laboratory communications
Acta Cryst. (2011). F67, 1015–1021 doi:10.1107/S1744309111028776 1015
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
The Protein Maker: an automated system for high-
throughput parallel purification
Eric R. Smith,a,b Darren W.
Begley,a,b Vanessa Anderson,a,b
Amy C. Raymond,a,b Taryn E.
Haffner,a,b John I. Robinson,b
Thomas E. Edwards,a,b Natalie
Duncan,c Cory J. Gerdts,c
Mark B. Mixon,a,b Peter Nollert,b
Bart L. Stakera,b and Lance J.
Stewarta,b,c*
aSeattle Structural Genomics Center for
Infectious Disease (http://www.ssgcid.org),
USA, bEmerald BioStructures, 7869 NE Day
Road West, Bainbridge Island, WA 98110, USA,
and cEmerald BioSystems, 7869 NE Day Road
West, Bainbridge Island, WA 98110, USA
Correspondence e-mail: lstewart@embios.com
Received 2 February 2011
Accepted 17 July 2011
The Protein Maker is an automated purification system developed by Emerald
BioSystems for high-throughput parallel purification of proteins and antibodies.
This instrument allows multiple load, wash and elution buffers to be used in
parallel along independent lines for up to 24 individual samples. To demonstrate
its utility, its use in the purification of five recombinant PB2 C-terminal domains
from various subtypes of the influenza A virus is described. Three of these
constructs crystallized and one diffracted X-rays to sufficient resolution for
structure determination and deposition in the Protein Data Bank. Methods for
screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to
upscaling expression and purification are also described. The Protein Maker
has become a valuable asset within the Seattle Structural Genomics Center for
Infectious Disease (SSGCID) and hence is a potentially valuable tool for a
variety of high-throughput protein-purification applications.
1. Introduction
The end goal of structural genomics is the rapid generation of three-
dimensional structures obtained from atomic level resolution studies
of pure proteins (Xiao et al., 2010; Elsliger et al., 2010; Watson et al.,
2007; Bonanno et al., 2005). Therefore, purifying protein targets in the
shortest amount of time maximizes the number of potential candi-
dates for further processing. Gravity-based, high-pressure and ‘fast
protein’ liquid-chromatography (HPLC and FPLC) methods have
been implemented on various scales to purify individual targets.
Although suitable for a research environment (Walls et al., 2011),
most standard instrumentation does not allow parallel purification
or testing to investigate a large number of purification conditions
simultaneously. To address this critical need on a structural genomics
scale, much research and development has gone into the creation of
pipelines designed to deliver the necessary high-quality materials at
a rapid pace (Kim et al., 2008; Cymborowski et al., 2010; Stols et al.,
2002; Dieckman et al., 2002; Steen et al., 2006). To facilitate rapid
purification of proteins for structural genomics, we have developed
the Protein Maker, a high-throughput parallel liquid-chromatography
system that is capable of purifying up to 24 protein targets in a single
unattended run (Fig. 1). The result is the purification of multiple
targets with identical or individualized buffer systems, decreasing the
time required for purification while potentially increasing protein
yields through time-efficient buffer optimization.
