Coding
DARPin929

Part:BBa_K3111011

Designed by: Matas Deveikis   Group: iGEM19_UCL   (2019-09-18)
Revision as of 15:15, 5 October 2019 by Matas deveikis (Talk | contribs) (Experimental Results)


DARPin929

This part encodes DARPin929 – a binding protein specific to HER2 receptor, that is similar to an antibody in terms of efficiency and specificity. However, due to its stability and ability to be expressed in bacteria, this DARPin can be used in a wider variety of applications (1). The part has had its start and stop codons removed for flexible fusion to N or C terminus.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Natural Ankyrin Repeat Proteins are versatile binding molecules which can bind to specific targets and subsequently trigger various molecular mechanisms (e.g. enzyme inhibition or protein anchoring) (2,3). Aiming to manipulate the immunologic potential of said repeat proteins for therapeutic applications, in 2003, Plückthun et al. genetically engineered designed DARPins (4). DARPins are small (14-21 kDa) single-domain binding molecules derived from natural ankyrin repeats (5). As natural repeat proteins, DARPins comprised a several structural motifs which stack to form the repeat protein domain (4,6). The assembly of highly expressed, stable and soluble DARPins happened by production of DARPin libraries using ribosome, phage display etc. indicated that these binding molecules had the potential to circumvent the limitations of monoclonal antibodies and empower new therapeutic approaches (4,6).

DARPin929 has been one of the most frequently used to evaluate probes for molecular imaging. It particularly targets human epidermal growth factor 2 (HER2) whose overexpression is associated with breast cancer and gastroesophageal cancer. Has been shown to bind down to nanomolar affinities to its target (7,8). DARPins have been shown to be very stable and show less tendencies for aggregation, unlike single chain variable fragments since they do not possess cysteines.

Functionalisation

BBa_K3111011 has been used along with BBa_K3111021 for initial investigation of binding to SK-BR-3 HER2+ breast adenocarcinoma cells. Moreover, it was fused with BBa_K3111003 and co-expressed with BBa_K3111032 to create the drug delivery vehicle containing cytotoxic cargo (BBa_K3111502).

Experimental Results

WIP. See BBa_K3111201, BBa_K3111202, BBa_K3111501 and BBa_K3111502.

Expression and Purification

Fusion to BBa_K3111003

Evaluation of binding

Performance in encapsulin-based drug delivery system

References

[1] Plückthun A. Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy. Annual Review of Pharmacology and Toxicology. 2015;55(1):489-511.

[2] Janeway CA, Medzhitov R. Innate Immune Recognition. Annu Rev Immunol. 1 de abril de 2002;20(1):197-216.

[3] Forrer P, Stumpp MT, Binz HK, Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett. 27 de marzo de 2003;539(1):2-6.

[4] Binz HK, Stumpp MT, Forrer P, Amstutz P, Plückthun A. Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins. J Mol Biol. 12 de septiembre de 2003;332(2):489-503.

[5] Stumpp MT, Binz HK, Amstutz P. DARPins: A new generation of protein therapeutics. Drug Discov Today. 1 de agosto de 2008;13(15):695-701.

[6] Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol. 1 de mayo de 2004;22(5):575-82.

[7] Siegler E, Li S, Kim YJ, Wang P. Designed Ankyrin Repeat Proteins as Her2 Targeting Domains in Chimeric Antigen Receptor-Engineered T Cells. Hum Gene Ther [Internet]. 2017 Sep 1 [cited 2019 Sep 14];28(9):726–36.

[8] Steiner D, Forrer P, Plückthun A. Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display. J Mol Biol [Internet]. 2008 Oct 24 [cited 2019 Sep 14];382(5):1211–27.

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