Part:BBa_K1582015

BFP+sJanus-m Fusion Protein

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 961
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 970
  

Usage

Admittedly the separation of proteins is difficult in biological experiments. More often than not, we use chromatography, but it is expensive. Extraction turns to be cheap, however, its low separation rate is really an obstacle. </br> Here comes the Janus, a kind of amphipathic protein, which is made into a fusion protein with the target and carry it to leave the bulk protein phase. We could use aqueous two-phase systems to make them separated from mixture, according to the property that Janus will direct to the phase of detergent in the system of detergent/polymer. BFP-inJanus fusion protein could be used to verify this purification system. </br>

Biology

mTagBFP is a monomeric protein with a narrow fluorescence emission spectrum with a maximum at 456 nm. It has a tyrosine-based chromophore, giving it substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescent proteins with a histidine in the chromophore. </br> sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. They shows thread-like structure, can be expressed in prokaryotic cells like E.coli, and are involved in transferring themselves to detergent phase spontaneously. </br> We did some mutations to it, and we call it sJanus-m.</br>

Reference

[1]Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands. Hydrophobins: multipurpose proteins. Annu Rev Microbiol. 2001;55:625-46.

Protein Expression

We insert the plasmid into competent C+ to make the fusion protein secreted.</br> Express the fusion protein:</br>

5ml tube with C+	37℃ 15h
1L culture bottle	37℃ 4h
induced by IPTG	500μl  16℃ 12-16h
Collect the bacterial
Break the bacterial

Purify the fusion protein:

0mM McAc	Resuspension
10mM McAc	Wash the impurity away
20mM McAc	Wash the impurity away
30mM McAc	Wash the impurity away
50mM McAc	Wash the impurity away
200mM McAc	Elute

Figure 19. BFP- sJanus-m: The target protein is about 40kDa. As is shown in the picture, the protein is purified through Ni-chelating affinity chromatography.