Coding

Part:BBa_K4022002

Designed by: Mines   Group: iGEM21_Mines   (2021-10-01)


TPA

Description

TPA (Tissue Plasminogen Activator) is a naturally occuring protien in the human blood stream produced by endothelial cells. It catalyzes the conversion of plasminogen to plasmin. Plasmin actively breaks down fibrin the primary component of blood clots. Free TPA has been used to the clinical treatment of blood clots; however, this can lead to blood thinning and potential hemorrhaging in the brain.
This part is designed to bind to DynaBeads for the localized treatment of blood clots. Initial work by Dr. Disharoon and David Marr at Colorado School of Mines has shown promising improvements on the rate of blood clot lysis using nonspecific binding to DynaBeads [1]. Through His Tag chemistry this part should improve the number of active tPA attached to a given DynaBead and further improve Lysis.

[1] T.O. Tasci, D. Disharoon, R.M. Schoeman, K. Rana, P.S. Herson, D.W.M. Marr, K.B. Neeves, “Enhanced Fibrinolysis with Magnetically Powered Colloidal Microwheels”, Small, 2017, 1700954. DOI:10.1002/smll.201700954.

[2] Martegani E, Forlani N, Mauri I, Porro D, Schleuning WD, Alberghina L. Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter. Appl Microbiol Biotechnol. 1992 Aug;37(5):604-8. doi: 10.1007/BF00240734. PMID: 1368914.

[3]Xiaoying Chen, Jennica L. Zaro, Wei-Chiang Shen, Fusion protein linkers: Property, design and functionality, Advanced Drug Delivery Reviews, Volume 65, Issue 10, 2013.


Sequence and Features

Because of complexity of TPA in part due to the multiple disulfide bridges the expression of active TPA in bacterial cultures is diffucult and limited. Previous papers [2], have indicated that active TPA can be produced in yeast cultures at reasonably high concentration. The amino acid sequence of TPA from Uniprot, was codon optimized for yeast, and two silent mutations were made to make the part RFC[1000] compatible. Homology for the p4XX yeast plasmid series was added for transformation. The alpha mating factor tag was added for the intracellular production of the protein. A flexible linker made up of the GGGGS motif was used to hopefully improve activity of the binded protein [3].



Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 31
    Illegal EcoRI site found at 633
    Illegal EcoRI site found at 781
    Illegal EcoRI site found at 1369
    Illegal XbaI site found at 1
    Illegal SpeI site found at 7
    Illegal PstI site found at 25
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 31
    Illegal EcoRI site found at 633
    Illegal EcoRI site found at 781
    Illegal EcoRI site found at 1369
    Illegal SpeI site found at 7
    Illegal PstI site found at 25
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 31
    Illegal EcoRI site found at 633
    Illegal EcoRI site found at 781
    Illegal EcoRI site found at 1369
    Illegal BglII site found at 1180
    Illegal BamHI site found at 13
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 31
    Illegal EcoRI site found at 633
    Illegal EcoRI site found at 781
    Illegal EcoRI site found at 1369
    Illegal XbaI site found at 1
    Illegal SpeI site found at 7
    Illegal PstI site found at 25
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 31
    Illegal EcoRI site found at 633
    Illegal EcoRI site found at 781
    Illegal EcoRI site found at 1369
    Illegal XbaI site found at 1
    Illegal SpeI site found at 7
    Illegal PstI site found at 25
  • 1000
    COMPATIBLE WITH RFC[1000]



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