Permeation associated with three-phase-partitioning method on release of green fluorescent protein.
Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were subjected to two methods of extraction: (1) freezing/thawing/sonication (FTS) cycles prior to the three-phase partitioning (TPP) method, or (2) directly to TPP extraction. The amount of GFPuv released by the FTS plus TPP method varied: 374 microg/mL (first cycle), 93-442 microg/mL (second cycle), 32-359 microg/mL (third cycle), 18-115 microg/mL (fourth cycle). The GFPuv yields by the second method (TPP only) were, 23-54 microg/mL for the first extract and 33-91 microg/mL for the second. The FTS plus TPP method released similar amounts of GFPuv to that extracted by TPP; and provided a better mixture elution through the hydrophobic interaction column: 13-63 microg/mL for FTS plus TPP methods, and 2.5-13 microg/mL for TPP. The results showed that although selective permeation is a more laborious methodology, it was more efficient for obtaining of GFPuv in relation to the direct extraction of the cells for TPP.
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Rua Antonio de Macedo Soares, 452, 04607-000, São Paulo/SP, Brazil. tcvpenna@usp.br
Thereza Christina Vessoni
TC
Penna
Eb
E
Chiarini
Adalberto Pessoa
AP
Junior
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D005285:Fermentation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014169:Transformation, Bacterial
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins