Part:BBa_K4829001
Codes for the CD33 signal peptide
This signal peptide, when appended to the beginning of a peptide sequence, promotes its secretion outside the cell. It does this via the Sec/Sp1 pathway of secretion in eukaryotes. This signal petide was selected from a list of signal peptides available here: https://novoprolabs.com/support/articles/commonly-used-leader-peptide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html From this list, what can be done is, we can append these sequences to the beginning of a peptide sequence and run the composite sequence through signal peptide 6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/) and compare the probabilities of secretion to choose the optimal signal peptide.
Usage and Biology
N-terminal signal sequences direct the placement of new secretory and membrane proteins to the endoplasmic reticulum (ER) through a process that relies on the signal recognition particle (SRP). These sequences have a three-part structure: a hydrophobic core region, with sections on both its ends known as the n- and c-region. The c-region contains a site where signal peptidase (SPase) acts. Typically, these sequences are removed during protein synthesis, resulting in the formation of signal peptides and the main protein. Despite their core function, signal sequences can vary greatly in length and composition. This variation can influence processes like ER targeting, protein placement, and SPase action.
Furthermore, this variability in signal sequences might have other functions beyond just targeting. Some signal peptides undergo further processing by a protease called signal peptide peptidase (SPP), leading to the release of fragments of the signal peptide into the cell's interior. In some cases, these peptides stay within the membrane and might be part of larger protein structures, while in other cases, they are freed from the ER membrane. These released peptides or fragments can serve multiple roles, either in conjunction with or separate from their associated mature proteins.
- Non-human proteins normally are not targeted to any particular cell organelles, much less secreted out of the cell. To ensure that the protein is secreted out of the cell and not simply degraded within the cytosol, we must append a signal sequence to the beginning of the scFv.
- Considering the wide variety of signal sequences that are normally used in mammalian cells to express non-mammalian proteins, we must select the best!
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
The signal peptide has been shown to work! We used the constructs, with 2ug of mRNA mixed with 2ul of Lipofectamine MessengerMAX, in each well of a 12-well plate, each well having 0.5 million cells. The expression was noticed 6 hours post-transfection in the supernatant. The cell lysate was not loaded, and the control was clear media. The blot results were as follows:
Figure 1. Western blot showing expression in supernatant 6hrs post transfection
The 2 lanes beside the bands are controls, the blot was done in duplicates. There was an unfortunate amount of background, yet the band at 31kDa is quite clear.
BBa_K4829002 does not have a standard signal peptide for secretion, so a chimeric protein must be made if this protein is to be secreted out of the cell. The list of most commonly used signal peptides as on (https://novoprolabs.com/support/articles/commonly-used-leader-peptide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html) is a rather long one, and to select the best one for our protein(the antibody), we used the software SignalPeptide6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/). The signal peptide of CD33 was selected after appending the various signal sequences listed on the page to the beginning of the antibody sequence. We noted that the probability of secretion of the antibody was highest with CD33.
Figure 2. Probability of cleavage and secretion via Sec/SP1 secretion pathway
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