Liquid-liquid extraction of a recombinant protein with reverse micelles.
Recombinant cytochrome b5 was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome b5 to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1-2 mol dm-3 NaCl) and low temperatures (4 degrees C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.
Pires
M J
MJ
Laboratório de Engenharia Bioquímica, Instituto Superior Técnico, Lisbon, Portugal.
Cabral
J M
JM
D000818:Animals; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D003580:Cytochromes / Q000235:genetics / Q000302:isolation & purification*; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000302:isolation & purification; D003902:Detergents; D004143:Dioctyl Sulfosuccinic Acid; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008823:Micelles; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D013501:Surface-Active Agents; D013696:Temperature
D002593:Cetrimonium Compounds; D003580:Cytochromes; D003902:Detergents; D008823:Micelles; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D004143:Dioctyl Sulfosuccinic Acid; D015786:Cytochromes b5; D000077286:Cetrimonium