Difference between revisions of "Part:BBa K1497032"

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.

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. The scaffold itself contains no other cysteine residues. Thus, coupling should be regiospecific.

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

Figure 2: p align="justify">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.

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