Project

UNIVERSITY OF PORTSMOUTH
SCHOOL OF BIOLOGICAL SCIENCES
BSc (Hons) Biochemistry
An Investigation into the Role of Temperature and
Crystal Seeding in Crystallography of Chimeric
Daphnia pulex Glycoside Family 7 Cellobiohydrolase
By
UP663120
2014/15 academic year
Supervisor: Dr J. E. McGeehan

Table of Contents
1.0 Abstract...................................................................................................................... 3
2.0 Introduction................................................................................................................ 3
2.1 Biofuels .................................................................................................................. 3
2.2 Biomass Recalcitrance............................................................................................. 5
2.3 Novel Cellulases...................................................................................................... 8
2.4 X-ray Crystallography............................................................................................ 11
2.5 Aims and Objectives .............................................................................................. 11
3.0 Materials and Methods............................................................................................... 12
3.1 Gallusgallus (chicken) Egg White Lysozyme, DpGH7, Terebellalapidaria GH9 and
Homo sapien Pro Matrix Metalloproteinase 1............................................................... 12
3.2 UV-vis Spectrophotometry..................................................................................... 12
3.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE).............. 13
3.4 Crystallisation Trials ............................................................................................. 14
3.5 Centrifugal Protein Concentration.......................................................................... 15
3.6 Temperature Optimisation of Crystallisation .......................................................... 15
3.7 Ligand Soaked DpGH7 Screen ................................................................................ 20
3.8 Seeding of Ligand Soaked Crystallisation Trials....................................................... 19
4.0 Results...................................................................................................................... 20
4.1 Spectrophotometry Analysis.................................................................................. 21
4.2 Identification of DpGH7......................................................................................... 21
4.3 Initial DpGH7 High-Throughput Crystallisation Trials ............................................. 22
4.4 Centrifugal Concentration of DpGH7 Solution ......................................................... 24
4.5 Secondary DpGH7 High-Throughput Crystallisation Trials....................................... 24
4.6 Temperature Controlled Crystallisation of Lysozyme .............................................. 24
4.7 Temperature Controlled Crystallisation of Novel Proteins. ...................................... 28
4.8 Seeding of Ligand Soaked DpGH7........................................................................... 32
5.0 Discussion................................................................................................................. 35
5.1 SDS....................................................................................................................... 36
5.2 Crystal Trials......................................................................................................... 36
5.3 Temperature Controlled Crystallisation Trials ........................................................ 37
5.4 Ligand Seeding Crystallisation Trials...................................................................... 38
6.0 Conclusions............................................................................................................... 39
6.1 Impact of Results of Biofuels Industry .............................Error! Bookmark not defined.
6.2 Further Study.................................................................Error! Bookmark not defined.
7.0 Bibliography ............................................................................................................. 41
Appendix........................................................................................................................ 51

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1.0 Abstract
Second generation biofuels have long been recognised as a potential solution for
the ever increasing worldwide energy demands. However, rate-limiting enzyme
hydrolysis steps in current industrial lignocellulosic deconstruction procedures
render the commercialisation of bioethanol cost-ineffective. For this reason
companies such as novozymes® and the National Renewable Energy Laboratories
(NREL) have focussed much of their attention towards prospecting the
environment for novel cellulases. Solving novel enzyme structures via X-ray
crystallography is crucial in cellulase research for understanding the relationship
between molecularstructure andenzymatic function. However, crystallographyis
limited to the ability to grow protein crystals suitable for X-ray diffraction. This
study explores the potential of temperature manipulation and crystal seeding in
optimising the crystallisation of chimerically expressed GH7 from the freshwater
crustacean Daphnia pulex (DpGH7) and other novel enzymes. A temperature
manipulation technique is proposed that could be extremely useful for defining
the temperature for optimum crystal size and nucleation in X-ray crystallography.
2.0 Introduction
2.1 Biofuels
Growing concerns for the ever increasing worldwide demand for energy have
fuelled the emergence of an ongoing pursuit for renewable, carbon neutral
alternatives to fossil fuels. It was reported that in 2010 fossil fuels accounted for
80.6% of the total world energy consumption and this figure has remained
significantly unchanged in recent years (Tanguay-Carel, 2015,
data.worldbank.org, 2015). The vast problems associated with the burning of
fossil fuels have been known and widely discussed for many years. The two most
predominantly recognised problems being that the worldwide use of burning
fossil fuels is both unsustainable and encompasses huge environmental impacts
with the release of greenhouse gases into the atmosphere. Moreover, it was
predicted in the annual energy outlook of 1999 that world energy consumption
would increase by as much as 54% between 2001 and 2025 (EIA. Annual energy

4
outlook 1999, 1998). Besides the discussed environmental effects, this forecast is
likely to createprogressivelyprominent economicand security anxieties formany
countries. This is due to countries such as the US that relied on the importation of
approximately 58% of its petroleum consumption from elsewhere in 2006, with a
predicted outlook of up to 70% by 2020 (Yergin, 2006). The energy dependence
of countries such as the US is a problem that has rapidly gained remarkable
political attention over the last decade.
“We’re convinced, for our own self-interest, that the way we use
energy, changing it to a more efficient fashion, is essential to our
national security, because it helps to reduce our dependence on
foreign oil, and helps us deal with some of the dangers posed by
climate change.”
– President Barack Obama
Address at the UN Climate Change Conference, Copenhagen,
Denmark, December 18, 2009
In response to these concerns, research into renewable energy through
biotechnology has been driven forward profusely on a global scale and as a result
bioethanol has now adopted the role of the most widely used liquid biofuel (Wang
et al, 2014). The production of first generation biofuels from organic materials
such as starch, oilseeds and animal fats has so far proven the most successful
conversion procedure. Bioethanol production from ethanol plants in the US has
now reached an all-time record with over 14.3 billion gallons of ethanol produced
in 2014 (2015 Ethanol Industry Outlook, 2015). However, there are many who
debate the use of agricultural produce for fuels against feeding the hunger (Pohit
et al, 2009,Ajanovic, 2010).Moreover,the potential fora long-term solution using
food ethanol as a transportation fuel has been rescinded after a publication from

