Part:BBa_K4497039

GFP-mCherry-Fc

This part encodes GFP-mCherry-Fc, GFP fused to mCherry and to an Ig antibody Fc domain. The Fc domain dimerizes through disulfide bonds and allows for easy purification using Protein A chromatography.

Design & Cloning

Design

This part is made of the following components:

• IL-11 Signaling Sequence (Part:BBa_K4497003): Signaling Sequence for secretion
• Flag-tag (Part:BBa_K4497012): Flag epitope tag that enables surface expression detection using immunofluorescence labeling
• mEGFP (Part:Bba_K4497034)
• mCherry(Part:Bba_K4497031)
• TEV recognition sequence (Part:Bba_K4497035: TEV cutting site for removing the Fc-domain from mEGFP after purification.
• Fc domain (Part:Bba_K4497036)

Cloning

We received the part in pcDNA3.1 from Prof. Scheller [1]: pcDNA3.1-spIL11R-GFP-Cherry-TEV-Fc

Purification

We expressed GFP-mCherry-Fc in ExpiCHO using the standard expression protocol (8days) in 25mL of media. The protein was purified from the cell supernatant using a Protein A column (iba-lifesciences) as per protocol of the column. Before application to the column the supernatant was dialyzed into Buffer A overnight. After purification relevant collected fractions were analyzed by SDS PAGE. The chosen fractions were pooled, concentrated and frozen using liquid nitrogen. Protein aliquots were kept in a -80 freezer.

Result

Figure 1. SDS Gel of GFP-mCherry-Fc protein purification. Proteins were purified with Protein A columns. Loading samples (L), waste samples (W) and fractions were loaded. The fractions were chosen with chromatography at λ = 280 nm. Reduced (reducing) samplessample using β-Mercaptoethanol and non reduced (oxidizing) protein samples using NEM were loaded. Marker (M) ‘PageRuler Plus Prestained’ was used.

The purification protocol was adapted from Mossner et al. [1], to be performed significantly faster (several months vs 10 days). While our purity is not as high as shown for the longer protocol, the results were fully sufficient for our ligand binding experiments with the MESA system (Part:Bba_K4497017). The yield was around 4mL with 117µg/mL, which provided plenty of protein for our experiments where we worked with 500ng/mL concentrations in cell media. We used 4 of 80 aliquots during our project.

We used the same protocol to purify GFP-Fc and GFP-mCherry-Fc:

• GFP-Fc: Part:Bba_K4497037
• mCherry-Fc: Part:Bba_K4497038

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 2182
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1576
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 94
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 2182
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 2182
  Illegal AgeI site found at 102
  
• 1000
  COMPATIBLE WITH RFC[1000]