Novel Affinity Chromatography Processes for the Purification of Plasmid DNA Using Small Aromatic ...

161 Pages·2013·9.74 MB·English

UNIVERSIDADE DA BEIRA INTERIOR
Ciências

Novel Affinity Chromatography Processes for the
Purification of Plasmid DNA Using Small Aromatic
Molecules

Catarina Caramelo Nunes

Tese para obtenção do Grau de Doutor em
Bioquímica
(3º ciclo de estudos)

Orientador: Prof. Doutora Cândida Ascensão Teixeira Tomaz
Co-orientadores: Prof. Doutor Paulo Jorge da Silva Almeida
Prof. Doutor João Carlos Nunes Marcos

Covilhã, Outubro de 2013
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Work supported by FCT, the Portuguese Foundation for Science and Technology
SFRH/BD/64918/2009, PTDC/QUI-QUI/100896/2008, and FCT-COMPETE PEst-
C/SAU/UI0709/2011.

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To my parents and sister

Somewhere, something incredible is waiting to be known.
Carl Sagan

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Acknowledgments

Acknowledgments

There are never enough words to express deep gratitude towards all of those persons
that were somehow involved in making this journey a true adventure of science and growth.

I gratefully thank my supervisors, Professor Cândida Tomaz, Professor Paulo Almeida
and Professor João Carlos Marcos for all the knowledge transmitted, unconditional support
and more importantly, for the friendship and kindness that made everything much more easy.
A special thank to Professor Cândida for following my work, and also my troubles up close,
always with a kind word of support.

I thank all my friends and colleagues for the help, friendship and all those good and
funny lab moments filled with work, science and laugh.
A very special thank to my friend Marta Gabriel for the unconditional help, friendship
and support. Thanks for always having my back. I also give a special thank to my friend José
Nunes for the friendship and all of those great and funny conversations. You both proved that
even in the middle of a storm, the rainbow can still always appear.

I give my deepest thanks to my family, my parents and my sister, for their
unconditional love and support. For the help and patience through so many troubled paths
and adventures. You truly are the best family one could ever have, and wish for.

I thank Fundação para a Ciência e a Tecnologia for the scholarship and financial
support.

Finally, I would like to thank the Universidade da Beira Interior.

Thank you all.

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List of publications

List of publications

Papers included in the thesis:

I. Aromatic ligands for plasmid DNA chromatographic analysis and purification: an
overview
C. Caramelo-Nunes, P. Almeida, J.C. Marcos, C.T. Tomaz
Submitted for publication (2013).

II. Specific berenil–DNA interactions: An approach for separation of plasmid isoforms
by pseudo–affinity chromatography
C. Caramelo-Nunes, T. Tente, P. Almeida, J.C. Marcos, C.T. Tomaz
Analytical Biochemistry (2011) 412: 153-158.

III. Purification of plasmid DNA from clarified and non-clarified Escherichia coli
lysates by berenil pseudo-affinity chromatography
C. Caramelo-Nunes, M. Gabriel, P. Almeida, J.C. Marcos, C.T. Tomaz
Journal of Chromatography B (2012) 904: 81-87.

IV. Negative pseudo-affinity chromatography for plasmid DNA purification using
berenil as ligand
C. Caramelo-Nunes, M.F. Gabriel, P. Almeida, J.C. Marcos, C.T. Tomaz
Submitted for publication (2013).

V. Specific recognition of supercoiled plasmid DNA by affinity chromatography using
the intercalator DAPP as ligand
C. Caramelo-Nunes, P. Almeida, J.C. Marcos, C.T. Tomaz
Journal of Chromatography B (2013) 928: 121-124.

VI. Dynamic binding capacity and specificity of 3,8-diamino-6-phenylphenanthridine-
Sepharose support for purification of supercoiled plasmid deoxyribonucleic acid
C. Caramelo-Nunes, D. Bicho, P. Almeida, J.C. Marcos, C.T. Tomaz
Journal of Chromatography A (2013) 1307, 91-98.

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List of publications

Patent included in the thesis:

I. Suporte cromatográfico de afinidade para purificação de DNA plasmídico
superenrolado utilizando 3,8-diamino-6-fenilfenantridina (DAPP) como ligando.
Portuguese patent application n. 106595.

Papers from scientific conferences:

I. Separation of plasmid DNA from non clarified lysate impurities using berenil as
ligand in pseudo-affinity chromatography
C. Caramelo-Nunes, M. Gabriel, P. Almeida, J.C. Marcos, C.T. Tomaz
Actas do Simpósio 2011: Transferência do Conhecimento e Tecnologia (2011) ISBN:
978-989-654-084-5, 41-43.

II. DAPP as affinity ligand: support characterization and chromatographic studies
with pDNA
C. Caramelo-Nunes, P. Almeida, J.C. Marcos, C.T. Tomaz
Actas do Simpósio 2012: Transferência do Conhecimento e Tecnologia (2012) In press.

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