Crystallisation via novel 3D nanotemplates as a tool for protein purification and bio-separation

Umang V. Shah 1, Niklas H. Jahn 1, Shanshan Huang1, Zhongqiang Yang2, Daryl R. Williams1, and Jerry Y. Y. Heng 1, *

1 Surface and Particle Engineering Laboratory (SPEL), Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.

2 Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China.

*Corresponding Author: jerry.heng@imperial.ac.ukPhone: +44-(0)207-594-0784. Fax: +44-(0)207-594-5700 Web: www.imperial.ac.uk/spel

Highlight:

Validation of a correlation between protein hydrodynamic and nucleant pore diameter

for crystallisation

Rational approach for crystallisation of a target protein from binary protein mixture

Surface preferential crystallisation of proteins using nucleants having specific pore

dimensions

Abstract:

This study reports an experimental validation of the surface preferential nucleation of proteins

on the basis of a relationship between nucleant pore diameter and protein hydrodynamic

diameter. The validated correlation was employed for the selection of nucleant pore diameter

to crystallise a target protein from binary, equivolume protein mixture. We report proof-of-

concept preliminary experimental evidence for the rational approach for crystallisation of a

target protein from a binary protein mixture on the surface of 3D nanotemplates with

controlled surface porosity and narrow pore-size distribution selected on the basis of a

relationship between the nucleant pore diameter and protein hydrodynamic diameter. The

outcome of this study opens up an exciting opportunity for exploring protein crystallisation as

a potential route for protein purification and bio-separation in both technical and

pharmaceutical applications.

Keywords: A.1 Crystallisation, B.1 Protein, B.1 3D nanotemplates, B.1 Protein mixture, A.1

Surface Porosity, A1. Purification

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1. Introduction

The rise of molecular biology in the past three decades has enabled both drug discovery

and development to improve the understanding of disease pathogenesis on a molecular level

and within their respective biological systems. From this and the success of the Human

Genome Project, the development of biopharmaceuticals has accelerated in the past decade

and protein-based therapeutics is rapidly entering the market. On the other hand, the demand

for proteins, especially technical enzymes, is growing in different industrial sectors including

biochemical, consumer packaged goods and food technology [1]. Upstream processing of

proteins with high value and availability from natural resources relies on recombinant

methods and can be prepared at considerably higher concentration compared to the natural

resources. Upstream processing using a recombinant route is relatively well developed and

industrially applied; most appropriate example of this success is recombinant insulin, which

is widely used for therapeutic applications [2].

The proteins prepared this way generally require several purification steps before they can

be integrated into their formulation matrix. In contrast to the majority of small-molecule

pharmaceuticals, the purification of therapeutic proteins typically relies on high cost, low-

throughput chromatographic methods which are one of the main factors responsible for the

high cost of biopharmaceutical products and one of the major bottlenecks in providing

improved biopharmaceutical healthcare [3]. Although macromolecular separation is

dominated by packed bed chromatography, its limitations have accelerated the search for

alternative low cost separation methods. Chromatographic techniques can be justified for

therapeutic proteins where purity is the unquestionable need. However, for protein production

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the cost factor of these processes demands the development of alternative purification

techniques [3].

Crystallisation is considered to be a potential alternative for purification. Crystallisation of

biological macromolecules continues to evolve from a large empirical technique [4]. High

throughput screening systems with robotic liquid handling and in-situ analysis techniques are

being developed enabling rapid screening for a broad ranges of condition [5, 6]. The

industrial scale developments of products ranging from enzymes like glucose isomerase to

approved biopharmaceuticals like insulin have demonstrated the capabilities of crystallisation

as a cost effective alternative method. However, the potential for crystallisation is yet under

demonstration, as a result of the empirical nature and poor reliability, in contrast to the small

molecular pharmaceutical products. Only 0.2% of biopharmaceutical products developed

are administered in the crystalline form currently. This highlights the acute need for

developing an understanding of the crystallisation process of biological macromolecules.

Nucleation is the first stage of formation of crystal and is reported to govern not only the

quality of crystals obtained, as required for structure determination but also size, shape and

polymorphic form, which is key from an industrial point of view [7]. In the past, efforts were

aimed towards developing systematic approaches to control nucleation of proteins,

employing different heterogeneous nucleation. Nucleant surfaces ranging from minerals [8]

to the porous gold [9] have been employed to control nucleation of proteins. Various control

mechanisms, ranging from surface epitaxy to surface texture, surface porosity, and specific

chemical interactions have been reported to be effective [10, 11]. To the best of our

knowledge, all reports using heterogeneous nucleants are exclusively considering the issue of

crystallisation and nucleation-enhancing surfaces for the purpose of structural determination

[12]. If we consider crystallisation as an alternative for purification, it may be not feasible

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with the surface driven nucleation mechanisms proposed for crystalogenesis aimed for

structural determination, to crystallise target molecules from a protein mixture.

