Part:BBa_K3815010

NOP1v-Mxe GryA intein-PT-linker-His tag

Description of this part

Targeted protein

This part is for the purification of NOP1v. This is made by adding a valine to the N-terminus of NOP1. The effect of it is the same as NOP1. We could not get NOP1 sufficiently when using BBa_K3815009. Then, we thought that considering N-end-rule, the N-terminus of NOP1 might have a negative effect on the recovery of peptides. Therefore, this part is made.

Purification system

In order to purify the peptide, we adopted the ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe GyrA intein, and PT linker. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the targeted protein.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 120
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 120
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 120
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 120
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 120
  Illegal NgoMIV site found at 553
  
• 1000
  COMPATIBLE WITH RFC[1000]

Purification

Fig1. SDS-PAGE of purified peptide

Expression

• Cells were grown in 1000ml LB media at 37℃ shaking at 180 rpm.
• when the OD exceeded 0.35, 1 M IPTG 200μL was added to induce the peptide expression.
• Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.

Purification

1.When this fused protein were produced, it was recovered by Ni chromatography.
2.Adding DTT to it, the targeted protein was cut out by the cleavage of intein.
3.SDSPAGE was performed in order to confirm the presence of it.

Fig1 shows the result of SDS-PAGE. Lane 5 and 11 are the result of NOP1v.
NOP1v is 1231Da, so this data shows that we could not confirm its production.

Reference

1.Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019). The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers. Sci. Rep. 9, 1287.
2.Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991). The N-end rule in bacteria. Science 254, 1374–1377.