The Seattle Structural Genomics Center for Infectious Disease
(SSGCID) has a mandate to generate approximately 100 protein
structures each year from a variety of Category A, B and C patho-
genic organisms, as listed by the National Institute of Allergy and
Infectious Diseases (NIAID; Van Voorhis et al., 2009; Myler et al.,
2009). The targets selected for study are proven or putative points of
therapeutic intervention and include a high-value subset of proteins
requested by external scientists from the infectious disease research
community. Multiple sequence variants are queued for most targets
to increase the likelihood that at least one gene product will express,
purify and become suitable for study by NMR spectroscopy or X-ray

laboratory communications
1016 Smith et al.  The Protein Maker Acta Cryst. (2011). F67, 1015–1021
crystallography. In this communication, we introduce the Protein
Maker and describe its use in purifying five C-terminal domain
subunits of the polymerase basic protein 2 (PB2) from two different
subtypes of influenza virus. Specific C-terminal domain mutations in
the PB2 component of the viral polymerase heterotrimer have been
associated with different levels of human virulence, giving structural
characterization of this protein a potentially important role in the
prevention of future influenza pandemics (Guilligay et al., 2008;
Subbarao et al., 1993; Tarendeau et al., 2008; Yamada et al., 2010). All
five C-terminal domain PB2 (CPB2) variants were purified in a single
run of the Protein Maker and three of them crystallized, yielding one
crystal system with sufficient quality for structure determination by
X-ray diffraction and subsequent deposition in the Protein Data
Bank (PDB; Berman et al., 2000, 2003). Another application of the
Protein Maker is in scouting for optimal cell-lysis buffer conditions,
an earlier step in the pipeline used to maximize protein yields during
scale-up procedures. This is of particular value for proteins which
exhibit high levels of expression but appear to be insoluble under
standard lysis-buffer conditions. In this report, we also detail scouting
experiments in which a single batch of cells was split into 12 pools and
lysed by sonication in 12 different buffer conditions, followed by
parallel purification with the Protein Maker. The protein in this case
was cytochrome P450 51 A1 (CYP51A1) from Coccidioides immitis,
the organism which causes coccidioidomycosis or San Juan Valley
fever (Crum et al., 2004; Hector & Laniado-Laborin, 2005; Galgiani,
1999). Both studies highlight the advantages in using a parallel
purification platform such as the Protein Maker in a high-throughput
structural genomics pipeline.
2. Experimental methods
2.1. Expression and purification of PB2 viral polymerase subunits
Five constructs were designed spanning the C-terminal domain of
influenza virus A polymerase basic protein 2 (CPB2), one component
of the heterotrimeric viral polymerase. These CPB2 constructs were
derived from two genetic sources: an H3N2 swine-flu subtype isolated
in Japan and an H7N7 equine subtype originating in the Czech
Republic (see Table 1). Each of the five constructs was cloned into
a modified pET28 vector system engineered to donate an amino-
Figure 1
The Protein Maker instrument with syringe valves, liquid-handling sample manifold and deep-well plate deck (a). In the depicted configuration, 15 of the 24 syringe valves
are at the front, with the remaining nine at the back (not shown). Also depicted is a schematic drawing of the plumbing for each individual nine-port valve (b) and a close-up
image of the sample-load and primary purification manifolds with 24  1.0 ml purification columns in place (c). All 24 valves are individually lined and independently
operated, thus allowing up to 24 sample-uptake lines and purification columns in a single run.

terminal 6His-Smt fusion tag (Mossessova & Lima, 2000) and a
site-specific protease cleavage site to the open reading frame using
Polymerase Incomplete Primer Extension (PIPE) cloning (Lorimer et
al., 2009). The N-terminal sequence of all five gene products was
MSHHHHHHSGEVKPEVKPETHINLKVSDGSSEIFFKIKKTTP-
LRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDME-
DNDIIEAHREQIGGS followed by the first amino acid of the
desired influenza Aviral protein construct. Each clone was expressed
in Escherichia coli BL21 (DE3) cells grown in autoinduction medium
(Terrific Broth plus Novagen Overnight Express System 1) using a
LEX bioreactor at 293 K for approximately 65 h. Cells were collected
by centrifugation at a relative centrifugal force (RCF) of 32 960g. The
supernatant was decanted and the cell paste was flash-frozen in liquid
nitrogen prior to storage at 193 K. The cell paste for each CPB2
protein was then thawed and resuspended in lysis buffer at a 1:5
mass:volume ratio. The lysis buffer (500 ml) consisted of 25 mM Tris
pH 8.0, 200 mM NaCl, 50 mM arginine, 10 mM imidazole, 0.02%
CHAPS, 0.5% glycerol, 1 mM Tris(2-carboxyethyl)phosphine
(TCEP), 100 mg lysozyme, 500 U Benzonase and one Complete
Protease Inhibitor Cocktail tablet (Roche). The cells were resus-
pended by vigorous stirring for 30 min at 277 K and mechanically
lysed on ice using a Misonix sonicator (70% power, 2 s on/1 s off
pulses, 3 min total). The crude lysate was clarified immediately after
sonication by centrifugation at 18 000g RCF for 35 min at 277 K. The
clarified supernatant was decanted and stored at 277 K prior to
purification with the Protein Maker.