Crystallisation of proteins from protein mixtures or impure protein solutions are reported

in the literature using difference in protein-protein interaction under varying solution

conditions [13], as well as empirical screening followed by spars matrix optimisation

methods to identify suitable crystallisation conditions [1, 14, 15]. Furthermore, crystallisation

of ovalbumin from frozen egg white has been reported using the industrial crystallisers at a

10kg scale [16]. However, few reports of protein crystallisation from protein mixtures as well

as fermentation broths have been reported [17], no systematic crystallisation method is

proposed. Intense multidisciplinary efforts are involved to develop understanding on protein

crystallisation and crystallisation scale up [18-23].

Recently Shah et al. employed surfaces with narrow pore size distribution to control

nucleation of proteins, and proposed a correlation between protein hydrodynamic diameter

and pore diameter to control surface preferential nucleation [24-26]. The proposed

relationship was validated by the crystallisation of seven different proteins varying in

hydrodynamic diameters ranging from approximately 4nm to 17nm and crystallising from

different crystallisation conditions. Shah et al. used nucleant which had a specific pore

diameters and narrow pore sizes, and can only be employed for crystallisation of proteins

with specific hydrodynamic diameter [24]. Due to its specificity, such nucleants

demonstrated the potential for crystallisation of proteins from a mixture of different proteins

varying in hydrodynamic sizes.

This study aims to validate the proposed correlation between protein hydrodynamic

diameter and nucleant surface pore diameter by changing the effective hydrodynamic

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diameter of the same molecule, while maintaining crystallisation condition. Experimental

validation of the relationships proposed in this study is aimed to normalise effect of

crystallisation conditions. This study also aims to utilise the relationship established between

the hydrodynamic diameter of protein molecules and surface porosity [24] to investigate

possibility of crystallisation of a target protein from a protein mixture.

2. Materials and Methods

2.1 Materials

Lysozyme, which is a well investigated protein for understanding crystallisation of

proteins was selected as a model molecule to validate the correlation reported between

protein hydrodynamic diameter and nucleant pore diameter to control crystallisation.

Pancreatic lipase, which is an enzyme found in pancreatic juices responsible for catalysing

the hydrolysis of triglycerides with a molecular weight of 46.0 kDa, and pancreatic

Ribonuclease (RNAse), which is an enzyme that catalyses the degradation of RNA with a

molecular weight of 13.7 kDa (RNAse type A), were selected as model compounds to

investigate crystallisation of target protein from the binary protein mixture. Lyophilised

lysozyme (90%), Crude (Steapsin) lipase (≥20%) which is a mixture of active lipase,

amylase and protease, and RNAse (≥70%) were purchased from Sigma Aldrich, Dorset, UK

and used without further purification. Sodium acetate (≥99%), acetic acid, hydrochloric acid

(37%), 2-methyl-1,3-propanediol (≥98%), and ammonium sulphate (≥99%) were obtained

from Sigma Aldrich, Dorset, UK, whereas, 2-amino-2-hydroxymethyl-propane-1,3-diol

(TRIS) (≥98%), and polyethylene glycol (M.W. 4000) were purchased from VWR BDH

Prolabo, Lutterworth, UK and used without further purification.

2.2 Methods

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2.2.1 Measurement of hydrodynamic diameter

Hydrodynamic diameter of the proteins was measured using Zetasizer Nano S (Malvern

Instruments Ltd., Malvern, UK), a dynamic light scattering equipment for sub-micron particle

or molecule size characterisation. Particle diameter calculated from the diffusional properties

of a molecule in a particular solvent condition is indicative of apparent diameter of dynamic

solvated particle, which represents both inclusion of solvent (hydro) and shape (dynamic)

effects. Hydrodynamics of very long or thin disk shape particles has been theoretically

extensively evaluated and models for three key parameters required to accurately calculate

hydrodynamic diameters, translational frictional co-efficient, rotational diffusion co-efficient,

and intrinsic viscosity of the medium has been presented [27]. To accurately calculate

hydrodynamic diameter from dynamic light scattering measurements for long or disk shaped

molecules (which is not the case in current study), appropriate theoretical models can be

employed.

