Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

Biotechnology and Bioengineering
P-O WahlundI Yu Galaev

Abstract

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N, N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production.

References

Mar 14, 1967·Journal of Molecular Biology·D E OlinsP H Von Hippel
Jan 1, 1995·Nature Medicine·R G Crystal
Jan 1, 1997·Annual Review of Immunology·J J DonnellyM A Liu
Feb 21, 1998·Trends in Biotechnology·P D RobbinsS C Ghivizzani
Mar 24, 1998·Immunology Today·H TigheE Raz
Apr 16, 1998·Nature·C L AndersonB Okamura
Jun 26, 1998·Journal of Chromatography. a·D M PrazeresC Cooney
Sep 22, 1998·Nucleic Acids Research·T Schluep, C L Cooney
Aug 3, 1999·Nature Biotechnology·J C MurphyR C Willson
Sep 2, 1999·Pharmaceutical Science & Technology Today·A V Kabanov
Dec 10, 1999·Nature Biotechnology·J L Fox
Dec 22, 1999·Infection and Immunity·J VasiB Guss
Jan 14, 2000·Nature Biotechnology·D Luo, W M Saltzman
Feb 17, 2000·Trends in Biotechnology·A Mountain
May 23, 2000·Biotechnology and Bioengineering·D W KahnM E Winkler
Jun 3, 2000·Annual Review of Immunology·S GurunathanR A Seder
Aug 16, 2000·Trends in Biotechnology·G N FerreiraJ M Cabral
Aug 8, 2001·Analytical Biochemistry·J C MurphyR C Willson
Nov 1, 2001·Biotechnology Annual Review·D M PrazeresJ M Cabral
Jan 10, 2002·The Journal of Gene Medicine·M M DiogoD M Prazeres
Apr 12, 2002·Biotechnology and Bioengineering·S C RibeiroD M F Prazeres
Sep 5, 2002·Biotechnology and Bioengineering·Russel J LanderAnn L Lee
Dec 31, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Raf LemmensJoachim Stadler
Jan 7, 2003·Biotechnology and Bioengineering·Matteo D CostioliRuth Freitag
Feb 5, 2003·Journal of Chromatography. a·Jason C MurphyRichard C Willson
Mar 1, 2003·Journal of Immunological Methods·Dan V MourichAnn B Hill

❮ Previous
Next ❯

Citations

Jul 15, 2005·Journal of Chromatography. a·Anja Hasche, Carsten Voss
Jun 5, 2007·Nature Protocols·U Loi LaoWilfred Chen
May 12, 2009·Journal of Biotechnology·Christina ParilAlois Jungbauer
Jan 20, 2009·IEEE Engineering in Medicine and Biology Magazine : the Quarterly Magazine of the Engineering in Medicine & Biology Society·Eugene R ArulmuthuHendrik K Versteeg
May 25, 2005·European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences·Tomás EtrychMichel Vert
Oct 14, 2014·Expert Review of Vaccines·Alex Xenopoulos, Priyabrata Pattnaik
Jul 2, 2011·Journal of Chromatography. a·Peter TiainenPer-Olof Larsson

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.