Team:Queens Canada/Guide/Protein Structure

In our chimeras we have linker sequences on either side of the chimeric insert sequence. The linkers we chose to use were inert, and flexible linker sequences, with different sequences on either side so that overlap extension will be successful.

FliC -GGGGS- insert -GGSGG- FliC

It is important to also consider the flexible regions of the protein in which you are making your insertion. Flexible regions may be able to accommodate a protein insertion, and would not require very long linkers. However, longer linkers may be required to introduce enough flexibility for the inserted protein to fold properly.

The protein database is possibly the most important tool in chimeric protein design. Without having any previous knowledge of your structure, it is impossible to really say where or how you would design your chimeric protein. In our case, the crystal structure for E. coli flagellin is not available on the protein database. However, the structure for the flagellin of S. typhymurium was available and is very similar to the structure of E. coli flagellin. The PDB ID is 1UCU.

Additionally, some general assumptions can be made when searching for a good place to make an insertion. For example, the site that we chose to make our variable domain insertions is:

...AVTVANDGTVTMATG...

The insertion was made in between AVT and TVT, replacing the amino acid sequence VANDG. We needed to have some spacing in between the overlap regions for PCR overlap extension, which is why VANDG was replaced.

The presence of several threonine, alanine, valine and glycine residues is indicative of a loop region, which would make a good spot for an insertion. Additionally, when we used PCR to make the overlapping regions for the insertion, we amplified off of the already existing linker used for ovarlapping as a deletion. Hence our total linker was:

...AVTTTGGGGS-insert-GGSGGTSTVT...