Difference between revisions of "Part:BBa K2959005"

 
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<partinfo>BBa_K2959005 short</partinfo>
 
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This composite part consists of a lacI regulated promoter, ribosome binding site, a coding sequence for <i>Pinus sylvestris</i> Defensin 1 as a fusion protein with a 6x His-Tag, and a double terminator. This construct allows the expression of PsDef1, an antifungal peptide from Scots pine seeds, in <i>E. coli</i>. Expression can be positively regulated by the addition of IPTG or lactose thanks to the lacl regulated promoter. The fusion of PsDef1 to a 6x His-Tag is intended for the purification of the peptide by immobilized metal affinity chromatography.
  
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This composite part consists of a lacI regulated promoter, ribosome binding site, a coding sequence for Pinus sylvestris Defensin 1 as a fusion protein with a 6x His-Tag, and a double terminator. This construct allows the expression of PsDef1, an antifungal peptide from Scots pine seeds, in E. coli. Expression can be positively regulated by the addition of IPTG or lactose thanks to the lacl regulated promoter. The fusion of PsDef1 to a 6x His-Tag is intended for the purification of the peptide by immobilized metal affinity chromatography.  
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Since PsDef1 is a peptide with four disulfide bonds<sup>1</sup>, a modification hard to replicate on prokaryotic expression systems, the construct allows for the co-expression of the peptide with a truncated version of Erv1p. This is a protein from Saccharomyces cerevisiae capable of catalyzing the formation of disulfide bonds<sup>2</sup>.  
  
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Since PsDef1 is a peptide with four disulfide bonds, a modification hard to replicate on prokaryotic expression systems, the construct allows for the co-expression of the peptide with a truncated version of Erv1p. This is a protein from Saccharomyces cerevisiae capable of catalyzing the formation of disulfide bonds.
 
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===Usage and Biology===
 
===Usage and Biology===
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<p align="justify">
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PsDef1 is present in  <i>Pinus sylvestris</i> seeds, vegetative and generative organs. The defensin encodes a protein of 83 amino acids in length, whose first 33 amino acids correspond to the ‘N-terminal signal peptide, it also has eight highly conserved cysteines residues that are predicted to form four disulfide bonds C3–C49, C14–C34, C20–C43, and C24–C45<sup>1</sup>, which stabilize its structure making it resistant to environmental changes<sup>2</sup>. Its conformation consists in a cysteine-stabilized αβ-motif (CSαβ) with a prominent α-helix and a triple-stranded antiparallel β-sheet that is stabilized by the 4 disulfide bonds<sup>1</sup>.
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Endogenous PsDef1 causes morphological changes in fungi mycelium  when interacting with the sphingolipid membrane on fungal cell, disrupting the membrane integrity. Antifungal activity towards phytopathogenic fungi <i>Botrytis cinerea</i>, <i>Fusarium oxysporum</i>, <i>Fusarium solani</i>, and <i>Heterobasidion annosum</i> has been demonstrated; as well as its antimicrobial activity against Gram-positive (<i>Bacillus pumilus</i>) and Gram-negative bacteria (<i>Pectobacterium carotovorum</i>, <i>Pseudomonas fluorescens</i>)<sup>1</sup>.
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For a successful expression in <i>E. coli</i>, it is indispensable to avoid interactions that result in the aggregation of folding intermediates. Disulfide bonds can be involved in the structural, catalytic and signaling roles of the protein, but the formation of disulfide bonds can have certain problems that can cause misfolding, aggregation and low yields during the production of the protein<sup>4</sup>. The usage of Erv1p has been proposed as a mechanism for the formation of disulfide bonds in recombinant proteins in prokaryotic expression systems. It has been shown that, thanks to its ability to form disulfide bonds <i>de novo</i>, co-expression of Erv1p allows the proper formation of disulfide bonded proteins in the cytoplasm of <i>E. coli</i><sup>5</sup>.
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The ERV1 gene from <i>Saccharomyces cerevisiae</i> encodes for a 189 amino acids protein, Erv1p, that is involved in different processes of mitochondrial biogenesis and maintenance<sup>2, 6</sup>. The protein shows a flavin-linked sulfhydryl oxidase enzymatic activity linked to the carboxy-terminal domain. Thus, Erv1p is capable of oxidizing thiol groups in proteins and catalyzing disulfide bond formation. A 15 kDa truncated version of Erv1p, consisting of the 117 amino acid residues carboxy-terminal domain, shows a similar or improved sulfhydryl oxidase activity compared to the full length protein<sup>2</sup>.
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K2959005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2959005 SequenceAndFeatures</partinfo>
  