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the United Nations showing an increase of 23% in agricultural commodity prices
in 2010 (United Nations, 2010).
Researchers are now showing considerable focus towards the possibility for the
commercialization of the second generation of biofuels, produced from the most
abundant biopolymer on the planet – cellulose. The potential for lignocellulosic
biomass asthe most abundant sourceoffixedcarbonhas long beenacknowledged
for over 85 years (Himmel, 2007). With waste cellulosic feedstock available from
agriculture, forestry and domestic sources, cellulosic ethanol production certainly
has the potential to displace fossil fuel consumption and significantly reduce the
affiliated concerns (Carere, 2008). There are currently as many as six pathway
technologies that have been established in which lignocellulosic biomass
conversion can be used as a source of sugars for fermentation into bioethanol
(Brown & Brown, 2013). In late 2014 two plants capable of generating over 20
million gallons peryear (mgpy)of cellulosic ethanol wereopened in Emmetsburg,
Iowa and Hugoton Kansas and plans for the opening of a 30 mgpy facility in
Nevada Iowa is set for 2015 (2015 Ethanol Industry Outlook, 2015). However,
thereare still many challenges that remain unsolvedthat render cellulosic ethanol
production cost-ineffective for long-term commercialisation. Therefore, it has
been suggested that the formulation of a cost-competitive lignocellulosic biomass
deconstructionprocedurecouldproveto further relievethe increasing worldwide
energy concerns.
2.2 Biomass Recalcitrance
Lignocellulosic biomass refers to dry plant matter composed of cellulose,
hemicellulose and lignin (Fig. 1). The primary structural component of
lignocellulose is cellulose, arranged as bundles of crystalline microfibrils (Kern et
al, 2013). Cellulose is a carbon-rich structural biopolymer that comprises up to
70% of plant cell walls (King et al, 2010). It exists as an organic polysaccharide
comprised of a linear arrangement of unbranched β-1,4-linked glucan chains
containing successive inversions of glucose residues (Taylor, 2008). This
successive inversion conformation of glucan chains in cellulose provides a strong
inter- and intra-molecular hydrogen bonding network (Nishiyama et al, 2002)

6
that gives cell wall cellulose a natural resilience to deconstruction. Its many
beneficial structural properties, including its crystallinity and insolubility can also
beheld responsibleforits vast abundance asaconservedbiomolecule synthesised
in every plant on the planet. However, it is for these very same properties that
cellulose is recalcitrant to carbonrelease (Taylor,2008).In regardsto thebiomass
conversion process, the extraordinary crystallinity of cellulose is the key feature
that inhibits enzymatic hydrolysis (Reinikainen et al, 1992). The associated cell
wall polymers of lignocellulose also pose difficulty in cellulose accessibility,
particularly lignin which has proven significantly difficult to process due to the
randomness of its bonding nature (Yinghuai, 2013). These are examples of a vast
number of complex natural mechanisms that reside within plant biomass found to
be responsible for the difficulty in biotechnical exploitation of lignocellulose. The
collective problems associated with the inaccessibility of mixed sugars from the
recalcitrant biopolymer matrix of lignocellulose has been broadly termed as
‘biomass recalcitrance’ (Chundawat et al, 2011). Lignocellulosic biomass
recalcitrance is a major concern in the effort for the commercialisation of second
generation biofuels.

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It is solely as a resultof biomass recalcitrance that many severely cost-ineffective
and rate limiting feedstock pre-treatment and enzymatic hydrolysis steps are
currently required in the biomass conversion process. The recalcitrant properties
of lignocellulose are accountable for both slow enzyme kinetics and the
requirement for thermochemical pretreatment (Himmel et al, 2007). The most
common chemical pretreatment is either acid or base combined with heat
(Chiaramonti et al, 2012, Haghighi Mood et al, 2013). However, upon
neutralisation these produce large concentrations of salt and other inhibitors,
such as carboxylic acids and phenolic compounds (Chiaramonti et al., 2012) that
significantly hinder the rate ofcellulolytic hydrolysis by halo-intolerant cellulases.
One of the foremost aspects of the current conversion process that must be
improved includes the significantly slow rate of conversion of crystalline cellulose
into fermentable sugars by currently established cellulases (Himmel et al, 2007).
However, the process by which cellulases act upon crystalline cellulose has
Figure 1: Structure of lignocellulose. The arrangement of hemicellulose and lignin
covalently bonded with cellulose microfibrils in bundle formations or ‘macrofibrils’ in
plant cell walls (image from Murphy & McCarthy, 2005).

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remained poorly understood but in recent years much research has since been
conducted in an effort to improve industrial cellulase performance. Companies
such as Novozymes and the National Renewable Energy Laboratory (NREL) have
recently given enormous attention focussed towards prospecting for novel
cellulases that may help to overcomethesechallenges andhelp to upturn the cost-
effectiveness of lignocellulosic biomass conversion.
2.3 Novel Cellulases
There is a widespread assortment of cases whereby lignocellulosic digestion has
been found to be harnessed as a source of energy by a variety of organisms. The
vast majority of such organisms that have been studied are terrestrial species
(King et al, 2010). Almost all animals observed have been found to rely on the
symbiotic relationships with microorganisms that are capable of producing many
of the required cellulolytic enzymes necessary for lignocellulosic deconstruction
(King et al, 2010).One ofthe mostcommonly studied examples is the wood-boring
termite that possesses a diverse microbial community of cellulolytic bacterial and
protist microbiota cultured within its hindgut (Warnecke et al, 2007, Todaka N, et
al, 2007). However, a small species of marine crustacean Limnoria
quadripunctata, or ‘gribble’, have recently drawn much attention due to their
unusual ability to digest lignocellulose despite their sterile digestive tract (King et
al, 2010). This would suggest that the gribble are capable of independently
translating the enzymes required for the digestion of lignocellulose. King et al
describes an expressed sequence tag (EST) transcriptome analysis of the gribble
digestive tract and collated results that shows an abundance of glycoside
hydrolase (GH) genes.These are cellobiohydrolasegenes that encodeforenzymes
responsibleforhydrolysing glycosidic linkages of crystalline cellulose into soluble
cellobiose units and glucose (King et al, 2010). It was found that 27% of
transcriptome ESTs corresponded with putative cellulase encoding genes (Fig. 2).

