Part:BBa_K3008005

CAHS 107838

Tardigrade-specific intrinsically disordered proteins (TDPs) allow tardigrades to survive extreme environmental conditions such as desiccation, radiation, and temperatures ranging between -200°C and 149°C. These genes increase desiccation tolerance when expressed in heterologous systems. TDPs form a non-crystalline glass-like structure (vitrify) upon desiccation. This vitrified state is believed to be what allows tardigrades to survive under extreme stress.

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 150
  Illegal AgeI site found at 625
  
• 1000
  COMPATIBLE WITH RFC[1000]

Purification of Large Scale

During the first phase of our project, we performed a large scale protein production and purification of native CAHS 94205. We also conducted large scale production of CAHS2 (D3), CAHS1 (D4), SAHS10 (G3), LEA1(A4), and rvLEAM (H4); all five of which contained an N-terminal FLAG-tag. After we received a product, we autoclaved them for 20 minutes to purify the proteins. The SDS-PAGE gel of the purified products shows that only CAHS D (CAHS 94205), CAHS 1 (CAHS 107838), SAHS 10 and LEA1 had good yield and withstood being exposed to a temperature of 121°C. As a result, we decided to continue site-directed mutagenesis with these four proteins. The ability of CAHS 107838 to withstand a temperature of 121°C is promising and shows that it can be subject to higher temperatures without degrading.

Figure 4: SDS-PAGE of Autoclaved Samples from Large Scale Protein Production SDS-PAGE gel of purified protein from large scale production. CAHS 94205, CAHS 107838, SAHS 10, and LEA1 have a significant band corresponding to the size of the protein produced.