Hydrophobic Interactions in Bioseparation - Applications of Modified Green Fluorescent Protein

Sammanfattning: This thesis describes interactions that are salt-promoted and mainly based on the hydrophobicity of proteins. A series of variants of the green fluorescent protein (GFP) was first constructed by site-directed mutagenesis. Several tags with various properties were then genetically fused to the N-terminus of GFP. The protein variants constructed had small characteristic differences in hydrophobicity, charge and metal affinity. These variants were then used to develop separation media and methods, and for the characterization of the proteins themselves for various applications. A mild pH-responsive hydrophobic medium was characterized, which was able to resolve the protein variants, down to just one difference in the amino acid sequence in hydrophobic interaction chromatography. The results were compared with theoretical calculations of hydrophobicity, and found to correlate well. Furthermore, a multifunctional purification tag was developed for use in protein isolation using various techniques. The tag, consisting of hydrophobic, charged, metal-binding amino acid residues, bound its protein host more strongly than the corresponding non-tagged protein when using a number of relevant isolation techniques. The isolation technique was scaled down to capillaries where variants differing in charge and hydrophobicity could be resolved using nanoparticles as the stationary phase in capillary electrochromatography. Finally, some of the GFP variants were tested regarding their metal affinity and the quenching of fluorescence by metal ions in the development of a biosensor. It was found that the accessibility of histidine residues on the surface of the protein is important for the quenching effect.