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This study also reported that glycoside hydrolase family 7 (GH7)
cellobiohydrolases are endogenously transcribed, a gene that had only previously
been reported in particular cellulolytic fungal species (Momeni et al, 2013) and
symbiont protists of the termite hindgut (Warnecke, 2007). These findings
instigated a successive investigation conducted by Kern et al in which they
explored the expression distribution and structural properties of the L.
quadripunctata GH7 cellulase (LqCel7B). In this study the molecular structure of
this enzyme was solved by X-ray crystallography and thus revealed that this
particular GH7 enzyme possessed a dominance of negative surface charge
residues compared with other fungal GH7 enzymes (Kern et al, 2013). It was
thought that this was most likely as a result of an evolutionary adaptation of
halotolerance to a marine environment. Enzyme assays confirmed that the
LqCel7Bcellulase remains stable and active at high salt concentrations(Kern et al,
2013). This is a feature that may render LqCel7B an extremely attractive
alternative to many currently established cellulases in lignocellulosic biomass
conversion either during or following pre-treatment steps. Further research must
be conducted in order to understand the relationship between the structural
Figure2: EST transcriptome analysis of L. quadripunctata hepatopancreas cDNA
library. (A) Pie-chart diagram showing a summary of the most common
sequences corresponding with ESTs detected in the hepatopancreas. (B)
Proportion of putative glycoside hydrolase encoding genes represented by
ESTs (others includes GH2, GH13, GH16, GH18, GH20, GH31, and GH38).
(Figure adapted from King et al, 2010)

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properties of novel cellulases and their desirable functional capabilities, such as
that of the salt-tolerance of the LqCel7b enzyme as described.
One method ofunderstanding the relationship between structureand function of
novel cellulases would be through comparison. In the case of the salt-tolerance
mechanisms of LqCel7B,comparisonwith an orthologousGH7cellulase expressed
by the well-studied freshwater crustacean relative, Daphnia pulex is an
appropriate step. Species within the genus Daphnia are characterised as
planktonic filter-feeders commonly referred to as ‘waterfleas’ (Ebert, 2005) (Fig.
3).
In recent years the Daphnia pulex became
the first crustacean to have its entire
genome sequenced and published
(Colbourne et al, 2011, wFleaBase Daphnia
Genome Project) in an effort to appoint
Daphnia as a central ecological genomic
model organism (Stollewerk, 2010). Upon
the analysis of the Daphnia genome many
putative cellulase encoding genes were
identified, including the glycoside family 7
cellobiohydrolase (DpGH7) (Colbourne et
al, 2011). Since the discovery of the DpGH7
gene, researchers at NREL have successfully
expressed the DpGH7 protein in a Trichoderma reesei expression system. This
fungal expression system typically adds a linker domain known as a carbohydrate
binding module (CBM) (Fig.4) uponcellulase expression (Chundawatetal, 2011).
This brings forward the opportunity for another angle of experimentation as the
CBM is a molecular feature that is of particular interest in current cellulase
research (Várnai et al, 2013). The CBM is a discretely folded amino acid sequence
possessing carbohydrate binding activity which is typically joined to a catalytic
domain by a flexible linker peptide (Várnai et al, 2013). The role of the CBM is to
increase the affinity of enzymes to insoluble polysaccharides, subsequently
Figure3: Daphnia pulex.
(Gewin, 2005) photographed
by Paul Hebert.

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increasing the local concentration of enzymes near the substrate surface (Bolam
et al, 2004, Kraulis et al, 1989).
2.4 X-rayCrystallography
Using X-ray crystallography as a means of solving the molecular structure of
proteins is a crucial technique in cellulase research. This has been shown in
countless studies whereby elucidating the structural properties of a novel protein
has proven paramount in obtaining interpretable three dimensional structural
data that underpins the understanding of the relationship between protein
structure and desirable enzyme properties (Kern et al, 2013).
2.5 Aims and Objectives
This paperexplores the potential of optimising crystallisation ofa novel chimeric
cellulase (DpGH7) through temperature manipulation and crystal seeding.
Temperature optimised crystallography is described with the aim of growing
suitable chimeric DpGH7 crystals for X-ray diffraction analysis and to compare
with TlGH9 and ProMMP1. Diffraction data may then be interpreted to determine
a three dimensional DpGH7 molecular structure which can thus be studied to
further understand the structural and functional properties of the halotolerance
of LqCel7B enzymes. Ligand soaks associated CBMs for lignocellulosic
Figure 4: GH family 7 cellobiohydrolase with family 1 CBM in complex with
crystalline cellulosesubstrate. The GH family 7 enzyme with a CBM attached at
the C-terminus of the catalytic domain (figure adapted from Sammond et al).

12
deconstruction. Investigations into the optimisation of protein crystallisation of a
range of novel proteins using temperature and concentration are also described
through the use various crystallography techniques.
3.0 Materials andMethods
3.1 Gallus gallus (chicken) egg white lysozyme, DpGH7, Terebella lapidaria
GH9 and Homo sapien Pro Matrix Metalloproteinase 1
The purified protein extract DpGH7 used in this study was kindly provided by
NREL under a material transfer agreement (MTA) to Dr. John McGeehan of the
University of Portsmouth, as was the Terebella lapidaria GH9 (TlGH9) from
novozymes®. Homo sapien Pro Matrix Metalloproteinase 1 (ProMMP1) was
provided by post-graduate researcher Nikul Khunti of the University of
Portsmouth. Chimeric DpGH7 was expressed using a Trichoderma reesei
expression system. TlGH9 was expressed in an Aspergillus oryzae expression
system. (ProMMP1) was expressed in E. coli BL21 (DE3) competent cells using a
DNA construct made by Dr Louise Butt of the University of Portsmouth. Chicken
eggwhite lysozyme was purchasedfromSigma-Aldrich (productnumber:L6876).
3.2 UV-vis Spectrophotometry
The Thermo Scientific NanoDrop1000 UV-vis full spectrum (220-750nm)
spectrophotometer wasused foranalysis ofproteinsolution concentrations. Prior
to each analysis the spectrophotometer was tested with distilled water to indicate
any error and blanked using the associated protein buffer. The molecular weight
and extinction coefficient of each protein (Table 1) were used for analysis.

