Part:BBa_K4223018

A protein expression system of Csm6

CRISPR-Cas bases can be divided into three main types: I-III. The characteristic protein of type I is Cas3, the characteristic protein of type II is Cas9, and the characteristic proteins of type III are Cas6 and Cas10. Among them, the type III CRISPR-Cas system is the oldest and most complex of all CRISPR-Cas systems found so far. The A subtype of the type III CRISPR-Cas system has an effector complex called Csm, which consists of multiple Cas proteins together with crRNA. Unlike other CRISPR effector complexes, the Csm complex not only cleaves target RNA complementary to crRNA, but the binding of the target RNA activates the Csm complex to generate two new enzymatic activities, namely the cleavage of ssDNA during transcription and the synthesis of cyclic oligoadenylate (cOA). cOA acts as a second messenger that activates Csm6 to non-specifically degrade RNA. specific degradation of RNA. type III CRISPR can produce two types of cOA: cA6 and cA4.

Csm6 is a dimeric RNA endoribonuclease from the type III CRISPR-Cas system with the potential to facilitate RNA detection based on its endogenous function in signal amplification. recognition of viral RNA by Csm or Cmr triggers synthesis of cyclic tetra- or hexaadenylate (cA4 or cA6), which is associated with the CRISPR-associated Csm6 CARF structural domain binds and activates the ribonuclease activity of its eukaryotic/prokaryotic nucleotide-binding (HEPN) structural domain.

Through screening, the researchers found that the oligonucleotide A4-U6 binds and stimulates the cleavage of the reporter protein by TtCsm6 and releases fluorescent molecules. Furthermore, activation of TtCsm6 by different concentrations of A4-U6 resulted in an increase in fluorescence within 20-30 minutes, followed by a plateau value, with the final fluorescence level proportional to the amount of Csm6 activator.

Protein purification

After fragmentation and precipitation of the BL21 protein expression vector and protein purification (Ni column), we tested the resulting csm6 protein using SDS-PAGE and saw obvious, clear, bands of about 51 kda in size, consistent with the theoretical size of csm6 protein. The experimental results showed that we obtained a relatively pure csm6 protein.

Figure 1. SDS-PAGE detection Csm6 Ni column purification

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1861
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1934
  Illegal PstI site found at 1861
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 338
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1861
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1861
  Illegal NgoMIV site found at 624
  
• 1000
  COMPATIBLE WITH RFC[1000]