Part:BBa_K2208001

A coding sequence of tPep-Lamp2b fusion protein and position it outside the membrane.

tPep is a white fat tissue targeting peptide. To enhance the accuracy of siRNA delivery system and improve targeting index of drugs, tPep was displayed on the surface of the exosomes containing our previously designed siRNAs, allowing us to target recipient cells in vivo efficiently. The adipocyte targeting capability of exosomes was conferred by engineering the human embryonic kidney 293 (HEK293) cells to express lysosome-associated membrane glycoprotein 2b (Lamp2b), a well-characterized exosomal membrane protein, fused with tPep (CKGGRAKDC) for integrin. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect tPep with Lamp2b by a glycine-linker, and promote the expression using cmv promoter. In that way, the tPep exosomes (tPep-Exos) are endowed with site-specific identification ability and were purified from cell culture supernatants and loaded with siRNA.

Usage and Biology

tPep is a white fat tissue targeting peptide. To enhance the accuracy of siRNA delivery system and improve targeting index of drugs, tPep was displayed on the surface of the exosomes containing our previously designed siRNAs, allowing us to target recipient cells in vivo efficiently. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect tPep with Lamp2b by a glycine-linker, and promote the expression using cmv promoter. In that way, the tPep exosomes (tPep-Exos) are endowed with site-specific identification ability and were purified from cell culture supernatants and loaded with siRNA.

Characterization

To prove the targeting property of tPep exosomes, we co-incubated them respectively with 3T3L1 cells (mouse adipose tissues) and C2C12 (mouse myoblasts). Also we set parallel experiments using exosomes without tPep-lamp 2b fusion protein. It was thrilling to see only tPep exosomes accumulated in the 3T3L1 cells while the other groups’ results were contrary. This fully illustrates tPep exosomes can target precisely to the adipose tissues.

<img src="http://2017.igem.org/File:NJU_China_iGEM_2017_test0000.jpeg" style="width:400px;height:auto;">

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1276
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 199