All clarified lysate samples were brought to 105.0 ml by addition of
buffer A (25 mM Tris pH 8.0, 200 mM NaCl, 50 mM arginine, 10 mM
imidazole, 0.25% glycerol and 1 mM TCEP). One 5.0 ml HisTrap FF
nickel-chelate column (GE Healthcare) for each CPB2 sample was
attached to a separate line on the gantry of the Protein Maker, with
the following steps conducted in parallel for all samples. Each puri-
fication step was conducted in 5.0 ml volumetric increments, utilizing
a flow rate of 1.5 ml min1. For the first run of the Protein Maker
(‘Nickel 1’), each column was first washed with 20.0 ml Milli-Q water
to remove the ethanol-based storage buffer. Each column was then
washed with 5.0 ml buffer B (25 mM Tris pH 8.0, 200 mM NaCl,
500 mM imidazole and 1 mM TCEP) followed by equilibration with
25.0 ml buffer A. Resuspended cell lysates were then loaded onto
individual columns followed by a 15.0 ml wash with buffer A. Each
protein sample was then eluted in three steps: 5.0 ml at 30 mM
imidazole, 5.0 ml at 206 mM imidazole and 10.0 ml at 500 mM
imidazole, using 96:4, 60:40 and 0:100 ratios of buffer A:buffer B,
respectively. These buffers were drawn from separate individual
stocks and actively mixed in the syringe valve prior to column loading
(Fig. 1). Elution fractions were collected separately in deep wells of a
Whatman 24-well plate positioned on the deck of the Protein Maker.
After the final elution step, all columns were re-equilibrated with
100.0 ml buffer A as a temporary storage condition. Elution fractions
were tested for the presence of protein using 4–12% Bis-Tris SDS–
PAGE gels (Invitrogen). Fractions containing the protein of interest
with a minimum of impurities were pooled and stored at 277 K.
Each of the five eluted and pooled CBP2 protein samples was
brought to 10.0 ml in buffer A and treated with 50 ml 1.0 mg ml1
6His-tagged ubiquitin-like protease 1 (Ulp1) overnight to cleave
the N-terminal 6His-Smt fusion tag. Ulp1 cleaves the protein
between the first residue of each viral target and the C-terminal
serine of the QIGGS tag sequence, leaving no remnant of the tag on
the protein. Each sample was then dialyzed against 2.0 l bufferA for a
minimum of 4 h to reduce the concentration of imidazole prior to
further purification on the Protein Maker. The second run on the
Protein Maker (‘Nickel 2’) also employed 5.0 ml volumetric incre-
ments, but at a reduced flow rate of 1.0 ml min1 for all ensuing steps.
Samples were loaded onto the Protein Maker and passed through the
same HisTrap FF columns as in the first run. During Nickel 2, the
cleaved 6His-Smt tag, the 6His-tagged protease and any un-
cleaved target proteins bound to the column, while fully cleaved
CPB2 eluted in the flowthrough. The columns were then washed with
3.0 ml buffer A followed by 5.0 ml buffer B to remove all 6His-
tagged and nonspecifically bound proteins from the column. Flow-
through, wash and elution fractions from Nickel 2 were collected
separately and analysed by SDS–PAGE for comparison with the
initial pooled protein from Nickel 1 (Fig. 2). All five influenza CBP2
proteins were successfully cleaved in this process and were pooled
and concentrated to a volume of 5.0 ml for subsequent size-exclusion
chromatography.
