Journal: Protein Engineering, Design and Selection
Article Title: PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins
Figure Lengend Snippet: Biophysical characterization of PAS#1(600)-hGH. ( A ) Mass spectrometric analysis of PAS#1(600)-hGH by ESI-MS, confirming the calculated molecular mass of 73121.65 Da as well as precisely monodisperse composition, without any indication of prematurely terminated gene products. ( B ) Hydrophobicity analysis of PASylated hGH by reverse-phase (RP) HPLC. HIC of the original recombinant hGH and its PASylated version was performed on a Resource RPC column using an elution gradient from 2% v/v acetonitrile, 0.065% v/v TFA to 80% v/v acetonitrile, 0.05% v/v TFA. Both profiles show a single homogeneous peak with slightly earlier elution of PAS#1(600)-hGH (51.8% acetonitrile) in comparison with the corresponding unfused protein (52.3% acetonitrile). ( C ) Thermal denaturation of hGH and PAS#1(600)-hGH as determined by far UV CD measurement. Thermal unfolding of a 8.2 µM protein solution in 50 mM K 2 SO 4 , 20 mM K-P i pH 7.5 was followed at a wavelength of 208 nm where maximal spectral change upon unfolding was observed and the normalized unfolded fraction, f(u), was plotted as a function of temperature. The melting temperatures ( T m ) of hGH and PAS#1(600)-hGH were 85.4°C and 88.6°C, respectively.
Article Snippet: Reverse-Phase HPLC The purified protein solution in PBS was adjusted to 2% v/v acetonitrile, 0.065% v/v trifluoroacetic acid (TFA) and 1 ml was applied to a Resource RPC 1 ml column (GE Healthcare) equilibrated with buffer A (2% v/v acetonitrile, 0.065% v/v TFA).
Techniques: Mass Spectrometry, High Performance Liquid Chromatography, Hydrophobic Interaction Chromatography, Recombinant