13
Table 1: Molecular weight and extinction coefficients of novel proteins. Table
showing the molecular weights and extinction coefficients of each protein
(Lysozyme and DpGH7 obtained via the ExPASy ProtParam server, TlGH9 and
ProMMP1 obtained via post-graduate student collaborations) analysed by UV-vis
spectrophotometry.
3.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS
PAGE)
DpGH7 was run against the BenchMarkTM protein ladder (Thermo Fisher
Scientific, product no: 10747-012) using SDS-PAGE (see appendix A1) to
determine the protein molecular weight. Sequence Gel-loading dye 3x (Thermo
FisherScientific, productnumber: BP639-1)was usedto prepareDpGH7samples.
Samples werespun using anEppendorfcentrifuge5415R anddenaturedusing the
Grant QBT heat block prior to loading. In order to optimise the SDS PAGE gel
analysis ten protein samples of increasing dilutions were made up to be used for
loading (Table 2). This allowed for the best quality band to be used for elucidation
of the protein molecular weight.
Protein Molecular Weight Extinction Coefficient
Lysozyme 14.313 kDa 37,970 M-1 cm-1
DpGH7 56.688 kDa 83,770 M-1 cm-1
TlGH9 47.31 kDa 128,430 M-1 cm-1
ProMMP1 51.914 kDa 71,405 M-1 cm-1

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Table2: SDS PAGE loading sample dilutions. Tableoftendilutions usedforsamples
in the SDS-PAGE protocol for the identification of DpGH7 in the protein solution
received from NREL.
3.4
Crystallisation Trials
Vapour diffusion crystallisation was performed (Fig. 3). High-throughput JCSG-
plus HT-96 (Molecular DimensionsTM, product no: MD1-40), PACT premierHT-96
(Molecular DimensionsTM, product no: MD1-36) and Morpheus® (Molecular
DimensionsTM, product no: MD1-46) crystallisation screens were used. Screens
were prepared by pipetting 1 mL of each buffer condition from each respective kit
accordingly into a 96 well block. Blocks were sealed to prevent evaporation and
stored in the refrigerator at 4oC. For both screens, 60 µL of each condition was
transferred into the corresponding reservoir wells of a 96 well MRC
Crystallisation PlateTM (Molecular DimensionsTM – product no: MD11-00). Using
the Digilab HoneybeeTM robot 200 nL two 1:1 protein-buffer droplets were
distributed into sitting drop wells for each condition. Plates were covered with an
adhesive clear plastic cover to prevent evaporation and refrigerated at 16oC.
Screens were photographed and analysed for signs of crystallisation frequently
using the Leica M212.5 stereomicroscope. Images were taken of any significant
changes in any droplet in each of the wells and were labelled accordingly. Crystal
growth was measured using a S1-Stage Micrometer 10 mm/0.1 mm Graticule
(Pyser-SGI, product no: 02A00400).
Enzyme Volume (µL) dH2O Volume (µL) Loading DyeVolume(µL)
10 0 5
9 1 5
8 2 5
7 3 5
6 4 5
5 5 5
4 6 5
3 7 5
2 8 5
1 9 5

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3.5 Centrifugal Protein Concentration
Protein solutions were concentrated using a Sartorius stedim biotech Vivaspin
230,000 Da molecular weight cutoff polyethersulfone (PES) membrane, 2 mL
ultrafiltration spin column (product no: VSO222). Protein solutions were spun at
1500 RCF through the concentrator in the Beckman Coulter Allegra 25R
centrifuge, 60Hz 208V (product no: BP-CE-369434) and analysed using UV-vis
spectroscopy (see 2.1) at 1 minute intervals.
3.6 Temperature Optimisation of Crystallisation
Temperature controlled vapour diffusion crystallography was performed using
the CENTEO Biosciences TG40 System (Fig. 5) controlled using Centeo C3
Software for defining temperature profiles and recording data on a computer. 40
µL of each buffer condition was transferred into a single reservoir well in each of
five TG40 disposable COC inserts (Molecular DimensionsTM, product no: MD5-
141) .1 µL of protein solution was pipetted into each sitting drop well. Added to
each droplet was 1 µL of buffer transferred from each neighbouring reservoir.
Clear adhesive seals were used to seal Microplate inserts. The TG40 System was
programmed to maintain inserts at various temperatures of 281.15 K (8oC),
285.15 K (12oC), 289.15 K (16oC), 293.15 K (20oC) and 297.15 K (24oC). Images
of each crystallisation condition were captured daily using the Leica M212.5
stereomicroscope. Two images of each well were taken; one to show the entirety
ofthe well andoneto show proteincrystals at full zoom. This was in orderto allow
for the ability to both quantify the number of nucleation events and to measure
the size and rate of growth of protein crystals. The buffer conditions used for
temperature controlled crystallisation experiments are shown in Table 3 and
Table 4.

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Table 3: Lysozyme crystallisation conditions. A range of six optimum buffer
conditions for the crystallisation of lysozyme, containing sodium acetate at pH 4.8
and ethylene glycol, with six variations of salinity.
Condition Salt Buffer (0.1 M) pH Precipitant
1 1.20 M NaCl Sodium
Acetate
4.8 250 mM Ethylene Glycol
Acetate
Acetate
Acetate
Acetate
Acetate

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Table 4: Protein abbreviations, concentrations and optimised protein crystallisation conditions. Table showing novel proteins used
for experiments outlining each wild-type species, each respective concentration used and five previously optimised buffer
conditions used for the temperature controlled crystallisation of novel proteins experiment.
Condition Species Protein Concentration Salt(s) Buffer(s) pH Precipitant(s)
Lys Gallus
gallus
Lysozyme 50 mg/mL 1.71 M NaCl Sodium
Acetate
4.8 4 M Ethylene
Glycol
DpGH7i Daphnia
pulex
Glycoside
hydrolase
family 7
2.90 mg/mL None 0.8 M
Ammonium
Sulphate
4.0 100 mM
Sodium
Citrate
DpGH7ii Daphnia
pulex
Glycoside
hydrolase
family 7
2.90 mg/mL None 1 M
Ammonium
Sulphate
4.0 50 mM
Sodium
Citrate
TlGH9i Terebella
lapidaria
Glycoside
hydrolase
family 9
5.68 mg/mL 100 mM NaCl 100 mM
Tris/HCl
7.0 1 M Sodium
Citrate
TlGH9ii Terebella
lapidaria
Glycoside
hydrolase
family 9
5.68 mg/mL 200 mM NaCl 100 mM
Tris/HCl
7.0 1 M Sodium
Citrate
MMP Homo
sapien
ProMMP1*
DS HC
5.90 mg/mL 100 mM NaCl, 5mM cobalt
chloride, 5mM
nickel chloride, 5mM
magnesium chloride, 5mM
cadmium sulphate,1 mM
Calcium Chloride
20 mM
Tris/HCl,
0.1M
HEPES
7.5 12% PEG
3350