A Sephacryl S-100 10/300 GL column (GE Healthcare) was
prepared by equilibration with 200 ml SEC buffer (25 mM Tris pH
8.0, 200 mM NaCl, 1.0% glycerol, 1 mM TCEP) at 0.5 ml min1 using
an A¨KTApurifier system (GE Healthcare). A set of 5  10.0 ml
superloops was used to stagger each sample injection over the SEC
column in one continuous run. AUV-absorbance trace at 280 nm was
used to analyze column eluents and determine the presence of
protein as well as potential contaminants. Samples were collected in
3.0 ml fractions, analysed by SDS–PAGE, pooled and concentrated
to approximately 20 mg ml1 using Amicon Ultra 10 kDa molecular-
weight cutoff centrifugation tubes. After reaching the target con-
centration, each sample was divided into 100 ml aliquots, flash-frozen
laboratory communications
Acta Cryst. (2011). F67, 1015–1021 Smith et al.  The Protein Maker 1017
Figure 2
SDS–PAGE results for InvaB.07055.c (lanes 2–5) and InvaC.07055.b (lanes 7–10)
during nickel-chelate chromatography purification on the Protein Maker. Lanes 1
and 6, molecular-weight markers (labeled on the left in kDa); lanes 2 and 7, pooled
protein from Nickel 1; lanes 3 and 8, flowthrough of cleaved protein in buffer A
from Nickel 2; lanes 4 and 9, buffer A wash from Nickel 2; lanes 5 and 10, removal
of 6His-Smt tag, 6His-tagged protease and uncleaved protein with buffer B
from Nickel 2.
Table 1
Constructs of the C-terminal domain of polymerase basic protein 2 (PB2) derived
from different subtypes of influenza virus.
Target database
ID Construct Residues Source Subtype Results
InvaB.07055.c D16 538–741 Yokohama 2017 (2003) H3N2 Crystals
InvaB.07055.c D17 538–753 Yokohama 2017 (2003) H3N2 PDB entry 3r2v
InvaC.07055.b D15 538–759 Prague 1 (1956) H7N7 Crystals
InvaC.07055.b D16 538–741 Prague 1 (1956) H7N7 Purified
InvaC.07055.b D17 538–753 Prague 1 (1956) H7N7 Purified
InvaA.07055.a† D16 538–741 Vietnam 1203 (2004) H5N1 PDB entry 3kc6
InvaA.07055.a† D15 538–759 Vietnam 1203 (2004) H5N1 PDB entry 3l56
InvaE.07055.a† D16 538–741 Mexico INDRE4487
(2009)
H1N1 PDB entry
3khw
† Results previously reported elsewhere (Yamada et al., 2010).

laboratory communications
1018 Smith et al.  The Protein Maker Acta Cryst. (2011). F67, 1015–1021
in liquid nitrogen and stored at 193 K prior to crystallization
experiments.
2.2. Expression and purification of cytochrome P450 51 A1 from
C. immitis
Cytochrome P450 51 A1 from C. immitis (CYP51A1) spanning
residues 36–490 (CoimA.07054.l) was cloned into a modified pET28
vector with an N-terminal 6His-Smt tag and cleavage site using
PIPE cloning as described above. CYP51A1 was then expressed and
the cells harvested and stored in the same manner as the CPB2
proteins described in the previous section. The cell paste for a single
batch of CYP51A1 was divided into 12 aliquots of 3.0 g each for lysis-
buffer testing. Each portion was first resuspended in 30.0 ml of a
different lysis buffer on ice (Table 2). Each aliquot was then stirred
for 30 min at 277 K followed by mechanical lysis on ice with a
Branson sonicator (70% power, 2 s on/1 s off pulses, 3 min total).
Crude lysates were spun at 32 960g for 35 min at 277 K to remove cell
debris. The clarified lysates were decanted and brought to 35.0 ml
total volume, followed by incubation at 277 K for 15 min, prior to
purification with the Protein Maker.
The following purification steps employ the same load, wash and
elution buffers for all 12 samples to allow direct comparison of
protein yields across different cell-lysis buffer components. 12 1.0 ml
HisTrap FF (GE) columns were attached to the Protein Maker
gantry, rinsed with 20.0 ml Milli-Q water, washed with 5.0 ml buffer D
[50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
pH 7.5, 200 mM NaCl, 500 mM imidazole] and equilibrated with
25.0 ml buffer C (50 mM HEPES pH 7.5, 200 mM NaCl, 10 mM
imidazole, 50 mM arginine, 1 mM TCEP, 0.25% glycerol) at
2.0 ml min1. All clarified lysates of CYP51A1 were then loaded,
followed by a wash with 30.0 ml buffer A at 2.0 ml min1. Each
sample was eluted in three steps at 0.75 ml min1: 5.0 ml at 30 mM
imidazole, 5.0 ml at 206 mM imidazole and 10.0 ml at 500 mM
imidazole, using 96:4, 60:40 and 0:100 ratios of buffer C:buffer D,
respectively. Elution fractions were collected separately in deep wells
of a Whatman 24-well plate positioned on the deck of the Protein
Maker (Fig. 1) and analysed by SDS–PAGE for optimal lysis-buffer
components (Fig. 3).
