Aqueous two-phase extraction for protein recovery from corn extracts.
Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5x enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35x higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na(2)SO(4) as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9x (germ) and 5x (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.
Gu
Zhengrong
Z
Department of Chemical and Biological Engineering, 2114 Sweeney Hall, Iowa State University, Ames, IA 50011-2230, USA.
Glatz
Charles E
CE
D002498:Centrifugation; D005591:Chemical Fractionation / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification; D010936:Plant Extracts / Q000737:chemistry*; D010940:Plant Proteins / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000302:isolation & purification; D013431:Sulfates / Q000737:chemistry; D014867:Water; D003313:Zea mays / Q000737:chemistry*
D010936:Plant Extracts; D010940:Plant Proteins; D013431:Sulfates; D014867:Water; C012036:sodium sulfate; D011092:Polyethylene Glycols; D045304:Cytochromes c; D012259:Ribonuclease, Pancreatic; D009113:Muramidase