Part:BBa_K1750001

YFP fused to CIB1 N-terminal domain

Yellow fluorescent protein, which produces yellow light (excitation 400nm, emission 530nm), fused to the carboxy-terminus of cryptochrome-interacting basic-helix-loop-helix1 (CIB1), a transcription factor derived from A. thaliana, in which it initiates flowering upon binding the photoreceptor cryptochrome 2 (CRY2).

We used the N-terminal domain of CIB1 (a.a. 1-170), that lacks the conserved basic helix-loop-helix domain which mediates dimerisation and DNA binding. This part is expressed strongly in the pETMCSII vector.

This device can be used as a reporter for binding to CRY2 fused to a second fluorescent protein, e.g. cyan fluorescent protein as detected by FRET assay. Expression in E. coli showed strong YFP solubility and fluorescence, however SDS-PAGE analysis suggested that a proportion of the CIB1 domains may be proteolysed in the cell, which would hinder its ability to bind CRY2.

We then ran an SDS-PAGE gel on the soluble fractions of CFP-CRY2 and the insoluble fraction of CFP-CRY2. For CIB1-YFP, a weak band at the expected size of 47kDa was observed, however much stronger bands at lower molecular weights were observed - this may indicate proteolytic products reflecting cleavage of likely of CIB1 (as cleavage of YFP would reduce vivid yellow colour of the purified protein, further supported by SDS-PAGE analysis of other CIB1 constructs). This suggests that while expression of the fusion protein may be strong, proteolysis may render the system unable to associate with CRY2 and thus be activated by light.

SDS-PAGE of FRET constructs. Lane 1: protein markers, 2: CIB1-YFP (soluble fraction). Stained with Coomassie Blue.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 718
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 718
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 718
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 718
  Illegal AgeI site found at 262
  
• 1000
  COMPATIBLE WITH RFC[1000]