18
Figure5: Temperature controlled vapour diffusion crystallography ofnovel proteins using
the CENTEO Biosciences TG40 System. (A)The TG40 System provides five rows of eight
wells in a temperature controlled microplate. Each column represents a specific protein
in a specific condition (Table 4). (B) Sitting drop vapourdiffusion system of a disposable
TG40 insert. (C) Schematic diagram illustrating the principle of vapour diffusion
crystallography. In an enclosed vapour diffusion crystallography chamber there is a
reservoir solution and a raised pedestal supporting a droplet of protein and reservoir
solution, usually at a 1:1 ratio. The reservoir solution contains salt, a buffer and one or
more precipitant species at a specific pH. As the solute concentration of the reservoir
solution is much higher than that of the protein droplet, the reservoir solution will have
a lower vapour pressure. The difference in vapour pressure initiates a net transfer of
water through the vapour phase from the protein droplet to the reservoir to maintain
equilibrium. The loss of water in the protein droplet causes the concentrations of both
protein and precipitant to increase thus saturating the protein pushing it past the
metastable point to nucleation (Forsythe et al, 2002). (D) Disposable TG40 Microplate
insert that provides eight vapour diffusion systems for a single row of the TG40
Microplate.

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3.8 Seeding of LigandSoaked Crystallisation Trials
Crystals for seeding were fished from the protein droplet using a loop and
transferred into a 2 µL drop of reservoir solution (Fig. 7). Crystals were
homogenised by agitating the protein crystals using the loop until sufficiently
small size crystal seeds were produced. Seeding was performed in accordance
with Figure 7.
Figure 7: DpGH7 Crystal Seeding. Schematic diagram illustrating the crystal
seeding procedure performed on ligand soaked DpGH7 crystallisation trials.
Crystal seeds were transferred from the reservoir solution drop by
submerging the loop amongst the crystal seeds (left) and subsequently
submerging the loop containing seeds into the ligand soaked crystallisation
protein drop, swiping through the drop to release the seeds (right).

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3.7 Ligand Soaked DpGH7 Screen
LigandsoakedDpGH7crystallisation trials were performedusingtwo previously
accomplished buffers from crystal screening and optimisation, with five different
cellulose ligands and two concentrations of DpGH7 protein (Fig. 6).
Figure6: LigandSoaked DpGH7 Crystallisation Trials. Diagram showing the layout of a 24-
well XRL Plate (Molecular DimensionsTM – product no: MD3-11) with columns labelled
with proteinconcentration, wells labelled with cellulose ligand andbuffercondition(Table
4). Ligands include cellobiose (Sigma-Aldrich – product no: C7252), cellotriose (Sigma-
Aldrich – product no: C1167), cellotetraose (Sigma-Aldrich – product no: C8286),
cellohexose (Carbosynth – product no: OC16452) and celloheptaose (Carbosynth –
product no: OC05241). Cellulose ligands were at a 5x molar excess. Protein drops were at
a 1:1 ratio. Ligand soaked DpGH7 crystallisation trials were prepared by post-graduate
researcher Ali Ebrahim of the University of Portsmouth on 11/12/2014.

21
4.0 Results
4.2 Identificationof DpGH7
The protein solution received from NREL was run against the BenchmarkTM
protein ladder to find the protein molecular weight (see section 2.1). The results
of this experiment have been interpreted from the observations of the SDS PAGE
gel shown in Fig. 8. It can be seen that there is a contaminating band of low
intensity observed at approximately 17 kDa.
4.1 Spectrophotometry Analysis
All protein concentrations were analysed using UV-vis spectrophotometry (see
section 2.1) prior to all following experiments (Table 5).
Table 5. Protein concentrations. Table of measured concentrations of novel
proteins used for subsequent experimentation.
MolecularWeight(kDa)
Volume of DpGH7 solution (μL)
Figure 8: SDS PAGE gel analysis of DpGH7 molecular weight. Gel showing wells
containing the BenchmarkTM protein ladder with bands representing peptides
of a defined molecular weight labelled (far left) and successive dilutions of the
DpGH7 protein solution. Individual bands are observed for wells with
decreasing intensity for each dilution. The approximate protein molecular
weight of 60 kDa was derived from furthermost side of the band from the well
containing 1 µL of protein.

22
4.3 Initial DpGH7 High-Throughput CrystallisationTrials
High-throughput JCSG and PACT crystallisation screens were initially
interrogated with the intention of elucidating any buffer environments that
provide the conditions for successful crystallisation of DpGH7 (see section 2.2).
After eight weeks of observation there was no sign of crystallisation in the JCSG
screen. Nevertheless, protein drops did exhibit certain characteristics that may be
interpreted to give indications of the reactions between the DpGH7 protein and
each condition (see appendix A4.1). On the other hand, crystal formation was
Protein Concentration
Lysozyme 50 mg/mL
Glycoside hydrolase family 7 2.90 mg/mL
Glycoside hydrolase family 9 5.68 mg/mL
ProMMP1* DS HC 5.90 mg/mL

23
observed in condition E1 of the PACT screen (see appendix A4.2). Crystal
nucleation was first observed on day 7 of crystallisation trials in both protein
droplets (Fig. 9). High-throughput crystallisation trials did not produce crystals
suitable for analysis in any other conditions.
Figure 9: Crystal growth in two protein drops of PACT premier HT-96 condition E1.
Uppermost images show the crystal growth in one protein droplet over 49 days; the
crystal on day 7 (circled in green) was measured using a graticule at 23 µm length by
11 µm width and the crystal on day 56 (circled in red) was measured at 75 µm length
by 45 µm. Images below show the crystal growth in the second protein droplet over
49 days; the crystal on day 7 (circled in orange) was measured using a graticule at 88
µm length by 11.5 µm width and the crystal on day 56 (circled in blue) was measured
at 117.5 µm length by 50 µm.

