A method for three-dimensional protein characterization and its application to a complex plant (corn) extract.
Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.
Gu
Zhengrong
Z
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011-2230, USA.
Glatz
Charles E
CE
D003198:Computer Simulation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007526:Isoelectric Point; D008956:Models, Chemical; D008958:Models, Molecular; D008970:Molecular Weight; D010936:Plant Extracts / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D055672:Static Electricity; D003313:Zea mays / Q000737:chemistry*
D010936:Plant Extracts; D010940:Plant Proteins