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Ion-exchange membranes for protein crystallization and protein crystals’ derivatization
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This PhD thesis is focused on the application of ion-exchange membranes for protein
crystallization and protein crystals derivatization. The experimental work is divided in
three parts. The first part of the work is focused on the understanding of the effect of
topography on nucleation. Soft lithography is used to modify the surface topography
of Nafion® membranes with target designs, avoiding changes of surface chemistry that
might mask the effect of topography on nucleation. The imprinted membranes are characterized
by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and
contact angle and tested for the crystallization of Trypsin from Bovine Pancreas. Nucleation
and crystals growth are followed over time by optical microscope. Experimental
results are compared with theoretical calculations of the ratio of change of Gibbs free
energy of heterogeneous to homogeneous nucleation. The second part of the work is
focused on the development of a method for performing a gentle derivatization of protein
crystals using ion-exchange membranes. Nafion® and Neosepta 01 were selected
after an initial screening of several membranes, due to their ability of promoting nucleation.
The kinetics of ion-transport for Br, PtCl2
4 and Hg2+ is evaluated and used for
modelling the transport in the derivatization set-up. Stability of crystals derivatized by
ion-exchange membranes over time is compared with the stability of crystals derivatized
by the conventional soaking method. The crystals derivatized by the help of the ionexchange
membranes are analysed by synchrotron and protein structure resolved with
the Isomorphous Replacement technique. The third part of the work involves the integration
of the ion-exchange membrane derivatization concept in a Polydimethylsiloxane
(PDMS) microdevice. A microdevice composed of two compartments, one with channels
and one with wells is designed and built by photolithography and soft-lithography.
Bonding of the membrane to the PDMS parts is done by grafting. Transport modelling
of water, NaCl and Hg2+ transport in the microdevice, crystallization experiments where supersaturation is achieved by osmosis and evaluation of the crystals’ diffraction quality
are performed.
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Palavras-chave
Crystallization Derivatization Protein Ion-exchange membranes