3. Results and discussion
3.1. High-throughput parallel purification with the Protein Maker
The Protein Maker (US Patent No. 6818060, Emerald BioSystems)
is a 24-channel parallel liquid-chromatography system developed
specifically for high-throughput protein purification and related
structural genomics pipeline applications. The instrument has 24
precision intake nozzles and syringe pumps, each with a nine-port
valve allowing up to six different intake solutions per line (Fig. 1).
Each port has a 5.0 ml syringe barrel to allow individualized step-
gradient buffer mixing, with a maximum flow rate of 20 ml min1 but
typically run at 0.5 to 2.0 ml min1 with the same flow rate in each
line. Each channel has a column and resin capacity of 1.0–25.0 ml,
allowing up to 250.0 ml lysate per line. The maximum load of protein
per line is approximately 100 mg and is limited mainly by the capacity
of the affinity resin used for capture. Elution fractions of purified
samples are collected in 24 deep-well plates on a deck which can hold
up to 20 plates. Samples are aspirated from the deck into each syringe
valve using the sample-load manifold and then passed through a
second set of tubing connecting the purification columns fitted in the
primary manifold (Fig. 1). Using customized software for program-
ming, one operator can input a minimum amount of information for
routine parallel purifications. For instance, one can enter the desired
elution-buffer concentrations, allowing the software to calculate the
Figure 3
SDS–PAGE analysis of 12 different cell-lysis buffer conditions described in Table 2 for CYP51A1. Lanes correspond to the load (L), wash (W) and elution (E) cycles
conducted in parallel on the Protein Maker, with the same molecular-weight standards throughout (labeled on the left in kDa). Cell-lysis buffer scouting resulted in two
conditions with optimal yields after affinity chromatography (red boxed bands).

appropriate ratios of buffers to mix in each syringe prior to column
loading. Tubing in the Protein Maker is made of chemically resistant
polyether ether ketone (PEEK) and fluorinated ethylene propylene
(FEP) polymers, allowing a wide range of pH buffers and organic
solvents to be moved through the lines without damaging the
instrument.
In comparison to single-line gravity-flow, HPLC or FPLC systems,
the Protein Maker greatly increases output owing to its parallel
plumbing and purification capability. Its speed, load capacity and
collection volumes make it ideal for reverse-phase, ion-exchange and
affinity-based chromatographic separations. In a typical run for
SSGCID proteins purified at Emerald BioStructures, immobilized
metal-affinity chromatography resin is washed and conditioned for
each channel used in the experiment. Protein lysates are typically
loaded in volumes of up to 250 ml, washed and eluted prior to
analysis by gel or capillary electrophoresis followed by fraction
pooling for direct use or subsequent purification steps. Wash buffer
containing a minimal amount of imidazole (usually 10 mM) assists in
removing nonspecifically bound protein from nickel columns and
tends to result in higher purity elutions than wash buffer with no
imidazole. For cleaved proteins, the 6His tag and the 6His-tagged
protease used to cleave it are typically removed by one subsequent
round of purification on the Protein Maker, collecting the target
protein in the initial flowthrough.
Adapting the standard SSGCID purification protocol for the
Protein Maker (see x2), a single person can purify as many as 48
proteins in an 8 h shift. Using the same protocol in the same amount
of time, a lone operator can purify at best four proteins with a single-
line FPLC system. For structural genomics work, this level of output
is essential for maintaining the high rate of target delivery to crys-
tallization trials and NMR studies. Proteins purified on the Protein
Maker for the SSGCID which have resulted in PDB structure
depositions include prokaryotic targets from Bartonella (PDB entry
3grp; J. Abendroth, T. E. Edwards, B. Sankaran, T. Arakaki & B. L.
