Difference between revisions of "Part:BBa K590087"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K590087 short</partinfo>
 
<partinfo>BBa_K590087 short</partinfo>
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[[Image:Washington_Bottle.jpg|right|borderless|175px]]
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A mutated [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590021 Kumamolisin-As] enzyme aimed to break down gluten by increased activity with the PQLP peptide, an antigenic epitope in gliadin.
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===Usage and Biology===
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This part (KumaMax) was constructed by the [http://2011.igem.org/Team:Washington 2011 University of Washington] iGEM team to break down gluten, the primary cause of Celiac's disease.  KumaMax was generated by making rational mutations to the active site of the enzyme, as detailed on our [http://2011.igem.org/Team:Washington/Celiacs/Results our wiki].  To test BBa _K590021, it was inserted into a protein expression vector, pET29b+. KumaMax (Kumamolisin-As_N219D, S354N, D358G, D368H) was then produced and purified as described in the [http://2011.igem.org/Team:Washington/Protocols/50mL_Scale 2011 iGEM Team's Small Scale Protein Expression and Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay 2011 UW iGEM Purified Enzyme Assay Protocol]. The resulting data is shown below.  We achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!
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[[Image:Washington_KumaMax.png|500px|left|thumb|The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour. Error bars represent a 95% confidence interval from triplicate data.]]
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Mutation(s)'''
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| align="center" style="background:#f0f0f0;"|'''''k<sub>cat</sub>''/''K<sub>M</sub>'' (M<sup>-1</sup> s<sup>-1</sup>)'''
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|-
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| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590023 '''N291D''']||(5.21 ± 0.11) x 10<sup>5</sup>
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|-
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| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590024 '''S354N, D358G, D368H''']||(2.45 ± 0.02) x 10<sup>5</sup>
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|-
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| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590022 '''G319S, D358G, D368H''']||(2.02 ± 0.02) x 10<sup>5</sup>
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|-
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| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590087 '''G319S, D358G, D368H, N291D (KumaMax)''']||(3.86 ± 0.04) x 10<sup>6</sup>
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|-
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| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590021 '''wt-Kumamolisin''']||(3.25 ± 0.05) x 10<sup>4</sup>
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|-
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| SC-PEP ||(4.96 ± 0.75) x 10<sup>3</sup>
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|-
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|}
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A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant is the best combinatorial mutant found and has point mutations at residues 291, 319, 358, and 368 from Asparagine to Aspartate, Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.
 
  
  
  
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===Usage and Biology===
 
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This part was constructed by the [http://2011.igem.org/Team:Washington 2011 University of Washington] iGEM team to break down gluten, the primary cause of Celiac's disease.  To test BBa _K590021, it was inserted into a protein expression vector, pET29b+. Kumamolisin-As_N219D, S354N, D358G, D368H was then produced and purified as described in the [http://2011.igem.org/Team:Washington/Protocols/50mL_UW 2011 iGEM Team's Small Scale Protein Expression and Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay 2011 UW iGEM Purified Enzyme Assay Protocol]. The resulting data is shown below.
 
  
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[[Image:Washington_BestCombMutant.png|750px|center|thumb|The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour. Error bars represent a 95% confidence interval from triplicate data.]]
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K590087 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K590087 SequenceAndFeatures</partinfo>

Latest revision as of 06:27, 1 November 2011

KumaMax: Kumamolisin-As_N291D, G319S, D358G, D368H

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A mutated Kumamolisin-As enzyme aimed to break down gluten by increased activity with the PQLP peptide, an antigenic epitope in gliadin.


Usage and Biology

This part (KumaMax) was constructed by the [http://2011.igem.org/Team:Washington 2011 University of Washington] iGEM team to break down gluten, the primary cause of Celiac's disease. KumaMax was generated by making rational mutations to the active site of the enzyme, as detailed on our [http://2011.igem.org/Team:Washington/Celiacs/Results our wiki]. To test BBa _K590021, it was inserted into a protein expression vector, pET29b+. KumaMax (Kumamolisin-As_N219D, S354N, D358G, D368H) was then produced and purified as described in the [http://2011.igem.org/Team:Washington/Protocols/50mL_Scale 2011 iGEM Team's Small Scale Protein Expression and Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay 2011 UW iGEM Purified Enzyme Assay Protocol]. The resulting data is shown below. We achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!


The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour. Error bars represent a 95% confidence interval from triplicate data.
Mutation(s) kcat/KM (M-1 s-1)
N291D (5.21 ± 0.11) x 105
S354N, D358G, D368H (2.45 ± 0.02) x 105
G319S, D358G, D368H (2.02 ± 0.02) x 105
G319S, D358G, D368H, N291D (KumaMax) (3.86 ± 0.04) x 106
wt-Kumamolisin (3.25 ± 0.05) x 104
SC-PEP (4.96 ± 0.75) x 103








Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1696
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 447
    Illegal NgoMIV site found at 720
    Illegal NgoMIV site found at 1248
    Illegal NgoMIV site found at 1494
    Illegal AgeI site found at 772
    Illegal AgeI site found at 1348
    Illegal AgeI site found at 1588
  • 1000
    COMPATIBLE WITH RFC[1000]