Part:BBa_K2074021

Cry4Aa(Codon optimization) - Extended FMDV

Cry4Aa is cloned from Bacillus thuringiensis BRC-LLP29. It shows specific toxicity to Culex by bioassay. The Cry protein is consisted of three functional domains. Domain I is a seven α-helices bundle. It can insert itself into a membrane by using its hydrophobic helices α4 and α5 to insert into the phospholipid bilayer. The pore formation occurs on its α3 helix. Domain II and domain III are two β-sheets which are involved in the receptor interactions. Domain II contains extremely variable loops, which are the binding site of the receptor. Domain III has the function of stabilizing the toxin. Cyt proteins have a single α-β domain which do not bind to receptors but can directly insert into the cell membrane and then form a pore causing cell death. Although Cry and Cyt proteins are two big families of δ-endotoxins, they are far related.Cyt1 and Cyt2 are two types of Cyt proteins found in Bti.. Generally, Cry proteins are believed to exert toxicity by interacting with the proteins on the brush border membrane and then insert into the membrane which takes multiple steps. At the beginning in mosquitoes' gut, the crystalline inclusions are cleaved at the disulfide bond to release the Cry pre-toxin. Then the soluble proteins are activated by being cleaved again by intestinal protease. When toxins reach to the brush border membrane microvilli, they bind to the proteins, or known as receptors on the membrane. The binding process takes two step. Firstly, the monomeric Cry toxin binds to cadherin, resulting in the formation of pre-pore oligomer as Figure2.

Figure.2 Figure1.The Mechanism of Cry and Cyt Toxins

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 986
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1378
  Illegal NgoMIV site found at 3631
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 31
  Illegal BsaI.rc site found at 184
  Illegal BsaI.rc site found at 2071
  Illegal BsaI.rc site found at 2611
  Illegal BsaI.rc site found at 2920
  Illegal BsaI.rc site found at 3487
  Illegal SapI.rc site found at 1188
  Illegal SapI.rc site found at 1230