24
4.4 Centrifugal Concentration of DpGH7 Solution
In an attempt to optimise high-throughput crystallisation trials, the DpGH7
protein solution was concentrated using centrifugal concentration (see section
2.5). The DpGH7 protein was concentrated from 2.90 mg/mL to 6.48 mg/mL after
5 minutes of centrifugation (Table 6) and was not concentrated furtherto prevent
a significant loss of protein.
Table 6: Centrifugal concentration of DpGH7 solution. Table of the measured
concentrations of DpGH7 at 1 minute intervals during centrifugal concentration.
It can be seen that with each protein concentration, concentration becomes less
effective. Concentration was stopped at 5 minutes as the DpGH7 solution was only
concentrated by 0.17 mg/mL, significantly less than prior concentration.
Time (minutes) Concentration (mg/mL)
0 2.90
1 4.14
2 5.18
3 5.87
4 6.31
5 6.48
4.5 SecondaryDpGH7 High-Throughput CrystallisationTrials
High-throughput crystallisation screening was performed once more using both
concentrated (6.48 mg/mL) and 2.90 mg/mL DpGH7 protein in an attempt to
optimise crystallisation (see section 2.2). After six weeks there was no
crystallisation observed in any condition (see appendix).
4.6 Temperature Controlled Crystallisation of Lysozyme
Crystallisation of lysozyme using the TG40 system (see section 2.6) was initially
performed in order to provide a control to compare against in following
temperaturecontrolled crystallisation experimentsof novelproteins, as lysozyme
has a tendency to grow many good quality crystals over a short period of time
(Durbin et al, 1986). Temperature controlled crystallisation of lysozyme trials
were monitored over eight days (Fig. 11). It can be seen in each condition that net

25
crystal growth was insignificant over eight days. There is a noticeable trend in
lysozyme nucleation and crystal growth according to temperature observed in all
conditions, representedbycondition 4 in Figure10. It can beseen that the number
of lysozyme crystals was reduced significantly with an increase in temperature.
The average size of lysozyme crystals had increased relative to the increase in
temperature between 8-16oC. At 20oC crystals were observed to have grown with
noticeably more disordered morphologies and at 24oC there was no crystal
growth observed.
Figure10: Crystallisation of Lysozymeat Increasing Temperatures in Condition4.
Series of images adapted from Figure 11 showing crystal growth morphologies
at increasing temperatures. No crystallisation was observed at 24oC.

26
Figure 11: Lysozyme Crystal Morphology at Different Temperatures. Wells at full zoom (except those with no crystallisation)
showing crystal morphology at day 1 and day 8 for six conditions (Table 3) at different temperatures and salinity.

28
4.7 Temperature controlled crystallisation of novelproteins.
Temperature controlled crystallography of DpGH7, TlGH9, ProMMP1 and
lysozyme using the TG40 system (see section 2.6) was performed. TlGH9 and
ProMMP1 were implemented for comparison of DpGH7 crystallisation with a
similar GH cellulase without a CBM (TlGH9). Similarly to lysozyme, ProMMP1 was
used to provide a further control and elucidate any difference in the effect of
temperature on crystallisation between proteins with different structure and
function. Protein droplets were monitored over eleven days (Fig. 12).
For condition Lys it can be seen that lysozyme crystal growth was apparent in
each droplet and had varied significantly at different temperatures. Lysozyme
crystals grown at 8oC were in abundance (~70 crystals) by day 3 and after eleven
days crystals of up to 73 µm in length with highly ordered crystal morphology
were observed. Many crystals appear to have grown from common nucleation
points causing some crystal cluster formation with overlapping crystal growth.
Lysozyme crystallisation at 12oC, 16oC and 20oC had all produced an enormous
abundance of lysozyme crystals no larger than 7.5 µm in length. All three
conditions contained some individual crystals however the majority of
crystallisation was clustered throughout each droplet. Four similarly large
lysozyme crystals were produced at 24oC, the largest measuring 262 µm. The net
growth of lysozyme crystals between day 3 and day 11 was insignificant in each
condition.
There was no crystallisation observed for condition DpGH7i at 8oC and 12oC. No
crystallisation was also observed at 16oC however a minute spherical structure
had appeared by day 11. On day 3 signs of crystallisation had appeared at 20oC.
This was observedasatranslucent structurewith ahighly disordered morphology
and remained unchanged by day 11 showing no observable growth.
Crystallography was unobservable for DpGH7i at 24oC due to excessive
condensation.
No signs of crystallisation were observed for condition DpGH7ii at temperatures
between 8-20oC. Crystallography was unobservable for DpGH7ii at 24oC due to
excessive condensation.

29
In conditions TlGH9i and TlGH9ii there was no crystallisation observed at any
temperature.However, liquid-liquid phaseseparationwasapparentin all droplets
and varied with temperature. For TlGH9i on day 11, the phase separation
observed at 8oC appeared as an ordered arrangement of few large separation
drops positioned on the edge of the protein droplet with many smaller drops
proceeding away from them. The phase separation observed at 12oC had
comparable features but separation drops appear larger, more prominent and
more ordered. At temperatures 16-24oC the phase separation was significantly
lesser and more disordered. For TlGH9ii phase separation showed the same trend
as described for TlGH9i however with greater separation at each temperature. A
large, translucent helical structurewas observedonday3 at 12oCbut showednow
significant growth by day 11.
For condition MMP there was also no crystallisation observed at any
temperature. A white precipitate was observed at each temperature on day 3,
however droplets showed no significant change by day 11.

30
Figure 12: Novel Protein Crystallography at Different Temperatures. Images of wells at full zoom (except those with no
crystallisation) showing the difference in crystal morphology at day 3 and day 11 for six various novel protein conditions at
different temperatures.

32
4.8 Seeding of ligand soaked DpGH7
Ligand soaks were performed with the aim of crystallising chimeric DpGH7 in
complex with a cellulose ligand to be used for X-ray diffraction analysis (see
section 3.7). After two months of incubation all protein droplets remained clear
and no crystallisation was observed in any condition.
Crystal seeding was subsequently performed on unsuccessful ligand soaked
DpGH7 crystallisation trials in an attempt to induce crystallisation. DpGH7
crystals, previously grown in condition DpGH7ii (Table 4) by postgraduate
researcher Ali Ebrahim, were provided for seeding (see section 2.8) (Fig. 13).
SeededDpGH7ligand soaksweremonitoredover19days. It canbe seenin Figure
14 that crystal growth (Fig. 15) was observed in 21 of 24 crystallisation
conditions, of which crystals that may be suitable for X-ray diffraction analysis
were obtained in four conditions (Fig. 16). Crystal growth in positive conditions
was apparent at day 2 and rapid growth was observed until day 13 whereafter
crystal growth had ceased. The crystal growth observed in seeded ligand soaked
DpGH7 crystallisation trials is represented by the example in Figure 14,
highlighting the extentofcrystal growth oversixdaysand showinga linear growth
pattern across the centre of the droplet. Crystals that may be suitable for X-ray
analysis, shown in Figure 16, were chosen as they were over 50 µm in length and
Figure13: DpGH7Crystal Seeds. The DpGH7crystals transferred from the initial
plate well into a reservoir solution drop to be homogenised and used for
seeding. Image shows the reservoir droplet containing DpGH7 crystals (left)
and a magnified view of crystals (right).

