Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins.
To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4 degrees C prior to incubation, agitation speed, isopropyl-beta-D-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfate polyacrylamide gel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37 degrees C, 24-h incubation).
Penna
Thereza Christina Vessoni
TC
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
Ishii
Marina
M
Junior
Adalberto Pessoa
AP
de Oliveira Nascimento
Laura
L
de Souza
Luciana Cambricoli
LC
Cholewa
Olivia
O
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D008164:Luminescent Proteins; D011506:Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D007544:Isopropyl Thiogalactoside