the G1 cyclomaltodextrin glucanotransferase (CGtase) of Bacillus sp., which allows the extracellular secretion of the bacterial enzyme. Conduction of proteins attached by G1M5 out of the cytosol is achieved by the Sec pathway, a very common secretion system seen in all three major domains of life: arachaea, prokaryote, and eukaryotes. Once the signal peptide, in this case G1M5 is synthesized, the protein chaperon SecB binds to the preprotein (that is attached to G1M5), and transfers the preprotein to the protein translocase SecA, of which binds to the membrane bound protein conducting channel SecYEG. Once bound to the membrane, SecA binds to a molecule of ATP, of which is hydrolyzed to conduct the protein through heterotrimer complex of SecYEG. A membrane bound SPaseI then, once enough of the preprotein had been conducted through the channel, will remove the SP and allow the preprotein to fold properly into the correct protein.[1]
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SUMO (small ubiquitin-related modifier) family proteins are not only structurally but also mechanically related to ubiquitin. As ubiquitin, SUMO is covalently linked to its substrates via amide (isopeptide) bonds. Linkage to SUMO increases solubility of the proteins expressed. The linkage between SUMO and its substrates is an isopeptide bond between the C-terminal carboxyl group of SUMO and the ε-amino group of a lysine residue in the substrate. A three-step enzyme pathway attaches SUMO to specific substrates, and other enzymes cleave SUMO off its targets. The enzymes include SUMO-activating enzymes, SUMO-conjugating enzymes, and SUMO ligases.[2]
  
 
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<partinfo>BBa_K5184022 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5184022 SequenceAndFeatures</partinfo>
  
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===Characterization===
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The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides. This combination is used for both the expression of MVPs and SVPs.
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Induced expression and SDS-PAGE analysis were carried out respectively after the construction of vectors, including pET-GNA-His, pET-His-GNA ,pET-PelB, pET-MalE, pET-PelB-SUMO, pET-MalE-SUMO, and pET-G1M5-SUMO. In which, only pET-G1M5-SUMO exhibit correct folding of venom peptides in the supernatant portion. Others are observed in the precipitate (P) after lysis, indicating inclusion bodies. Thus, the results suggest that only expressions of proteins including the usage of G1M5-SUMO tag are capable of demonstrating correct expressions.[figure 1]
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/image-1.png" width="600"/></html></center>
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<center><b>Figure 1: SDS-PAGE of S: supernatant and P: precipitate (A) SP-rCtx4-GNA-His and SP-SUMO-rCtx4-GNA-His, (B) rCtx4-GNA-His, (C) G1M5-SUMO-rCtx4-GNA-His (D) His-rCtx4-GNA; all with complete proteins of BL21(DE3) as control</b></center>
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===Reference===
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[1]Liu, Changjun, et al. ‘A Secretory System for Extracellular Production of Spider Neurotoxin Huwentoxin-I in Escherichia Coli’. Preparative Biochemistry & Biotechnology, vol. 53, no. 8, Sept. 2023, pp. 914–22. DOI.org (Crossref), https://doi.org/10.1080/10826068.2022.2158473
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[2]Jürgen Dohmen, R. ‘SUMO Protein Modification’. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, vol. 1695, no. 1–3, Nov. 2004, pp. 113–31. DOI.org (Crossref), https://doi.org/10.1016/j.bbamcr.2004.09.021
  
 
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Revision as of 02:50, 2 October 2024


G1M5-SUMO-tag

Abstract

Since both SVPs and MVPs are both cysteine-rich proteins that are easily folded incorrectly while using E. coli as chassis, we aim to find out a method that can solve this problem. We discovered that through utilizing the G1M5-SUMO tag, solubility of the proteins expressed can be increased and successful expression is enabled. This investigation hence has reference value in E. coli tag selection for expression of cysteine-rich peptides for the iGEM community.

Usage and Biology

G1M5 is the mutated, less hydrophobic version of the secretion signal peptide of the G1 cyclomaltodextrin glucanotransferase (CGtase) of Bacillus sp., which allows the extracellular secretion of the bacterial enzyme. Conduction of proteins attached by G1M5 out of the cytosol is achieved by the Sec pathway, a very common secretion system seen in all three major domains of life: arachaea, prokaryote, and eukaryotes. Once the signal peptide, in this case G1M5 is synthesized, the protein chaperon SecB binds to the preprotein (that is attached to G1M5), and transfers the preprotein to the protein translocase SecA, of which binds to the membrane bound protein conducting channel SecYEG. Once bound to the membrane, SecA binds to a molecule of ATP, of which is hydrolyzed to conduct the protein through heterotrimer complex of SecYEG. A membrane bound SPaseI then, once enough of the preprotein had been conducted through the channel, will remove the SP and allow the preprotein to fold properly into the correct protein.[1] SUMO (small ubiquitin-related modifier) family proteins are not only structurally but also mechanically related to ubiquitin. As ubiquitin, SUMO is covalently linked to its substrates via amide (isopeptide) bonds. Linkage to SUMO increases solubility of the proteins expressed. The linkage between SUMO and its substrates is an isopeptide bond between the C-terminal carboxyl group of SUMO and the ε-amino group of a lysine residue in the substrate. A three-step enzyme pathway attaches SUMO to specific substrates, and other enzymes cleave SUMO off its targets. The enzymes include SUMO-activating enzymes, SUMO-conjugating enzymes, and SUMO ligases.[2]

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
    COMPATIBLE WITH RFC[1000]

Characterization

The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides. This combination is used for both the expression of MVPs and SVPs. Induced expression and SDS-PAGE analysis were carried out respectively after the construction of vectors, including pET-GNA-His, pET-His-GNA ,pET-PelB, pET-MalE, pET-PelB-SUMO, pET-MalE-SUMO, and pET-G1M5-SUMO. In which, only pET-G1M5-SUMO exhibit correct folding of venom peptides in the supernatant portion. Others are observed in the precipitate (P) after lysis, indicating inclusion bodies. Thus, the results suggest that only expressions of proteins including the usage of G1M5-SUMO tag are capable of demonstrating correct expressions.[figure 1]

Figure 1: SDS-PAGE of S: supernatant and P: precipitate (A) SP-rCtx4-GNA-His and SP-SUMO-rCtx4-GNA-His, (B) rCtx4-GNA-His, (C) G1M5-SUMO-rCtx4-GNA-His (D) His-rCtx4-GNA; all with complete proteins of BL21(DE3) as control

Reference

[1]Liu, Changjun, et al. ‘A Secretory System for Extracellular Production of Spider Neurotoxin Huwentoxin-I in Escherichia Coli’. Preparative Biochemistry & Biotechnology, vol. 53, no. 8, Sept. 2023, pp. 914–22. DOI.org (Crossref), https://doi.org/10.1080/10826068.2022.2158473 [2]Jürgen Dohmen, R. ‘SUMO Protein Modification’. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, vol. 1695, no. 1–3, Nov. 2004, pp. 113–31. DOI.org (Crossref), https://doi.org/10.1016/j.bbamcr.2004.09.021