33
were not significantly clustered. All crystals grew in thin plate morphology and
most formed in small crystal clusters.

34
Figure 15: Crystal Growth In Seeded 7 mg/mL DpGH7i Cellotriose Soak. Crystal
nucleation is apparent one day following seeding (day 2) and it can be seen that
crystal growth is significantly prominent by day 6.
Figure14: Ligand Soaked DpGH7 Seeding Results. Diagram of ligand soaked DpGH7
crystallisation trials (Fig. 6) showing the conditions in which it was observed that
DpGH7 crystal seeding had initiated significant crystal growth (orange) and
conditions that produced crystals suitable for X-ray diffraction analysis (green).
There was no significant crystal growth observed in conditions coloured white.

35
5.0 Discussion
The commercialisation of second generation biofuels can only become a reality if
rate-limiting enzyme hydrolysis steps are improved in industrial lignocellulosic
deconstruction procedures Prospecting the marine environment for novel
cellulases is a current strategy for tackling this issue and has identified LqCel7B
with halotolerant properties that may assist the formulation of a cost-efficient
process. To understand the structural halo-tolerant properties of LqCel7B a
comparison with the structure of the freshwater DpGH7 cellulase is appropriate.
The three dimensional structure analysis of novel cellulases through X-ray
Figure16: Sevensuitable crystalsforX-ray diffractioninseededligand soaked DpGH7.
There were (A) three crystals formed in 3 mg/mL DpGH7i cellohexose, (B) one
crystal formed in 3 mg/mL DpGH7 cellotriose, (C) one crystal formed in DpGH7ii (3
mg/mL DpGH7) no ligand (protein only). (D) and two crystals formed in DpGH7ii (7
mg/mL DpGH7) no ligand (protein only).

36
crystallography is key to allow for a structural platform for protein engineering of
s. For this reason the optimisation of DpGH7 crystallisation through temperature
and crystal seeding described in this paper could help to provide DpGH7 crystals
for 3D structure determination needed for the comparison through homology
modelling.
5.1 SDS
It can be seen that the sample dilutions have proved successful in optimisating
the interpretation of gel (Fig. 8). The well containing the least amount of protein
(1 µL) has produced the optimum results where the interpretation of molecular
weight is significantly more precise compared to overloaded wells (3-10 µL). The
interpreted molecular weight of approximately 60 kDa is close to the actual
molecular weight of DpGH7 (56.688 kDa), suggesting that DpGH7 is present
however there may be an unknown factor that is impeding the migration of
protein across the gel. This exaggerated molecular weight may be the result of
DpGH7 glycosylation as it has been known that fungal GH7 enzymes are
susceptible to post translational glycosylation (Boer et al, 2000). As there is only
one band of significant intensity that was observed on the gel it can be assumed
that the DpGH7 solution has a high purity. However, the presence of a low
intensity band at approximately 17 kDa shows that there may be a small amount
of contamination in the DpGH7 protein solution.
5.2 Crystal Trials
Interrogation of high-throughput crystallisation trials had elucidated a single
condition (condition E1 of the PACT-premier screen) in which nucleation of
crystallisation had occurred. However, it is unclear to whether or not the crystals
grown are DpGH7 crystals as salt crystal formation is a common incidence in most
crystallography (Luft et al, 2011). This may have been determined through X-ray
diffraction analysis however as trials only produced crystallisation in one
condition it was decided that optimisation of crystallisation would be attempted
through protein concentration. High-throughput crystallisation trials using
concentrated DpGH7 did not however optimise crystallisation as there was no
crystallisation observed in any condition. Trials may have required a longer

37
crystallisation period as protein crystallography can take a long time before
showing any positive results (Luft et al, 2011).
5.3 Temperature Controlled Crystallisation Trials
Temperature optimisation of crystallising chimeric DpGH7, TlGH9 and ProMMP1
was unsuccessful in all conditions as no crystallisation was observed after
nineteen days. Therefore, the aim of optimising the crystallisation of chimeric
DpGH7 to compare with TlGH9 and ProMMP1 has not been achieved. This was
likely dueto the time limitations in which crystallisation trials had been conducted
as protein crystallization is known to be an unpredictable process that can extend
over a long period of time before showing any positive results (Luft et al, 2011).
Additionally, there were positive signs of crystallisation observed for TlGH7 as
liquid-liquid phase separation was observed in all conditions. The presence of
liquid-liquid phase separation shows high-levels of protein supersaturation that
has been suggested to provide an interface effect with the immiscible low-protein
concentration surroundings that promotes initiation of crystal nucleation (Haas &
Drenth, 2000). From this it can be assumed that observation of temperature
controlled crystallisation of proteins over a longer time period may have been
required in order to allow for crystal nucleation to occur.
On the other hand, the significance of temperature manipulation on protein
crystallisation was clearly demonstrated in the temperature controlled
crystallisation oflysozyme. It was shownthat lysozyme nucleation events reduced
significantly with an increase in temperature allowing for the growth of larger
crystals. This trend suggests that the temperature of crystallisation is a factor that
directly affects the amount of nucleation events that occur within the protein
droplet. It can also be seen that the crystallisation of lysozyme has a temperature
thresholdat the optimum of 16oCwhereaftercrystal growth morphologybecomes
more disordered at 20oC until no crystallisation observed at 24oC. However, this
trend is not consistent with the results of the lysozyme control in the temperature
controlled crystallisation of novel proteins experiment. In order to gain more
reliable results temperature controlled crystallography must be repeated.
Nevertheless, these results indicate that a similar temperature manipulation trial
procedure can be applied for finding the temperature for optimal crystal size and

