Immobilisation of a repressor protein for binding of plasmid DNA

Hasche, Anja; Voss, Carsten

doi:10.1016/j.chroma.2005.05.025

Cited by 23 publications

(19 citation statements)

References 19 publications

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“…However, previous studies using gel-shift assays indicate that nonspecific interactions can occur between the full length Lac repressor and pET20b(þ) DNA which lacks the operator sequence. 29 This nonspecific binding was limited to interactions between the full Lac repressor sequence and double stranded DNA and was not observed for RNA molecules. Hence, our experiments confirm that no binding occurs with molecules other than pDNA containing lac region.…”

Section: Ftir Analysismentioning

confidence: 96%

“…28 Hasche and Vob found that interaction between repressor molecules and RNA was not detectable when interaction with double stranded DNA in the form of a short operator sequence and pDNA occurred. 29,30 Previous affinity approaches have also involved a sequence-specific zinc finger protein and triplex DNA formation, and are not cost-effective. In addition, the triple helix interaction is a commonly used binding method, 31 but has a slow binding kinetics.…”

Section: Introductionmentioning

confidence: 99%

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Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

2008

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in Wiley InterScience (www.interscience.wiley.com).Peptides constructed from a-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to ''biomimics'' of the DNA binding domain of LacI. Equilibrium dissociation constants (K D ), association (k a ), and dissociation rates (k d ) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 6 1.3 3 10 210 M, 7.2 6 0.6 3 10 210 M, 4.5 6 0.5 3 10 28 M, and 6.2 6 0.9 3 10 26 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operonrepressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

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Section: Ftir Analysismentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

2008

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“…The immobilization of the lac repressor and binding of short DNA fragments to this ligand demonstrated that this affinity interaction can also be exploited for nucleic acid purification [60]. Non-covalent immobilization allowed Hasche and Voß to screen different chromatographic supports for affinity separation [61]. Comparable experiments have been conducted with an immobilized zinc finger protein [62,63], but in both cases, binding capacities were low.…”

Section: Affinity Separationmentioning

confidence: 97%

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Voss

2008

Chem Eng & Technol

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Plasmid DNA is used as a cloning vector to deliver recombinant genetic information into microorganisms. Since the 1990s, this principle has also been applied for the delivery of therapeutic genes in gene therapy and genetic vaccination. This non-viral gene delivery is afflicted with fewer safety concerns in comparison to viral systems. Processes for the production of high-quality plasmid DNA at multiand kilogram scale are necessary to meet the needs of clinical trials as well as future therapeutics. Cell disruption, the separation of structurally-related impurities and analytical techniques for process and quality control are the main challenges for bioengineering. This review summarizes the development in these fields over the past recent years.

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“…Im Bereich der Proteinaufreinigung haben sich hier besonders immobilisierte Metallchelataffinitätsverfahren im Labormaßstab [54] [61]. Vergleichbare Untersuchungen wurden auch mit immobilisierten Zinkfinger-Proteinen durchgeführt [62,63], jedoch war hier, genauso wie bei dem zuvor beschriebenen lac-Repressor-System, die Bindungskapazität äußert gering, sodass die beschriebenen Studien lediglich modellhaften Charakter besitzen und darstellen konnten, dass diese Affinitätswechselwirkung prinzipiell einsetzbar ist.…”

Section: Affinitätsverfahrenunclassified

Aufarbeitung von Plasmid‐DNA für Gentherapie und genetische Impfung

Voss

2008

Chemie Ingenieur Technik

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Plasmid‐DNA wird als Klonierungsvektor zum Einbringen rekombinanter Gene in Mikroorganismen genutzt. Seit den 1990er Jahren wird dieses Prinzip auch zur Übertragung therapeutischer Gene in der Gentherapie und der genetischen Impfung verwendet. Diese Form der nicht viralen Gentherapie stellt eine deutlich risikoärmere Variante im Vergleich zur viralen Gentherapie dar. Zur Deckung des Bedarfs an Plasmid‐DNA in klinischen Studien und für in Zukunft zugelassene Therapeutika sind Produktionsverfahren erforderlich, die eine Herstellung im Multigramm‐ bis Kilogramm‐Maßstab möglich machen. Besondere Hersausforderungen werden hier im Bereich des Zellaufschlusses, der Abtrennung strukturell verwandter Verbindungen und der Prozess‐ und Produktanalytik gestellt. Dieser Übersichtsbeitrag fasst die Entwicklungen der letzten Jahre zusammen.

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Immobilisation of a repressor protein for binding of plasmid DNA

Cited by 23 publications

References 19 publications

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Aufarbeitung von Plasmid‐DNA für Gentherapie und genetische Impfung

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