I have a fusion protein which has a hydropathy (GRAVY - averaged Kyte-Dolittle hydropathy) score of -0.589. It is solubilised from bacterial inclusion bodies in buffer containing 2M urea, pH8.5 (without salt) and is isolated using IMAC before elution and digestion.
When cleaved to completion under these conditions, the tag has a hydropathy score of -0.78, while the removed protein of interest has a score of -0.15.
Does this mean that the protein of interest is more or less hydrophobic than the tag? I was hoping to use HIC resin to separate the tag differentially from the protein and am trying to work out the conditions to trial, but this will only work easily if the tag is more hydrophobic than the protein. The proteins already interact with Q-sepharose with closely overlapping parameters, and the detached protein also interacts non-specifically with Ni2+ resin used to capture removed tag, so I cannot use either chromatography method to separate them satisfactorily.
Many thanks - I'm having a slow brain day.
Hi Adam,
thanks for confirming that - it was a definite "not enough caffeine" moment a couple of days ago in spite of having read various resources that all suggested what you have confirmed was the case. It just doesn't square with the supposed characteristics of the tag, so I was questioning myself.
Hi Robin, since proteins are not linear peptide chains the use of an hydropathy score to describe the hydrophobicity of a certain protein is often not the best way to go. Information concerning the secondary and tertiary structure of a protein will give more info about the location of amphipathic residues in the 3D structure of a protein and if the surface residues of this proteins are more hydrophobic or not. An average score of all AA hydropathies makes no sense if i.e. the hydrophobic residues will be arranged in the inner parts of the folded protein whereas the hydrophillic residues are outside on the surface of the protein determining the "real" interaction hydrophathy of this protein.
Best, Murat
Murat makes a good point. Hydrophobicity plots came into use as a way to help identify the continuous sequences that make up transmembrane alpha-helices in membrane proteins.
Thank you both. My protein of interest is fused to a tag including GFP. GFP is reported widely in the literature as having HIC-binding properties, but it appears that the ligand (once cleaved off) is more hydrophobic (based on above scoring method). I think some actual wet bench science is needed here at this point rather than my own poor application of misinterpreted theory.
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