Difference between revisions of "Part:BBa K1497031"

Revision as of 00:14, 15 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).

Functional Parameters

The Scaffold-His protein was cloned into the plasmid vector pet21a+ for expression and purification . The test expression showed a successful production of the scaffold protein (figure 3(a)). The amount of the scaffold protein increased quickly, reaching a plateau 1-2 h after induction. The SDS PAGE (figure 3(b)) for the analysis of the purification of the scaffold showed, that the supernatant of the centrifugation contained the majority of the produced protein, although a distinct band was visible also for the pellet. The protein binds efficiently to the column. Therefore, the scaffold band is not visible in the flow-through. The protein was eluted at an imidazole concentration of 100 - 200 mM (figure 4). A Nanodrop measurement resulted in a yield of 3 mg of protein in both fractions. This leads to a total yield of 6 mg/L for the described method, whereas a quantitative estimation of the remaining amount in the pellet was not possible.

The influences of urea, KCl and pH changes on the stability of the scaffold were observed (figure 5). Urea does not cause denaturation at concentrations below 500 mM at low temperatures. The protein remains stable in 50 mM Tris at pH 8 at < 26°C (figure 5(a)). The additionally tested concentrations of 5 and 8 M are not presented since the protein was already denatured at 15°C. During the experiment with KCl, it became clear that the protein gained stability with increasing concentration (figure 5(b)). While denaturation started at 25°C with low KCl concentration, the stability rapidly increased, approaching a denaturation temperature of 40°C asymptotically when the KCl concentration was > 500 mM. A pH optimum was found at about 7 - 7.5 (figure 5(c)). Below pH 7, the thermal stability decreased rapidly. The protein was not correctly folded at pH 5.5 – 6 even below 15°C, while pH values above 7.5 showed similar, yet less drastic results.

The results match an expected behavior. The denaturing urea lowers the scaffold’s stability at higher concentrations while high salt concentrations support its structure. The stability curve at different pH values is similar to the expectation. The pI of the molecule lies between 5.8 and 6.2, at these pH values the protein is exprected to show the minimal solubility. Accordingly there is less solved protein to interact with the dye and a lower or non-existent intensity through denaturation. At pH > 7.5 the stability of the protein drops, since the amount of negatively charged amino acid rests increases and the amount of positively charges decreases respectively. This leads to stronger repulsion inside the molecule and lowers the energy that is necessary for denaturation. The experiments showed that the scaffold protein is heat labile, which explains the suggested expression temperature of 30°C from Dueber and could be a clue, that lower temperatures might prevent the formation of inclusion bodies and thereby increase the yield.

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.

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 <span style="font-size:1.2em"><b>Functional Parameters</b></span>
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-For expression and purification the Scaffold-His protein was cloned into the plasmid vector pet21a+. The test expression showed a successful production of the scaffold protein (figure 3(a)). The amount of the scaffold protein increased quickly, reaching a plateau 1-2 h after induction.
+The Scaffold-His protein was cloned into the plasmid vector pet21a+ for expression and purification . The test expression showed a successful production of the scaffold protein (figure 3(a)). The amount of the scaffold protein increased quickly, reaching a plateau 1-2 h after induction.
-The SDS PAGE (figure 3(b)) for the analysis of the purification of the scaffold showed, that the supernatant of the centrifugation contained a majority of the produced protein, although a distinct band was visible also for the pellet. The protein binds efficiently to the column. Therefore, the scaffold band is not visible in the flow-through. The protein was eluted at an imidazole concentration of 100 - 200 mM (figure 4). A Nanodrop measurement resulted in a yield of 3 mg of protein in both fractions. This leads to a total yield of 6 mg/L for the described method, whereas a quantitative estimation of the remaining amount in the pellet was not possible.
+The SDS PAGE (figure 3(b)) for the analysis of the purification of the scaffold showed, that the supernatant of the centrifugation contained the majority of the produced protein, although a distinct band was visible also for the pellet. The protein binds efficiently to the column. Therefore, the scaffold band is not visible in the flow-through. The protein was eluted at an imidazole concentration of 100 - 200 mM (figure 4). A Nanodrop measurement resulted in a yield of 3 mg of protein in both fractions. This leads to a total yield of 6 mg/L for the described method, whereas a quantitative estimation of the remaining amount in the pellet was not possible.
 </br></br>
 The influences of urea, KCl and pH changes on the stability of the scaffold were observed (figure 5). Urea does not cause denaturation at concentrations below 500 mM at low temperatures. The protein remains stable in 50 mM Tris at pH 8 at < 26°C (figure 5(a)). The additionally tested concentrations of 5 and 8 M are not presented since the protein was already denatured at 15°C. During the experiment with KCl, it became clear that the protein gained stability with increasing concentration (figure 5(b)). While denaturation started at 25°C with low KCl concentration, the stability rapidly increased, approaching a denaturation temperature of 40°C asymptotically when the KCl concentration was > 500 mM. A pH optimum was found at about 7 - 7.5 (figure 5(c)). Below pH 7, the thermal stability decreased rapidly. The protein was not correctly folded at pH 5.5 – 6 even below 15°C, while pH values above 7.5 showed similar, yet less drastic results.
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 <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>
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