Staker, unpublished work), Brucella (PDB entries 2l3v, 3fq3, 3grk,
3ix6, 3jst, 3oce and 3ocf; R. Barnwal & G. Varani, unpublished work;
J. Abendroth, T. E. Edwards, B. Sankaran, T. Arakaki & B. L. Staker,
unpublished work; T. E. Edwards, J. Abendroth, B. Sankaran, A. S.
Gardberg, T. L. Arakaki & B. L. Staker, unpublished work),
Burkholderia (PDB entries 3ecd and 3i4e; J. Abendroth, T. E.
Edwards, B. Sankaran, T. Arakaki & B. L. Staker, unpublished work;
T. E. Edwards, J. Abendroth, T. L. Arakaki & B. Staker, unpublished
work), Mycobacterium (PDB entries 3gwc and 3hzg; J. Abendroth,
T. E. Edwards, B. Sankaran, T. Arakaki & B. L. Staker, unpublished
work) and Rickettsia (PDB entries 3d53 and 3emj; T. E. Edwards,
J. Abendroth, T. L. Arakaki & B. Staker, unpublished work), and
eukaryotic proteins from Encephalitozoon (PDB entry 3kgb; J.
Abendroth, T. E. Edwards, B. Sankaran, T. Arakaki & B. L. Staker,
unpublished work), Babesia (PDB entries 3i3r, 3k2h, 3kjr and 3nrr; Li
et al., 2010; Begley et al., 2011) and Entamoeba (PDB entries 3lqw,
3sia, 3sib and 3sjs; T. E. Edwards, J. Abendroth, B. Sankaran, A. S.
Gardberg, T. L. Arakaki & B. L. Staker, unpublished work; A. S.
Gardberg, T. E. Edwards & B. L. Staker, unpublished work), as well
as viral proteins from rabies (PDB entry 3oa1; T. E. Edwards,
J. Abendroth, B. Sankaran, A. S. Gardberg, T. L. Arakaki & B. L.
Staker, unpublished work) and several subtypes of influenza A virus,
as described here and elsewhere (Yamada et al., 2010).
3.2. Parallel purification of viral proteins with the Protein Maker
Pandemic outbreaks of influenza have caused millions of deaths
throughout history and remain very real threats, as active subtypes
currently residing in birds and in swine and other mammalian species
possess the potential to infect humans around the globe (Christman et
al., 2011; Sencer, 2011; Taubenberger & Morens, 2006; Neumann et
al., 2009). Point mutations in the polymerase basic protein 2 (PB2) of
the heterotrimeric viral polymerase and their structural consequences
have been linked to host-species specificity and virulence factors in
humans (Guilligay et al., 2008; Mehle & Doudna, 2009; Subbarao et
al., 1993). This link between PB2 point mutations and transmissibility
has prompted further studies of proteins from different subtypes of
influenza, including the nomination and approval of various PB2
constructs for study by the SSGCID. Among a wider set of targets, we
have characterized the C-terminal PB2 domain (CPB2) from the
H1N1 and H5N1 influenza A virus subtypes (Yamada et al., 2010).
H5N1 originated in birds and is responsible for most avian influenza-
based fatalities in humans (Li et al., 2004), while H1N1 caused the
outbreak of swine flu in 2009 and is closely related to the 1918
pandemic Spanish flu virus (Taubenberger & Morens, 2006; Neumann
et al., 2009). Initial success with H5N1 and H1N1 CPB2 proteins
informed the design and cloning of genes arising from two other
influenza subtypes: H3N2, which was responsible for the 1968 and
1969 Hong Kong flu epidemics, and H7N7, a subtype with wide
zoonotic transmissibility and potential for high pathogenicity
(Coleman et al., 1968; Jackson et al., 2010; Kemink et al., 2004;
Fouchier et al., 2004). H3N2 is already an increasingly dominant
subtype in the annual flu season in North America and is also
endemic among livestock pigs in southern China (Gramer et al., 2007;
Richt et al., 2003). Since pigs can be co-infected with multiple
zoonotic influenza A virus subtypes, H3N2 in swine has the potential
to emerge in a more virulent form with transmissibility into human
populations through genetic reassortment.
