HYDROPHOBIC PEPTIDE TAGS - As tools in bioseparation and investigation of recombinant proteins

Sammanfattning: This thesis describes the possibility of using hydrophobic peptide tags as tools for bioseparation and for the investigation of recombinant proteins. The hydrophobicity of a protein can easily be changed by minor modifications, such as fusions of tyrosine-containing peptides. The fusion tags may also increase both stability and expression of the fusion protein. Furthermore, a protein library with randomised pentapeptides was created, and the variants exhibited significant differences in relative hydrophobicity, although the pentapeptides only caused minor alterations in primary structure, size and isoelectric point of the fusion proteins. The changes in relative hydrophobicity were measured with aqueous two-phase systems (ATPSs) and hydrophobic interaction chromatography (HIC), and the results indicated the sensitivity of both methods to slight variations in the primary structure of proteins and their surface hydrophobicity. The results of the techniques correlated well, and the correlation coefficient, R2, was 0.89. Upon comparing the relative hydrophobicity contributions from seventeen LDH variants from thirty-four hydrophobicity scales they were found to correlate fairly well with experimental results from ATPSs and HIC. However, the results from such scales should only be used as a complement to experimental studies, for example, to indicate tag exposure. A novel HIC adsorbent, HBVE/BVE, was developed with polymer grafting techniques. The advantage of the HBVE/BVE medium is the possibility to alter the hydrophobic properties of the medium by only changing the reaction ratio of the two monomers in the grafting procedure. The hydrophobicity can be tuned to improve protein purification.