Biomedical subjects

T Schluep

Publications and source records attributed to T Schluep.

3 recordsLinked to original sources

PubMed1999

Immobilization of oligonucleotides on a large pore support for plasmid purification by triplex affinity interaction.

Triplex affinity interaction provides a new process for the purification of plasmid DNA, which is especially suited to meet the demands of a gene therapy use. We developed a method for the functionalization of a large pore affinity support suitable for this application. A 5'-modified DNA oligonucleotide containing an aldehyde group was coupled to adipic acid hydrazide functionalized Sephacryl beads with a yield of 31% (over all immobilization yield 22.6% from starting oligonucleotide). The resulting selective and covalent immobilization of the ligand via a 16 atom, hydrophilic linker arm enables the oligonucleotide bases to freely bind to the target sequence. The proposed method provides affinity supports that might be used in large scale affinity purification of plasmid DNA.

Base Sequence↗

PubMed1998

Purification of plasmids by triplex affinity interaction.

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.

DNA, Bacterial↗

PubMed1998

Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography.

Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-exchange chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-M(r) RNA, cDNA and plasmid variants. A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 62 +/- 8% yield. Loadings higher than 40 micrograms/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.

Anions↗