Part:BBa_K4159013
This is one potential AIP-binding DARPin sequence. AIP's are the quorum sensing signalling molecules in gram-positive S.epidermidis bacteria, and DARPins are designed ankyrin repeat proteins, that possess a high affinity towards peptides.
We aim to design and optimise an efficient DNA library of different DARPin molecules by modifying and randomising specific amino acid positions so that biofilm formation in Staphylococcus epidermidis (S. epidermidis) is arrested. The designed library is used in ribosome display to select the DARPin with the highest affinity towards the target AIP1 peptide, which in turn will inhibit the quorum sensing mechanism of S. epidermidis. Our construct was based on three key settings: (1) previous and current state-of-the-art scientific research on designed DARPin molecules, (2) most successful DARPins that bound to their intended targets, and (3) most successful randomised IR modules that resulted in successful outcomes. With this strategy, we expected DARPins with excellent biophysical properties as well as high affinity and stability (figure 1).
First, we looked at the strategy employed by Seeger et al (2013) focused on the construction of a DARPin library with reduced hydrophobicity in order to generate each DARPin efficiently and more soluble. In our project, we decided for our final design, to still randomise 8 amino acid positions of each IR module and fix both capping repeats (N- and C- caps). To select our DARPin library for ribosome display testing, we developed an algorithm able to randomise the positions of each IR as well as fixed capping repeats and bridge amino acid connections to provide us with several different and optimised DARPin molecules. Finally, our DARPins are 157 amino acids long (471 base pairs) upon which the N-cap comprises 31 amino acids while each IR of 33 amino acids and finally the C-cap with 27 amino acids.
Each internal repeat underwent randomization on positions 1, 2, 3, 5, 10, 13, 14, and 26. Based on the work by Seeger et al. (2013), we still allowed all amino acids except G, P, C, V, L, I, M, and F at positions 2, 3, 5, 13, and 14 while position 1 were only allowed D, N, S, and T due to the excellent chemical properties of the DARPins. In addition, on position 10 we permitted the amino acids A, S, T, V, or L, and position 26 only three: N, H, or Y. Finally, we changed two fixed positions (21 and 25) to E and K, respectively. The choice for both E and K were based on the fact that the majority of previously reported and successful DARPins had these two fixed amino acids such as the novel LoopDARPins allowing for the selection of low picomolar binders with only one round of ribosome display (Schilling et al., 2014) and the reported GFP-binding DARPin (Hansen et al., 2017). Finally, we decided to optimise the bridge amino acids between N-cap and the first IR, the first IR to the second, the second IR to the third, and finally the third IR to the beginning of the C-cap. Finally, we decided to construct our DARPins with amino acids with polar uncharged side chains (for higher solubility) between the N-cap and the first IR as well as the last (third module) to the C-cap for stability purposes of the protein which was Q (Glutamine) and A (Alanine) between IR’s.
In our final design, we chose not to randomise the N- and C- caps (flanking sequences able to shield the hydrophobic core of each IR) but to optimise the amino acids of each so that the stability of our DARPin molecules would increase along with binding chances. Importantly, the main function of said terminal capping repeats is to make the protein more stable and to fold in an efficient manner by protecting and sealing the hydrophobicity of the internal repeats (IR) – the so-called hydrophobic core (Binz et al., 2003). For both the N-cap and C-cap and in contrast to the modifications performed on it with the reduced hydrophobicity DARPin library, we did not randomise the first three amino acids and fixed the whole sequence as proposed by the construct of Schilling et al (2014) and Hansen et al. (2017) (figure 2.).
If you are interested in using this part, please contact us at team@aaltohelsinki.com for more information, as the final NGS data analysing is still under work. This sequence is also just one example from our library, and to see more information about the others visit our wiki page.
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