Customizing a protein for a specific application starts at engineering the protein's sequence and structure for that intended application. While some situations may call for only the natural and unchanged protein, most custom proteins will be modified versions of their natural counterpart. Examples for engineered changes include:
- Addition of peptide tags to N- or C-terminus of target protein for purification, detection and surface binding purposes; among several used the hexahistidine sequence (or His-tag) is the favorite tag used at DIARECT
- Fusion of the target protein to a second protein with additional functional, enzymatic, fluorescence, ligand binding or surface binding properties
- Changes to intrinsic functional activity, e.g. pin-pointed removal of enzymatic activity by mutagenesis of important amino acid residues in the active center
- Changes of domain structure by domain removal
- Expression of isolated domains
- Conversion of a transmembrane protein's extracellular part to a secreted protein
- Intracellular targeting elements
The design stage is based on structural information for a target protein when available, for instance from crystal structures or alignments of homologous and related protein sequences. Output of protein design is a protein sequence for which subsequent gene design will have to provide a coding cDNA and expression construct. The requirements of the custom target protein will direct the choice of expression system, baculovirus / insect cell vs. E.coli bacteria, already at this protein design stage.