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===References===
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1. Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., ... & Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1865(8), 1085-1094.
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<br>
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2. Lee, J. E., Hofhaus, G., & Lisowsky, T. (2000). Erv1p from Saccharomyces cerevisiae is a FAD‐linked sulfhydryl oxidase. FEBS letters, 477(1-2), 62-66.
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<br>
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3. Kovalyova, V. A., Gout, I. T., Kyamova, R. G., Filonenko, V. V., & Gout, R. T. (2007). Molecular cloning and characterization of defensin 1 from Scots pine. Biopolymers and Cell,23(5), 398-404.
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<br>
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4. Veggiani, G., & de Marco, A. (2011). Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase. Protein expression and purification, 79(1), 111-114.
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<br>
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5. Hatahet, F., Nguyen, V. D., Salo, K. E., & Ruddock, L. W. (2010). Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microbial cell factories, 9(1), 67.
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<br>
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6. Lisowsky, T. (1996). Removal of an intron with unique 3′ branch site creates an amino‐terminal protein sequence directing the scERV1 gene product to mitochondria. Yeast, 12(15), 1501-1510.
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Revision as of 02:47, 17 September 2019


Expressible Pinus sylvestris Defensin 1 with Erv1p

This composite part consists of a lacI regulated promoter, ribosome binding site, a coding sequence for Pinus sylvestris Defensin 1 as a fusion protein with a 6x His-Tag, and a double terminator. This construct allows the expression of PsDef1, an antifungal peptide from Scots pine seeds, in E. coli. Expression can be positively regulated by the addition of IPTG or lactose thanks to the lacl regulated promoter. The fusion of PsDef1 to a 6x His-Tag is intended for the purification of the peptide by immobilized metal affinity chromatography. Since PsDef1 is a peptide with four disulfide bonds1, a modification hard to replicate on prokaryotic expression systems, the construct allows for the co-expression of the peptide with a truncated version of Erv1p. This is a protein from Saccharomyces cerevisiae capable of catalyzing the formation of disulfide bonds2.

Usage and Biology

<p align="justify"> PsDef1 is present in Pinus sylvestris seeds, vegetative and generative organs. The defensin encodes a protein of 83 amino acids in length, whose first 33 amino acids correspond to the ‘N-terminal signal peptide, it also has eight highly conserved cysteines residues that are predicted to form four disulfide bonds C3–C49, C14–C34, C20–C43, and C24–C451, which stabilize its structure making it resistant to environmental changes2. Its conformation consists in a cysteine-stabilized αβ-motif (CSαβ) with a prominent α-helix and a triple-stranded antiparallel β-sheet that is stabilized by the 4 disulfide bonds1.


Endogenous PsDef1 causes morphological changes in fungi mycelium when interacting with the sphingolipid membrane on fungal cell, disrupting the membrane integrity. Antifungal activity towards phytopathogenic fungi Botrytis cinerea, Fusarium oxysporum, Fusarium solani, and Heterobasidion annosum has been demonstrated; as well as its antimicrobial activity against Gram-positive (Bacillus pumilus) and Gram-negative bacteria (Pectobacterium carotovorum, Pseudomonas fluorescens)1.


For a successful expression in E. coli, it is indispensable to avoid interactions that result in the aggregation of folding intermediates. Disulfide bonds can be involved in the structural, catalytic and signaling roles of the protein, but the formation of disulfide bonds can have certain problems that can cause misfolding, aggregation and low yields during the production of the protein4. The usage of Erv1p has been proposed as a mechanism for the formation of disulfide bonds in recombinant proteins in prokaryotic expression systems. It has been shown that, thanks to its ability to form disulfide bonds de novo, co-expression of Erv1p allows the proper formation of disulfide bonded proteins in the cytoplasm of E. coli5.


The ERV1 gene from Saccharomyces cerevisiae encodes for a 189 amino acids protein, Erv1p, that is involved in different processes of mitochondrial biogenesis and maintenance2, 6. The protein shows a flavin-linked sulfhydryl oxidase enzymatic activity linked to the carboxy-terminal domain. Thus, Erv1p is capable of oxidizing thiol groups in proteins and catalyzing disulfide bond formation. A 15 kDa truncated version of Erv1p, consisting of the 117 amino acid residues carboxy-terminal domain, shows a similar or improved sulfhydryl oxidase activity compared to the full length protein2.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 580
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

1. Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., ... & Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1865(8), 1085-1094.
2. Lee, J. E., Hofhaus, G., & Lisowsky, T. (2000). Erv1p from Saccharomyces cerevisiae is a FAD‐linked sulfhydryl oxidase. FEBS letters, 477(1-2), 62-66.
3. Kovalyova, V. A., Gout, I. T., Kyamova, R. G., Filonenko, V. V., & Gout, R. T. (2007). Molecular cloning and characterization of defensin 1 from Scots pine. Biopolymers and Cell,23(5), 398-404.
4. Veggiani, G., & de Marco, A. (2011). Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase. Protein expression and purification, 79(1), 111-114.
5. Hatahet, F., Nguyen, V. D., Salo, K. E., & Ruddock, L. W. (2010). Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microbial cell factories, 9(1), 67.
6. Lisowsky, T. (1996). Removal of an intron with unique 3′ branch site creates an amino‐terminal protein sequence directing the scERV1 gene product to mitochondria. Yeast, 12(15), 1501-1510.