Selective Precipitation of DNA by Spermine during the Chemical Extraction of Insoluble Cytoplasmic Protein

Abstract

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.

Coupling of chemical extraction and expanded‐bed adsorption for simplified inclusion‐body processing: Optimization using surface plasmon resonance

“…Enzymatic degradation of the nucleic acids (Barnfield Frej et al, 1994;Clemmitt et al, 2000a) is not possible due to the strongly denaturing environment (8 M urea). Selective precipitation of DNA by spermine during extraction has been shown to precipitate >80% of released DNA without affecting protein solubility (Choe and Middelberg, 2001b). The pellet is easily separable by low-speed centrifugation or, under careful handling, by gravitational settling.…”

Releasing Biopharmaceutical Products from Cells

“…At these high cell concentrations, DNA release signifi cantly increases broth viscosity causing handling diffi culty. Suitable precipitants can selectively remove DNA during the extraction stage if necessary (e.g., 35 mM spermine followed by low -speed centrifugation [25] ).…”

“…For cytoplasmic inclusion bodies, a refolding operation must follow the extraction operation and protein is often purifi ed using a chromatographic or capture method after extraction. For example, the viral protein studied by Choe et al has been recovered using an immobilized metal affi nity capture step in expanded -bed or magnetic -bead capture mode, to give recovery yields approaching 90% and purifi cation factors of 10 -20 [25] .…”

Recent advances in biomolecular process intensification

“…EBA, HGMS). Addition of a stoichiometric amount of spermine during the extraction (10 mg-spermine per mg-DNA) resolved this issue by inducing almost 90% of released DNA to form an insoluble DNA-spermine complex in a highly specific manner without affecting the target protein solubility (Choe and Middelberg, 2001b). The resulting DNA-spermine complex could be easily removed by low speed centrifugation to give a post-extraction suspension compatible with immobilised metal chromatography operated in expanded bed mode (IMAC-EBA).…”