Seitenverantwortliche/r: Jan

Protein targeting

Essential to a protein's function is not only its activities or binding properties, but also its presence at the right time, at the right place. Just like in real estate, the motto here is 'location, location, location'.

One of the most ubiquitous elements for protein targeting is the signal peptide, which targets proteins to the ER, from where their journey continues. Proteins containing a signal peptide include transmembrane proteins, secreted proteins, proteins of the ER itself, proteins of the Golgi apparatus and several more.

The signal peptide

At the beginning of the 1970s, later Nobel Prize laureate Guenther Blobel made the discovery that some in vitro translated proteins would turn out slightly longer than their counterparts found in cells. This observation led to the discovery of the signal peptide, a short N-terminal protein sequence that is required for the targeting of proteins into the endoplasmic reticulum (ER) where it is cleaved off afterwards. This mainly hydrophobic sequence, being both required and sufficient for membrane proteins to be translocated to their place of action, allows targeting of proteins to the ER and to their specific location in the membrane - in the case of receptors to the cell surface. The signal peptide is a short peptide (up to 30 amino acids) that is recognized by the so-called signal recognition particle upon translation at the rough ER, which mediates the co-translational translocation of the protein’s peptide chains into the ER via a translocon called the Sec-complex.

During this project, three different peptides are being tested. The EGFR signal peptide was taken from the iGEM parts registry (BBa_K157001) and combined with the CMV-promoter via the RFC10 cloning standard, while CMV-BM40-SP and CMV-IgKappa constructs were designed manually and synthesized, and combined with a BioBrick containing the CMV promoter sequence via restriction cloning. As shown in figure X, the EGFR signal peptide taken from the parts registry is constructed in a way that the signal peptide ORF immediately follows the cloning scar after the CMV promoter, thus resulting in a very short 5’ untranslated region (UTR) of the transcribed mRNA. The CMV-signal peptide constructs created for the project on the other hand, which contain the BM40 and IgKappa signal peptides, were designed to have a considerably longer 5’ UTR, additionally allowing them to contain a full Kozak consensus sequence. The Kozak sequence is recognized by the ribosome as a translational start site; this element missing or deviating from the consensus sequence may considerably decrease translation efficiency.12 For the EGFR signal peptide construct, a Kozak sequence is not present, as it would have to preceed the start codon ATG - a position which is occupied by the RFC10 cloning scar. Since both the distance from the promoter to the open reading frame as well as the Kozak consensus sequence are considered crucial parameters for expression levels, the BM40 and IgKappa constructs were designed to potentially increase expression levels of the receptor.