These experiments were conducted under the crystallisation conditions for each protein

system under investigation (Table-1). Moreover, in order to find the influence of PEG on the

hydrodynamic diameter of lysozyme, 1% (w/v) and 10% (w/v) PEG were added to the

protein solutions and protein solution so prepared were mixed in equal volume with

precipitant solution and used for hydrodynamic diameter measurement. In this study, each

solution was measured at least 3 times.

2.2.2 Crystallisation of proteins

The hanging drop vapour diffusion protein crystallisation method, well documented in

the literature was used for crystallisation of all proteins [5]. Crystallisation conditions for

each protein system under investigation reported in the literature were used and can be

referred from Table-1. 3D nanotemplates with pore diameter 3.5 ± 1.0nm, 5.5 ± 1.5nm, 11.0

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± 3.0nm were used as nucleant as well as non-porous glass slide was used as a control

surface. Equal-volume droplet of protein and precipitant solution were prepared on glass

coverslips containing nucleant particles. Cover slips were carefully inverted and placed on

top of the reservoir filled with precipitant solution, pressed gently to relieve pressure and

twisted carefully to ensure proper seal. The crystallisation set up was then carefully placed in

a temperature controlled incubator and the crystallisation plates were monitored using a

microscope.

2.2.3 Preparation and characterisation of 3D nanotemplates

3D nanotemplates with specifically engineered pore diameter was selected on the

basis of hydrodynamic diameters determined for particular proteins in crystallisation

conditions and characterised by nitrogen sorption based methods for obtaining quantitative

information related to pore size and pore size distribution. Detailed methods for preparation

and characterisation of 3D nanotemplates with specific pore diameters, and narrow pore size

distribution and have been reported in the literature and can be referred elsewhere [26].

3. Results and Discussion

3.1 Validation of preferential crystallisation of proteins using 3D nanotemplates

To verify the relationship established between the nucleant surface pore diameter and a

protein’s hydrodynamic diameter, the effectively hydrodynamic diameter of lysozyme was

modified using polyethylene glycol (PEG) as a swelling agent. PEG is known to alter the

hydrodynamic diameter of a protein molecule by substitution of hydration water within the

molecules. Figure 1 shows the hydrodynamic radii measured for the lysozyme molecule in

solution as a function concentration of PEG 4000. It is evident from Figure 1 that the

hydrodynamic diameter of lysozyme increases with increasing PEG concentration over the

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studied concentration range and at given temperature. Lysozyme solutions with different

concentrations of PEG and hence with different hydrodynamic radii of protein molecule,

were used in our crystallisation studies.

Crystallisation trials of lysozyme with altered hydrodynamic radii were carried out on the

surfaces of a set of 3D nanotemplates with pore diameters 3.5 ± 1.0nm, 5.5 ± 1.5nm, and 11.0

± 3.0nm respectively. Narrow pore size distribution of different 3D nanotemplates employed

in this study is evident from Figure 2.

First crystals of lysozyme with 1% (w/v) PEG 4000 were observed on the surfaces of 3D

nanotemplates with pore diameter 5.5 ± 1.5nm whilst over similar time scale all other 3D

nanotemplate surfaces remained clear. The protein crystals were obtained on all other 3D

nanotemplate surfaces as well as non-porous soda lime glass surface within two weeks of

incubation. Macroscopic crystals of lysozyme observed on the surfaces of the 3D

nanotemplates are shown in Figure 3.

Lysozyme with 10% (w/v) PEG 4000 was observed to be first crystallising on the surfaces of

3D nanotemplates with a pore diameter of 11.0 ± 3.0nm. At similar time scales, surfaces with

3D nanotemplates with other pore diameters and non-porous glass surfaces remained clear.

Crystals on all other surfaces were observed within the experimental time. It is important to

note at this point that under similar crystallisation conditions without using PEG as swelling

agent, first crystals of lysozyme were obtained on the 3D nanotemplate surfaces with pore

diameter 3.5 ± 1.0nm.

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Addition of PEG is known to effect the crystallisation induction time, protein concentration

required for obtaining crystals, and crystal habit [28]. Control experiments were performed to

avoid any effect of PEG on the protein concentration required for obtain crystals. All the

experiments were repeated at four different protein concentrations, ranging from 2.5 mg/ mL

to 15.0 mg/ mL, while maintaining other crystallisation conditions.

At all four protein concentrations, protein solutions with 1% (w/v) PEG were found to be

preferentially crystallising on the surfaces of 3D nanotemplates with 3.5 ± 1.5nm pore

diameter, whereas protein solutions with 10% (w/v) PEG concentration were found to be

preferentially crystallisation on 3D nanotemplates with 11.0 ± 3.0nm pore diameter.

