Part:BBa_K1497032

Scaffold (G-S-P-His-Cys)

Usage and Biology

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 consists of three different protein binding domains namely the GBD (BBa_K1497024), SH3 (BBa_K1497025) and PDZ (BBa_K1497026) domain. 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.

In this BioBrick, a cysteine residue was added directly behind the His-tag. The thiole group of the cysteine can be used for covalent crosslinking of the scaffold by reactions with maleimids and haloacetamids for example. The scaffold itself contains no other cysteine residues. Thus, coupling should be regiospecific.

Functional Parameters

A test experiment was performed in order to provide the basis for a successful production of the Scaff-His-Cys variant. For that purpose, the product was examined by PAA gel electrophoresis (see figure 3). Onto the gel, next to the marker, a sample of the pre-culture, the cells prior, and several hours after induction were applied. A clear band was visible at the height above 30 kDa. The scaffold with His-tag has a size of 31.17 kDa. As a consequence, the band appearing after induction can be considered as the correct protein. Due to the presence of the scaffold protein in the lane of the pre-culture (VK), it can be concluded that the inducible promoter is not entirely locked. A clear overproduction of the scaffold protein is only reached after an incubation time of four hours.

The construction of the scaffold GSP-His-Cys is nearly identical to the scaffold GSP-His (BBa_K1497031). Consequently, the measurements taken with scaffold without an additional Cys could also be a good approximation of the behavior of the protein with the mutation.

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.

Figure 3: SDS-PAGE of the expression control of the scaffold protein with cysteine in E. coli BL21 cells. Next to the marker (BLUEplus perstained Protein Ladder, Biomol), cell lysates were applied of the pre-culture and of cells before and after several hours of induction. A clear overproduction of the protein is present at 4h after induction. The scaffold protein has a size of about 31.17 kDa.

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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