In one run of the Protein Maker, five channels were used to purify
five different CPB2 constructs (Table 1) in parallel with other pipe-
line proteins using our standard SSGCID protocol (see x2). In this
mode, buffer solutions were aspirated from large common reservoirs
into each individual syringe valve, while samples were loaded from 24
deep-well plates using the sample-intake manifold (Fig. 1). All five
constructs were successfully purified from cell lysates as described
above (see x2), generating highly pure protein (Fig. 2) for crystal-
laboratory communications
Acta Cryst. (2011). F67, 1015–1021 Smith et al.  The Protein Maker 1019
Table 2
Condition grid for cell-lysis buffer testing of recombinantly expressed fungal cytochrome P450 (CYP51A1 from C. immitis; CoimA.07054.l).
A B C D
1 50 mM MES, 250 mM NaCl,
5% glycerol, 0.5 mM TCEP pH 6.0
50 mM MES, 1 M NaCl,
5% glycerol, 0.5 mM TCEP pH 6.0
50 mM MES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% CHAPS pH 6.0
50 mM MES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% BOG pH 6.0
2 50 mM HEPES, 250 mM NaCl,
5% glycerol, 0.5 mM TCEP pH 7.5
50 mM HEPES, 1 M NaCl,
5% glycerol, 0.5 mM TCEP pH 7.5
50 mM HEPES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% CHAPS pH 7.5
50 mM HEPES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% BOG pH 7.5
3 50 mM Tris, 250 mM NaCl,
5% glycerol, 0.5 mM TCEP pH 8.0
50 mM Tris, 1 M NaCl,
5% glycerol, 0.5 mM TCEP pH 8.0
50 mM Tris, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% CHAPS pH 8.0
50 mM Tris, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% BOG pH 8.0

lization testing using sparse-matrix screens. Three of the five purified
CPB2 proteins yielded crystals (not shown) and a 1.3 A˚ resolution
structure was generated from one of them by X-ray diffraction. This
structure was determined by molecular replacement, refined using
standard SSGCID protocols (as reported elsewhere in this volume)
and deposited in the Protein Data Bank under accession code 3r2v
(T. E. Edwards, A. S. Gardberg, B. Sankaran & B. L. Staker, un-
published work), for which this communication serves as the first
report in the literature. Although previous purifications of CPB2
from H5N1 and H1N1 yielded crystals for 204-residue and 222-
residue variants, the only protein which provided well diffracting
crystals in this effort was a construct consisting of 216 amino acids
(Table 1). Despite the high sequence identity (>95%) across all
constructs, including three previously crystallized CPB2 proteins
(Yamada et al., 2010), only one (InvaB.07055.c.D17) of the five tested
led to rapid growth of crystals that diffracted to sufficiently high
resolution for structure solution and refinement. Thus, we eliminated
the 80% chance of failure (or the fivefold increase in bench time)
associated with serial processing of five targets for one successful
outcome via parallel purification on the Protein Maker.
3.3. Scouting lysis-buffer conditions with the Protein Maker
In addition to being a vital asset for high-throughput purification,
the Protein Maker can be an invaluable tool for rapid testing of cell-
lysis conditions for a wide variety of protein targets. The majority of
SSGCID proteins are initially lysed by sonication, followed by nickel-
affinity chromatography using genetically engineered histidine tags to
bind the protein to the resin (see x2). Some targets are insufficiently
soluble in the standard cell-lysis buffers for further processing despite
high levels of expression. To keep these targets moving through the
pipeline, it is often necessary to conduct optimization tests for puri-
fication in a rapid parallel fashion. An example of this is cytochrome
P450 51 A1 (CYP51A1) from C. immitis, a pathogenic fungus that is
endemic to North America (Crum et al., 2004; Galgiani, 1999; Hector
& Laniado-Laborin, 2005). Inhibition of CYP51A1 with a variety of
azole compounds causes accumulation of methylated sterol precur-
sors in fungi, leading to an imbalance in cell-wall stability and
reduced fungal growth (Kale & Johnson, 2005; Sheehan et al., 1999).
Although human CYP51A1 has been structurally characterized
(Strushkevich et al., 2010), together with those of Mycobacterium
tuberculosis (Podust et al., 2004) and several protozoan parasites, no
CYP51A1 structures from fungal species are available in the PDB.