Lysozyme crystal habit observed in the current study is elongated rod shape crystal habit,

which aggress with the crystal habits previously reported for lysozyme, particularly when

PEG was used to aid crystallisation [28]. Each set of experiments was repeated at least four

times to establish experimental reproducibility.

The experimental finding of this study clearly demonstrates surface induced crystallisation of

proteins with a direct correlation between protein hydrodynamic diameter under

crystallisation conditions and nucleant pore diameter.

3.2 Application of 3D-nanotemplates for preferential crystallisation of proteins from protein mixtures

Correlation between nucleant pore diameter and protein hydrodynamic diameter, validated in

this study, was employed for selection of 3D nanotemplates required for preferential

crystallisation of target proteins, particularly from a mixture of proteins.

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To avoid the argument regarding the crystallisation of a specific protein from the mixture

being dominated by the solution conditions or protein source rather than effect of surface

porosity, two technical enzymes Lipase and RNAse were selected as model compounds

which are obtained from the same source and known to crystallise under identical solution

conditions. However, both of the proteins have different hydrodynamic diameters. To enable

the selection of appropriate pore sizes for the used 3D nanotemplates, the hydrodynamic

diameters of Lipase and RNAse under crystallisation conditions were measured and are

presented in Figure 4.

Considering the relationship between nucleant pore diameter and hydrodynamic diameter of

proteins proposed by Shah et al. [24], and validated in this study, 3D nanotemplates with pore

diameter 3.5 ± 1.0nm and 5.5 ± 1.5nm were selected as nucleants for crystallisation of

RNAse and lipase, respectively.

First crystals of RNAse were observed on the surface of 3D nanotemplates with pore

diameter 3.5 ± 1.0nm, which is the lowest 3D nanotemplate pore diameter available, after 24

hours of incubation time. Lipase was observed to crystallise on the surface of 3D

nanotemplates with pore diameter 5.5 ± 1.5nm after 48 hours of setting up of an experiment,

suggesting preferential crystallisation of the protein on the surface of 3D nanotemplates. The

solution remained clear on the surface of non-porous glass coverslips for the duration of

experiment. The crystals of lipase and RNAse obtained on the surfaces of 3D nanotemplates

are shown in Figure 5.

According to the mechanisms proposed in literature [25, 29, 30], proteins have attractive

or repulsive interaction with the surface. Charge behaviour of the proteins of interest in this

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study, lipase and RNAse, have been reported in a recent publication suggesting that both

lipase and RNAse have positively charged surfaces [31, 32]. The surface charge of the 3D

nanotemplates with hydroxyl functional group was measured and found to be negative.

Considering the opposite charge of the protein and nucleant surface, it is postulated that the

protein is attracted towards the surface and forms a high density liquid phase in the close

vicinity of the 3D nanotemplate surfaces, such high dense liquid phase is rich in solute

molecule [33].

The high dense liquid phase, which is in equilibrium with the bulk solution, is argued to be

metastable due to the attractive interaction between the protein molecule and surface

functional group. Within the high dense liquid phase, supersaturation is sufficiently high so

that the probability of every molecule to be an embryo of the crystalline phase increases

[34]. In such cases, the free energy barrier for the formation of the crystalline phase from a

high dense liquid phase is lower than the thermal energy of the molecules [35]. In the phase

diagram for nucleation of one fluid in another fluid, the line at which the thermodynamic

nucleation barrier vanishes is known as spinodal phase line. At the spinodal phase line, the

rate of generation of a new phase is limited only by slow growth kinetics of ordered clusters

from high dense liquid droplets and is independent of change in free energy [36].

The protein mixture, which has constituents crystallising from the same crystallisation

conditions, surface with narrow pore size distribution is argued to play an important role.

The pore with the diameter equivalent to the hydrodynamic size of target protein stabilises

individual molecule by limiting the possible structural fluctuations once the molecule has

entered the pore. Hence, they play a critical role in controlling structural fluctuations of target

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molecules and inducing preferential nucleation of a protein molecule from a protein mixture.

Recent experimental finding by Dey et al. on heterogeneous crystallisation of calcium

phosphate on organic monolayer strongly supports with the arguments proposed [37]. The

proteins in the high dense liquid phase in the close vicinity of the porous surface may also

enter pores via capillary rise resulting in local immobilisation. Such immobilisation results in

reduction in free energy barrier required for formation of nucleus and ultimately resulting in

nucleation.

