electronic reprint
Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications
ISSN 1744-3091

Editors: H. M. Einspahr and M. S. Weiss

The Protein Maker: an automated system for high-throughput
parallel purification
Eric R. Smith, Darren W. Begley, Vanessa Anderson, Amy C. Raymond,Taryn E. Haffner, John I. Robinson, Thomas E. Edwards, Natalie Duncan,
Cory J. Gerdts, Mark B. Mixon, Peter Nollert, Bart L. Staker and Lance J.
StewartActa Cryst. (2011). F67, 1015–1021

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ISSN 1744-3091

Volume 67
Part 1January 2011

Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications
Editors: H. M. Einspahr and M. S. Weiss

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Acta Cryst. (2011). F67, 1015–1021

Smith et al. · The Protein Maker

laboratory communications
Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications

The Protein Maker: an automated system for highthroughput parallel purification

ISSN 1744-3091

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,bThomas E. Edwards,a,b Natalie
Duncan,c Cory J. Gerdts,cMark B. Mixon,a,b Peter Nollert,bBart 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

Acta Cryst. (2011). F67, 1015–1021

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 inparallel 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 forstructure determination and deposition in the Protein Data Bank. Methods forscreening 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 avariety of high-throughput protein-puriﬁcation applications.

1. Introduction
The end goal of structural genomics is the rapid generation of threedimensional structures obtained from atomic level resolution studiesof 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 candidates for further processing. Gravity-based, high-pressure and ‘fast
protein’ liquid-chromatography (HPLC and FPLC) methods havebeen 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 puriﬁcationor 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 ofpipelines 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-chromatographysystem 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 thetime 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 pathogenic 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 oftherapeutic intervention and include a high-value subset of proteinsrequested by external scientists from the infectious disease researchcommunity. 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
doi:10.1107/S1744309111028776

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laboratory communications
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 structuralcharacterization of this protein a potentially important role in theprevention 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 theProtein 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 understandard lysis-buffer conditions. In this report, we also detail scouting
experiments in which a single batch of cells was split into 12 pools andlysed by sonication in 12 different buffer conditions, followed byparallel puriﬁcation with the Protein Maker. The protein in this casewas 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 componentof 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 intoa 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.

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terminal 6His-Smt fusion tag (Mossessova & Lima, 2000) and asite-speciﬁc protease cleavage site to the open reading frame using
Polymerase Incomplete Primer Extension (PIPE) cloning (Lorimer etal., 2009). The N-terminal sequence of all ﬁve gene products was
MSHHHHHHSGEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGS followed by the ﬁrst amino acid of thedesired inﬂuenza A viral protein construct. Each clone was expressedin 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 collectedby 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 resuspended by vigorous stirring for 30 min at 277 K and mechanicallylysed 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, withthe following steps conducted in parallel for all samples. Each puriﬁcation step was conducted in 5.0 ml volumetric increments, utilizing
a ﬂow rate of 1.5 ml min1. For the ﬁrst run of the Protein Maker(‘Nickel 1’), each column was ﬁrst washed with 20.0 ml Milli-Q waterto remove the ethanol-based storage buffer. Each column was thenwashed 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

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, removalof 6His-Smt tag, 6His-tagged protease and uncleaved protein with buffer B
from Nickel 2.

Acta Cryst. (2011). F67, 1015–1021

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
InvaB.07055.c D16
InvaB.07055.c D17
InvaC.07055.b D15
InvaC.07055.b D16
InvaC.07055.b D17
InvaA.07055.a† D16
InvaA.07055.a† D15
InvaE.07055.a† D16

538–741
538–753
538–759
538–741
538–753
538–741
538–759
538–741

Subtype Results

Yokohama 2017 (2003) H3N2
Yokohama 2017 (2003) H3N2
Prague 1 (1956)
H7N7
Prague 1 (1956)
H7N7
Prague 1 (1956)
H7N7
Vietnam 1203 (2004) H5N1
Vietnam 1203 (2004) H5N1
Mexico INDRE4487
H1N1(2009)

Crystals
PDB entry 3r2v
CrystalsPuriﬁedPuriﬁedPDB entry 3kc6
PDB entry 3l56
PDB entry
3khw† Results previously reported elsewhere (Yamada et al., 2010).

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 wasbrought to 10.0 ml in buffer A and treated with 50 ml 1.0 mg ml1
6His-tagged ubiquitin-like protease 1 (Ulp1) overnight to cleave
the N-terminal 6His-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 buffer A for aminimum of 4 h to reduce the concentration of imidazole prior tofurther puriﬁcation on the Protein Maker. The second run on theProtein Maker (‘Nickel 2’) also employed 5.0 ml volumetric increments, but at a reduced ﬂow rate of 1.0 ml min1 for all ensuing steps.
Samples were loaded onto the Protein Maker and passed through thesame HisTrap FF columns as in the ﬁrst run. During Nickel 2, thecleaved 6His-Smt tag, the 6His-tagged protease and any uncleaved 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 6Histagged and nonspeciﬁcally bound proteins from the column. Flowthrough, wash and elution fractions from Nickel 2 were collected
separately and analysed by SDS–PAGE for comparison with theinitial 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 min1 using
an ÄKTApuriﬁer 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. A UV-absorbance trace at 280 nm wasused to analyze column eluents and determine the presence ofprotein as well as potential contaminants. Samples were collected in
3.0 ml fractions, analysed by SDS–PAGE, pooled and concentrated
to approximately 20 mg ml1 using Amicon Ultra 10 kDa molecularweight cutoff centrifugation tubes. After reaching the target concentration, each sample was divided into 100 ml aliquots, ﬂash-frozen

