3D Nanotemplates for Protein Crystallisation
Author(s)
Shah, Umang Vinubhai
Type
Thesis
Abstract
This thesis presents the first experimental investigations into the combined effect of nanoscale
surface porosity, narrow pore size distribution and surface chemistry, on nucleation and
crystallisation of proteins. Co-operative self assembly of surfactants and silica was applied to prepare
3D nanotemplates with pore diameter 3.5±1.0nm, 5.5±1.5nm, 11.0±3.0nm, 16.0±3.0nm and
22.0±5.0nm. Post synthesis functionalisation was used to graft surface with -OH, -NH2, -CF3, -C6H5, -
Cl and -CH3 functional groups.
The relationship between 3D nanotemplate, N2 sorption and TEM pore diameter, XPS surface
composition, wettability, surface charge and protein physicochemical properties was investigated,
resulting in a coherent understanding of the combined effect of nucleant surface porosity and surface
chemistry on protein crystallisation. The protein systems investigated for crystallisation include
lysozyme, thaumatin, trypsin, albumin, concanavalin A, catalase and ferritin; varying in molecular
weight from 14kDa – 450kDa and hydrodynamic diameter ~3-20nm.
Crystallisation of proteins was found to be strongly dependent on 3D nanotemplate pore size.
The 3D nanotemplate with a pore diameter similar to the protein’s hydrodynamic diameter (Dh) was
found to be successful in preferential crystallisation, e.g. albumin (Dh=~5nm) and catalase
(Dh=~10nm) was crystallised only on 5.5±1.5nm and 11.0±3.0nm 3D nanotemplates respectively.
Here, we report a direct correlation between protein hydrodynamic diameter and 3D
nanotemplate pore diameter, key for controlling protein nucleation. The correlation has been
experimentally validated for all protein systems investigated.
The concept of preferential crystallisation was further developed to investigate the
applicability of this methodology for the separation of a target protein from a protein mixture. A
solution of two proteins; porcine pancreatic lipase and RNAse was selected for this separation by
crystallisation. Lipase (Dh=~4.5nm) and RNAse (Dh=~1.5nm) crystals were preferentially obtained
with 5.5±1.5nm and 3.5±1.0nm 3D nanotemplates respectively. Furthermore, crystallisation of lipase
from a commercially available crude source, (purity ~20%) containing a mixture of lipase, amylase and protease, was also achieved on the surface of the 5.5±1.5nm 3D nanotemplate. The significance
of the work here is the demonstration that only specific nucleant surfaces with narrow pore size
distribution can preferentially crystallise a target protein from a protein mixture, which is not
possible with nucleants of broad pore size distribution, reported to be most suitable candidate for
“universal nucleant”.
Crystals of four out of the seven well studied proteins (lysozyme, albumin, concanavalin A
and catalase) were obtained at lower protein concentration, whilst thaumatin was crystallised at par
protein concentration but 3× lower precipitant concentration, on the surface of 3D nanotemplates
functionalised with -OH, -CH3 and -NH2 functional groups. Here, we report that a reduction of 50-
92% protein concentration compared to the lowest reported literature values was achieved with the
use of the 3D nanotemplates. As crystallisation was achieved for all seven protein systems at protein
concentrations in the range of 2-20mg/mL, this approach can be used to engineer surfaces for
preferential crystallisation of a target protein directly from industrial bioreactor broths.
In summary, the findings of this thesis offer a first systematic approach for controlling the
nucleation and crystallisation of biological macromolecules. The use of 3D nanotemplates offers the
possibility of crystallising complex proteins (e.g. enzymes, antibodies, protein complexes, DAbs,
MAbs) for structural determination and also novel crystallisation routes for downstream
bioseparations.
surface porosity, narrow pore size distribution and surface chemistry, on nucleation and
crystallisation of proteins. Co-operative self assembly of surfactants and silica was applied to prepare
3D nanotemplates with pore diameter 3.5±1.0nm, 5.5±1.5nm, 11.0±3.0nm, 16.0±3.0nm and
22.0±5.0nm. Post synthesis functionalisation was used to graft surface with -OH, -NH2, -CF3, -C6H5, -
Cl and -CH3 functional groups.
The relationship between 3D nanotemplate, N2 sorption and TEM pore diameter, XPS surface
composition, wettability, surface charge and protein physicochemical properties was investigated,
resulting in a coherent understanding of the combined effect of nucleant surface porosity and surface
chemistry on protein crystallisation. The protein systems investigated for crystallisation include
lysozyme, thaumatin, trypsin, albumin, concanavalin A, catalase and ferritin; varying in molecular
weight from 14kDa – 450kDa and hydrodynamic diameter ~3-20nm.
Crystallisation of proteins was found to be strongly dependent on 3D nanotemplate pore size.
The 3D nanotemplate with a pore diameter similar to the protein’s hydrodynamic diameter (Dh) was
found to be successful in preferential crystallisation, e.g. albumin (Dh=~5nm) and catalase
(Dh=~10nm) was crystallised only on 5.5±1.5nm and 11.0±3.0nm 3D nanotemplates respectively.
Here, we report a direct correlation between protein hydrodynamic diameter and 3D
nanotemplate pore diameter, key for controlling protein nucleation. The correlation has been
experimentally validated for all protein systems investigated.
The concept of preferential crystallisation was further developed to investigate the
applicability of this methodology for the separation of a target protein from a protein mixture. A
solution of two proteins; porcine pancreatic lipase and RNAse was selected for this separation by
crystallisation. Lipase (Dh=~4.5nm) and RNAse (Dh=~1.5nm) crystals were preferentially obtained
with 5.5±1.5nm and 3.5±1.0nm 3D nanotemplates respectively. Furthermore, crystallisation of lipase
from a commercially available crude source, (purity ~20%) containing a mixture of lipase, amylase and protease, was also achieved on the surface of the 5.5±1.5nm 3D nanotemplate. The significance
of the work here is the demonstration that only specific nucleant surfaces with narrow pore size
distribution can preferentially crystallise a target protein from a protein mixture, which is not
possible with nucleants of broad pore size distribution, reported to be most suitable candidate for
“universal nucleant”.
Crystals of four out of the seven well studied proteins (lysozyme, albumin, concanavalin A
and catalase) were obtained at lower protein concentration, whilst thaumatin was crystallised at par
protein concentration but 3× lower precipitant concentration, on the surface of 3D nanotemplates
functionalised with -OH, -CH3 and -NH2 functional groups. Here, we report that a reduction of 50-
92% protein concentration compared to the lowest reported literature values was achieved with the
use of the 3D nanotemplates. As crystallisation was achieved for all seven protein systems at protein
concentrations in the range of 2-20mg/mL, this approach can be used to engineer surfaces for
preferential crystallisation of a target protein directly from industrial bioreactor broths.
In summary, the findings of this thesis offer a first systematic approach for controlling the
nucleation and crystallisation of biological macromolecules. The use of 3D nanotemplates offers the
possibility of crystallising complex proteins (e.g. enzymes, antibodies, protein complexes, DAbs,
MAbs) for structural determination and also novel crystallisation routes for downstream
bioseparations.
Date Issued
2012-09
Date Awarded
2012-12
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
License URL
Advisor
Heng, Jerry
Publisher Department
Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)