Difference between revisions of "Part:BBa K1497031"
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The protein scaffold consists of three different protein binding domains namely the GBD (<a href="/Part:BBa_K1497024">BBa_K1497024</a>), SH3 (<a href="/Part:BBa_K1497025">BBa_K1497025</a>) and PDZ (<a href="/Part:BBa_K1497026">BBa_K1497026</a>) domains. The domains can be linked together in any number and in any order. Therefore, a broad variety of scaffold proteins can be constructed from the initial domains depending on the application. The scaffold protein shown here consists of all domains in the order GBD1SH31PDZ1 or GSP for short. The coding sequence was optimized for the expression in E. coli and revised for the usage as a BioBrick. Additionally, a C-terminal His-tag was added allowing easy purification of the protein.
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<span style="font-size:1.2em"><b>Usage and Biology</b></span>
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The protein scaffold consists of three different protein binding domains namely the GBD (<a href="/Part:BBa_K1497024">BBa_K1497024</a>), SH3 (<a href="/Part:BBa_K1497025">BBa_K1497025</a>) and PDZ (<a href="/Part:BBa_K1497026">BBa_K1497026</a>) domains. The domains can be linked together in any number and in any order. Therefore, a broad variety of scaffold proteins can be constructed from the initial domains depending on the application. The scaffold protein shown here consists of all domains in the order GBD<sub>1</sub>SH3<sub>1</sub>PDZ<sub>1</sub> or GSP for short. The coding sequence was optimized for the expression in <i>E.&nbsp;coli</i> and revised for the usage as a BioBrick. Additionally, a C-terminal His-tag was added allowing easy purification of the protein.
 
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The protein scaffold is an assembly platform for ligand coupled target enzymes. It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffold mimic naturally occurring catalysation cascades. In these, reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects like unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009).
 
The protein scaffold is an assembly platform for ligand coupled target enzymes. It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffold mimic naturally occurring catalysation cascades. In these, reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects like unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009).
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   3D-structure of the protein scaffold, created with PHYRE2 based on structure-homology-modeling  </span></p>
 
   3D-structure of the protein scaffold, created with PHYRE2 based on structure-homology-modeling  </span></p>
 
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<p class="MsoCaption" align="justify"><span lang="EN-US"><b>Figure 2:</b></span></a><span lang="EN-US">
 
<p class="MsoCaption" align="justify"><span lang="EN-US"><b>Figure 2:</b></span></a><span lang="EN-US">
 
Model of a scaffold´s function. The domains are connected with a linker. They are able to build up a tight bound with enzymes assigned with a proper ligand. The educt is channeled through the enzymes and converted to the product.  </span></p></center>
 
Model of a scaffold´s function. The domains are connected with a linker. They are able to build up a tight bound with enzymes assigned with a proper ligand. The educt is channeled through the enzymes and converted to the product.  </span></p></center>
 
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Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza; Moon, Tae Seok; Petzold, Christopher J.; Ullal, Adeeti V. et al. (2009): Synthetic protein scaffolds provide modular control over metabolic flux. In Nat. Biotechnol. 27 (8), pp. 753–759. DOI: 10.1038/nbt.1557.
 
Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza; Moon, Tae Seok; Petzold, Christopher J.; Ullal, Adeeti V. et al. (2009): Synthetic protein scaffolds provide modular control over metabolic flux. In Nat. Biotechnol. 27 (8), pp. 753–759. DOI: 10.1038/nbt.1557.
  

Revision as of 23:31, 14 October 2014

Scaffold (G-S-P-His)



Usage and Biology

The protein scaffold consists of three different protein binding domains namely the GBD (BBa_K1497024), SH3 (BBa_K1497025) and PDZ (BBa_K1497026) domains. The domains can be linked together in any number and in any order. Therefore, a broad variety of scaffold proteins can be constructed from the initial domains depending on the application. The scaffold protein shown here consists of all domains in the order GBD1SH31PDZ1 or GSP for short. The coding sequence was optimized for the expression in E. coli and revised for the usage as a BioBrick. Additionally, a C-terminal His-tag was added allowing easy purification of the protein.

The protein scaffold is an assembly platform for ligand coupled target enzymes. It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffold mimic naturally occurring catalysation cascades. In these, reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects like unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009).

Figure 1: 3D-structure of the protein scaffold, created with PHYRE2 based on structure-homology-modeling


Figure 2: Model of a scaffold´s function. The domains are connected with a linker. They are able to build up a tight bound with enzymes assigned with a proper ligand. The educt is channeled through the enzymes and converted to the product.


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 865
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 76
    Illegal AgeI site found at 199
  • 1000
    COMPATIBLE WITH RFC[1000]


References


Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza; Moon, Tae Seok; Petzold, Christopher J.; Ullal, Adeeti V. et al. (2009): Synthetic protein scaffolds provide modular control over metabolic flux. In Nat. Biotechnol. 27 (8), pp. 753–759. DOI: 10.1038/nbt.1557.