Selective precipitation of DNA by spermine during the chemical extraction of insoluble cytoplasmic protein.
The direct chemical extraction of recombinant L1 protein (the major capsid protein of human papillomavirus type 16) from the cytoplasm of E. coli HMS174(DE3) has recently been demonstrated at high cell density (to OD(600) = 160) without the use of reducing agent (1). Coextraction of DNA at high concentration prevents direct coupling to postextraction recovery operations including expanded bed adsorption. In this study, spermine is used to selectively precipitate DNA during chemical extraction. Highly efficient and selective DNA precipitation was achieved. An approximate 10-fold increase in the specific spermine concentration (mg of spermine/mg of DNA) was required to precipitate DNA when 8 M urea was added to the extraction buffer. EDTA (3 mM), required for effective chemical extraction, does not significantly inhibit DNA precipitation. Precipitation selectivity was demonstrated in a bovine serum albumin spiking test, with almost complete recovery of the spiked protein. During studies on the direct extraction of L1 protein from cells at OD(600) = 80, high DNA removal efficiency (>85%) and negligible L1 protein coprecipitation were achieved. This selective precipitation technique simply requires the addition of spermine to the chemical extraction buffer and therefore does not increase technique complexity. This modification enhances the method's general applicability and enables direct coupling to downstream recovery units following chemical extraction at high cell and product concentrations.
Choe
W S
WS
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, U.K.
Middelberg
A P
AP
D036022:Capsid Proteins; D003593:Cytoplasm / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000302:isolation & purification; D005285:Fermentation; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents; D013096:Spermine / Q000737:chemistry*; D014508:Urea / Q000737:chemistry; D014764:Viral Proteins
D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; D011506:Proteins; D011994:Recombinant Proteins; D012997:Solvents; D014764:Viral Proteins; D013096:Spermine; C091560:L1 protein, Human papillomavirus type 6; D014508:Urea; D004247:DNA