From the existing literature it is evident that different pore attributes, i.e. pore size, shape,

and pore depth plays a crucial role in controlling protein crystallisation. Shah et al. have

demonstrated the role of pore size on nucleation of proteins, and also discussed the role of

pore depth [24]. The importance of the nucleant pore architecture on crystallisation of

proteins is also highlighted [38]. The ordered structure of the pores with diameters in the size

similar to the size of protein molecules within the high dense liquid phase is argued to

provide a template for optimised ordered arrangement of protein molecules resulting in the

formation of 2D planar arrangements. In such case, the protein molecule is argued to be

stabilised by the nanoscale surface porosity and facilitating 2D planar arrangement of

molecules. Recent experimental findings by Yau et. al. on the observation of formation and

growth of critical nuclei for apoferritin via atomic force microscopy agrees with the proposed

mechanism herein above. The apoferritin critical nuclei was found to be a 2D planar arrays of

one or two monomolecular layers having arrangement of constituent molecules identical to

the one found in the apoferritin protein crystal [39]. Figure 6 schematically depicts the role

of 3D nanotemplates in preferential crystallisation of protein from protein mixtures.

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4. Conclusions

The findings of this study provide a clear evidence of surface preferential crystallisation,

validating the correlation proposed between protein hydrodynamic diameter and nucleant

pore diameter to control crystallisation of proteins. Increasing the concentration of PEG as a

swelling agent resulted in increasing the hydrodynamic diameter of a target protein in the

crystallisation solution. Protein hydrodynamic diameter demonstrated dependence on the

concentration of swelling agent, and the pore dimension for surface preferential

crystallisation. Furthermore, the relationship between protein hydrodynamic diameter and 3D

nanotemplates pore diameter, validated in this study, was utilised for the crystallisation of a

target protein from binary protein mixture. The results reported in this study show the

potential to address the critical issue of biological macromolecule crystallisation from the

perspective of the acutely emerging need for low-cost protein purification and bio-separation.

Acknowledgments

The authors acknowledge Dr. Rajeev Dattani for assistance with hydrodynamic diameter

measurements of protein systems investigated in this study. Z. Yang acknowledges funding

from National Natural Science Foundation of China, 21474059 for this work.

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Table 1 Conditions under which the proteins are crystallised in the presence of 3D nanotemplates.

Protein Buffer Solution (Solvent water)

Precipitant Solution(Solvent Buffer Solution)

Final Protein Concentration

(mg/mL)Lysozyme 20 mM Acetate buffer

(pH=4.8) 0.5M Sodium Chloride 2.5 - 15.0

Lipase 5 mM Tris-HCl(pH = 8.1) 1.8 M Ammonium

Sulphate10% v/v MPD

50.0RNAse 25 mM Sodium acetate trihydrate, 50% v/v glycerol

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0

10

20

30

40

50

60

0 1 2 3 4 5 6 7 8 9 10

% V

olum

e

Hdrodynamic Radius (nm)

Lysozyme (w\o PEG-4000)

Lysozyme (1% PEG-4000)

Lysozyme (10% PEG-4000)

Figure 1 Effect of PEG concentration on hydrodynamic radius of lysozyme under crystallisation conditions.

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0 5 10 15 20 25 300.00

0.10

0.20

0.30

0.40

0.50

Type-IType-IIType-III

Pore Diameter (nm)

Pore

Vol

ume

(cm

³/g)

Figure 2 Pore size distribution of the 3D nanotemplates obtained using the BJH model.

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Figure 3 Lysozyme crystals with different concentration of PEG 4000 on surfaces of 3D nanotemplates (a) crystals with 1% (w/v) PEG 4000 obtained on 3D nanotemplate with pore diameter 5.5 ± 1.5nm (b) crystals with 10% (w/v) PEG 4000 obtained on 3D nanotemplate with pore diameter 11.0 ± 3.0nm (Scale bar 200μm).

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(a) (b)

0 1 2 3 4 5 6 7 8 9 100

10

20

30

40RNAse

Hydrodynamic Radius (nm)

Vol

ume

(%)

Figure 4 Hydrodynamic radius of lipase and RNAse.

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Figure 5 Crystallisation of proteins (a) lipase crystals obtained on the surface of Type-II templates (5.5±1.5nm) and (b) RNAse crystals obtained on the surface of Type-I templates (3.5±1.0nm) (Scale bar 150 μm).

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(a) (b)

dh-a

ϕ

dh-b

Selective Crystallisation, when ϕ ≈ dh-a

Figure 6 Schematic representation of the preferential crystallisation phenomenon discussed using 3D nanotemplates.

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