Owing to its potential for structure-guided development of novel
antifungals, and as a community-request target, CYP51A1 from
C. immitis (target ID CoimA.07054.l) was deemed to be a high-value
target.
Initial attempts with CYP51A1 showed excellent recombinant
protein expression in bacteria, but nearly all of the CYP51A1
remained insoluble using our standard SSGCID cell-lysis methods.
We therefore initiated scouting experiments to see if altering the cell-
lysis buffer components might provide higher yields from crude
lysate. After conducting individual small-scale rapid cell lysis on 12
samples from the same batch of protein, each sample was loaded onto
the Protein Maker using a 24 deep-well plate and the sample-intake
manifold (Fig. 1). Keeping the purification buffers identical across all
samples allowed a comparative analysis of the different lysis-buffer
components used to lyse the cells. SDS–PAGE analysis shows little to
no protein in the soluble fraction after lysis, suggesting that CYP51A1
is only soluble under certain conditions. The most promising results
were obtained with lysis buffer containing 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate (CHAPS) or n-octyl--d-
glucopyranoside (BOG) with 500 mM salt at the lowest pH tested
(Fig. 3). CHAPS and BOG are nonionic detergents that are favored
in membrane-protein extraction, partly owing to the relative ease of
their removal further downstream in the purification. BOG is espe-
cially favored when working with membrane proteins because of
its ability to soften the phospholipid layer (Markovic Housley &
Garavito, 1986; Garavito & Ferguson-Miller, 2001). In this case, the
detergents are likely to have contributed to the breakdown of cell
membranes, releasing greater amounts of recombinant protein into
solution (Fig. 3). With a putative transmembrane helix N-terminal to
the canonical P450 domain of our construct, it is also possible that
detergents are necessary to help solubilize the target protein. Thus,
results from this test have provided optimal cell-lysis buffer condi-
tions for further work on scale-up and purification of CYP51A1 and
have led to further work in gene optimization.
4. Conclusions
As more genomes are sequenced, the range of uncharacterized
biological targets of interest expands, increasing the need to rapidly
clone, express and purify targets for structural and functional geno-
mics. With more than 5000 targets now selected for investigation by
the SSGCID, it has become essential to utilize automated laboratory
systems capable of producing large quantities of pure crystallization-
grade protein. A single person using a single-channel FPLC system in
our facility can fully process up to four protein targets in a week from
lysis to size-exclusion chromatography following standard SSGCID
practices and protocols. The same individual can process up to 40
proteins per week with the Protein Maker using the same protocols
for individual sample lysis and staggered-loop consecutive injections
for single-line size-exclusion chromatography. This tenfold increase
in sampling capacity allows the processing of many more constructs
per target, thereby improving the chance that a protein of interest
becomes structurally characterized. Without the Protein Maker,
serial purification of five unique CBP2 constructs would be likely to
have taken two weeks of effort, fully occupying the instrument and a
single researcher for that duration. In such a scenario, construct
design may have been limited to residue lengths which previously
worked for CBP2 from H1N1 and H5N1, constructs which to date
have not generated crystals for H3N2. Although only two conditions
appeared to increase the yield of our CYP51A1 protein target, this
information was sufficient to improve lysis conditions for milligram-
scale purification. The identification of key components in the cell-
lysis buffer also prompted a more thorough investigation of the
construct and has led to second-generation deletion mutants to
obtain the P450 domain of this important antifungal drug target.
Whether for method development or high-throughput processing, the
parallel purification options afforded through the Protein Maker has
made it a valuable asset for the SSGCID pipeline and the determi-
nation of structures for infectious disease research.
The authors wish to thank Robin Clark, as well as everyone at
Emerald BioSystems, whose input aided in the design, assembly and
functionality of the Protein Maker. We also thank the entire team at
SSGCID, without whom this work would not have been possible.
This research was funded under Federal Contract No.
HHSN272200700057C from the National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Department of
Health and Human Services.
laboratory communications
1020 Smith et al.  The Protein Maker Acta Cryst. (2011). F67, 1015–1021

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laboratory communications
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