38
abundance in protein crystallography. This is a technique that could prove
extremely useful for defining the optimum temperature for crystallising specific
proteins thus improving crystallography efficiency for producing diffraction-
quality crystals.
5.4 Ligand Seeding Crystallisation Trials
The attempt to instigate crystallisation in cellulose ligand soaked chimeric
DpGH7 crystallisation trials has proven to be extremely successful, with rapid
crystal growth observed in 87.5% of seeded conditions and seven crystals
obtainedthat may be suitable for X-ray diffraction. However,due to the lack ofany
controlconditions it could bearguedthat the crystallisation observedaftercrystal
seeding may have been due to other extraneous variables, such as contamination.
Nevertheless, it can be seen in Figure 14 that the crystal growth has grown in
accordance with the action of crystal seeding suggesting that crystal enlargement
has nucleated from crystal seeds. This is evidence to show that the crystal seeding
is in fact accountable for successful optimisation of ligand soak crystallisation
trials.
It is unclear to whether the crystallisation observed was indeed chimeric DpGH7
in complex with the cellulose ligand without 3D structural determination through
X-ray diffraction analysis. However, due to the thin plate morphology of crystals
obtained (Fig. 16), crystals may prove to be of poor diffraction quality. Given that
protein crystals are damaged by X-ray radiation (Teng & Moffat, 2000), there is a
high probability of crystal deterioration under a high intensity X-ray beam during
diffraction dueto this morphology,compared with high quality three-dimensional
crystals such as those grown for lysozyme (Fig. 10). Moreover, ligand soaked
crystallisation trials were carried out for over two months prior to crystal seeding
meaning that it is possible that significant ligand deterioration may have occurred
causing uncertainty in the protein ligand-crystallisation. The crystals are also all
relatively small in size (ranging between 50-100 µm) and therefore require an X-
ray beam of higher quality than available using the in-house X-ray diffractometer.
Crystals may be transported to a synchrotron, such as the Diamond Light Source
facility, for diffraction analysis on a high quality X-ray beamline in an attempt to
obtain a high resolution 3D structure. However, this may be undesirable due to

39
the poor quality of crystals and the uncertainty of protein-ligand crystallisation.
Therefore, performing further seeding experimentation to obtain better quality
crystals for diffraction analysis would be appropriate for future study. New ligand
soak trials prepared with further fine-tuned crystallisation conditions may be
required to produce diffraction-quality protein-ligand crystals.
6.0 Conclusions
 Temperature controlled crystallography has not proven successful in
optimising the crystallisation of chimeric DpGH7, TlGH9 or MMP1 as no
crystallisation was observed in any condition.
 Temperature manipulation has established potential as a technique that could
be extremely useful for defining the temperature for optimum crystal size and
nucleation when crystallising specific proteins in attempts at producing
diffraction-quality crystals for high-resolution structure determination by X-
ray crystallography.
 Crystal seeding of ligand soaked chimeric DpGH7 crystallisation trials has
successfully instigated an abundance of crystal nucleation in 87.5% of
conditions. However, crystals that were obtained were relatively small and of
thin plate morphology that renders them poor quality for X-ray diffraction.
6.1 Further Study
To obtain high quality diffracting chimeric DpGH7 protein crystals further
optimisation of the experiments described in this paper must be carried out. Fine-
tuning optimisation of the crystallisation buffer conditions would improve the
chances of crystallisation (Luft et al, 2011). Upon the production of a high quality
chimeric DpGH7 crystals high-resolution structure determination by X-ray
diffraction can be performed. The three dimensional structure of DpGH7 and CBM
can be studies through molecular simulation studies and homology modelling to
understanding relationship between structure and function. Comparison with the
crystal structure of LqCel7B could subsequently be conducted to investigate the
structural properties of halotolerance to apply in cellulase engineering.

40
Acknowledgements
I would firstly like to thank postgraduate researcher Ali Ebrahim for his
invaluable support and encouragement throughout my project. I would also like
to thank John McGeehan for who provided the advice and support that I needed to
complete this write up.
Word Count: 5,487
Declaration
I declare that the work submitted here is my own. Any material used from other
sources (e.g. data, ideas, images) is clearly identified as such with the author(s)
of the material cited.
Signed:
Date:

41
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Appendix
A1 SDS PAGE
A1.1 Polyacrylamide Gel Recipes. Table containing recipes of resolving and
stacking gels used in SDS PAGE.
Resolving Gel Stacking Gel
- 3.35 mL ProtoGel [30% (w/v) Acrylamide:
0.8% (w/v) Bis-Acrylamide stock solution
(37.2:1). Protein and sequencing
electrophoresis grade (gas stabilised)]
- 2.00 mL ProtoGel Buffer [1.5 M Tris-HCl,
0.384% SDS, pH 8.8]
- 2.60 mL dH2O
- 45 µL 10% APS w/v
- 5 µL TEMED [(N, N, N’, N’-
tetramethylethylenedianine) Electrophoresis
grade]
- 0.75 mL ProtoGel
- 1.25 mL ProtoGel Stacking Buffer
[0.5 M Tris-HCl, 0.4% SDS, pH 6.8]
- 3.00 mL dH2O
- 30 µL 10% APS w/v
- 10 µL TEMED
A2.2 Buffer Recipes. Table containing recipes for both loading and running
buffers used in SDS PAGE.
50 ml Loading Buffer (LB)
1 L 10x Tris-Glycerine SDS Running
Buffer pH 8.6
- 9% SDS (4.5 g)
- 15% Mercaptoethanol (7.5 mL)
- 15% w/v Glycerol (4.5 g)
- 0.03% Bromophenol Blue (15 mg)
- 375 mM Tris-HCl pH 6.8 [1 M] (18.75 mL)
- Diluted to 50 mL with dH2O
- 0.25 M Tris (30.3 g)
- 1.92 M Glycine (144 g)
- 1% SDS (10 g)

52
A3. CrystallisationScreen Conditions.
A3.1 JCSG-plus HT-96. Table of salt, buffer and precipitate conditions for each
well.

54
A3.2 PACT premier HT-96. Table of salt, buffer and precipitate conditions for
each well.

56
A3.3 Morpheus®. Table of ligand, salt, buffer and precipitate conditions for each
well.

58
A6. Crystallography
A5.1 Characterising Protein Drops in Crystallisation. Series of images showing varied
examples of protein drop analysis from DpGH7 crystallisation trials data.
Observational characterisations of protein drops during crystallisation trials shows
examples of (A-F) Liquid-liquid phase separation (G) Skin formation. (H-J)
Contamination. (K-L) Denaturation. (Luft et al, 2011)

A7.1 Temperature Controlled Crystallography of Lysozyme Condition 1

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