Coding

Part:BBa_K4829009:Design

Designed by: Aditya Kamath Ammembal   Group: iGEM23_IISc-Bengaluru   (2023-10-05)


Sequence coding for a dAb against IL6


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 13
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part has not been tested out by methods like BLI. However, it does give very good results in silico. We also provide here, a method to make multiple such amino acid sequences by following a simple pipeline to get a good enough starting point to make a good, well-functioning sequence. The things to be kept in mind are:

  • In each of the two variable domains of the scFv, there are three distinct regions known as complementary determining regions (CDRs) that are interconnected by framework regions (FRs).

The CDRs play a key role in binding to antigens, with their structure tailored to match the epitope. On the other hand, the FRs serve primarily as a support structure and show minimal variability compared to the CDRs. Notably, each CDR contributes differently to antigen binding. For example, the heavy chain's CDR3 is especially vital, contributing to 29% of the binding specificity, whereas the CDR2L's contribution is a mere 4%.

  • In the conventional VH region, the FR2 consist of four highly conserved hydrophobic amino acids (Val37, Gly44, Leu45, and Trp47)that in contribution with Gln39, Gly44, Tyr91, and Trp103 form a conserved hydrophobic interface of ~700 Å 2 to facilitate VL joining.

The way to 'camelise' would be as follows: Remove the entirety of the sequence (except the VH)

  • Replace these four hydrophobic residues (Val37, Gly44, Leu45, and Trp47) with more hydrophilic amino acids (Phe37, Glu44, Arg45, and Gly47) to avoid the exposure of such a sizeable hydrophobic region to solvent.
  • In addition to this substitution, residues adjacent to this interface have rotated their side chains without deforming the Cα backbone to increase the VHH surface's hydrophilicity. Furthermore, the CDR3 domain of VHH folds over this interface to shield the amino acids formerly covered by the VL partner. These alterations elucidate the augmented solubility of VHHs in comparison to the single VH domain and scFvs.

This simple change is all it takes to get a preliminary dAb

Source

This sequence is not from any living organism to the best of our knowledge.

References