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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 6His-Smt tag and cleavage site using
PIPE cloning as described above. CYP51A1 was then expressed andthe 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 lysisbuffer testing. Each portion was ﬁrst resuspended in 30.0 ml of adifferent lysis buffer on ice (Table 2). Each aliquot was then stirredfor 30 min at 277 K followed by mechanical lysis on ice with aBranson 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 celldebris. 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 andelution buffers for all 12 samples to allow direct comparison ofprotein 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 min1. All clariﬁed lysates of CYP51A1 were then loaded,followed by a wash with 30.0 ml buffer A at 2.0 ml min1. Eachsample was eluted in three steps at 0.75 ml min1: 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 wellsof 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-portvalve allowing up to six different intake solutions per line (Fig. 1).
Each port has a 5.0 ml syringe barrel to allow individualized stepgradient buffer mixing, with a maximum ﬂow rate of 20 ml min1 but
typically run at 0.5 to 2.0 ml min1 with the same ﬂow rate in eachline. 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 capacityof the afﬁnity resin used for capture. Elution fractions of puriﬁed
samples are collected in 24 deep-well plates on a deck which can holdup to 20 plates. Samples are aspirated from the deck into each syringe
valve using the sample-load manifold and then passed through asecond set of tubing connecting the puriﬁcation columns ﬁtted in the
primary manifold (Fig. 1). Using customized software for programming, one operator can input a minimum amount of information forroutine 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).

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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

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

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 MES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% CHAPS pH 6.0
50 mM HEPES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% CHAPS pH 7.5
50 mM Tris, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% CHAPS pH 8.0

50 mM MES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% BOG pH 6.0
50 mM HEPES, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% BOG pH 7.5
50 mM Tris, 500 mM NaCl,
5% glycerol, 0.5 mM TCEP,
1% BOG pH 8.0

appropriate ratios of buffers to mix in each syringe prior to column
loading. Tubing in the Protein Maker is made of chemically resistantpolyether 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 andcollection volumes make it ideal for reverse-phase, ion-exchange andafﬁnity-based chromatographic separations. In a typical run for
SSGCID proteins puriﬁed at Emerald BioStructures, immobilized
metal-afﬁnity chromatography resin is washed and conditioned foreach channel used in the experiment. Protein lysates are typicallyloaded 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 inremoving nonspeciﬁcally bound protein from nickel columns andtends to result in higher purity elutions than wash buffer with no
imidazole. For cleaved proteins, the 6His tag and the 6His-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 theProtein 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 amountof time, a lone operator can purify at best four proteins with a singleline FPLC system. For structural genomics work, this level of output
is essential for maintaining the high rate of target delivery to crystallization 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, unpublishedwork), Mycobacterium (PDB entries 3gwc and 3hzg; J. Abendroth,T. E. Edwards, B. Sankaran, T. Arakaki & B. L. Staker, unpublishedwork) and Rickettsia (PDB entries 3d53 and 3emj; T. E. Edwards,
J. Abendroth, T. L. Arakaki & B. Staker, unpublished work), andeukaryotic 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; Liet al., 2010; Begley et al., 2011) and Entamoeba (PDB entries 3lqw,
Acta Cryst. (2011). F67, 1015–1021

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 etal., 2011; Sencer, 2011; Taubenberger & Morens, 2006; Neumann etal., 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 inhumans (Guilligay et al., 2008; Mehle & Doudna, 2009; Subbarao etal., 1993). This link between PB2 point mutations and transmissibility
has prompted further studies of proteins from different subtypes ofinﬂuenza, including the nomination and approval of various PB2
constructs for study by the SSGCID. Among a wider set of targets, wehave characterized the C-terminal PB2 domain (CPB2) from theH1N1 and H5N1 inﬂuenza A virus subtypes (Yamada et al., 2010).
H5N1 originated in birds and is responsible for most avian inﬂuenzabased fatalities in humans (Li et al., 2004), while H1N1 caused theoutbreak 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 otherinﬂuenza subtypes: H3N2, which was responsible for the 1968 and1969 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 humanpopulations 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 pipeline proteins using our standard SSGCID protocol (see x2). In thismode, 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 describedabove (see x2), generating highly pure protein (Fig. 2) for crystal-electronic reprint

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laboratory communications
lization testing using sparse-matrix screens. Three of the ﬁve puriﬁed
CPB2 proteins yielded crystals (not shown) and a 1.3 Å 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, unpublished 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 222residue 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 highresolution for structure solution and reﬁnement. Thus, we eliminatedthe 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 celllysis conditions for a wide variety of protein targets. The majority of
SSGCID proteins are initially lysed by sonication, followed by nickelafﬁnity chromatography using genetically engineered histidine tags tobind the protein to the resin (see x2). Some targets are insufﬁciently
soluble in the standard cell-lysis buffers for further processing despitehigh levels of expression. To keep these targets moving through thepipeline, it is often necessary to conduct optimization tests for puriﬁcation 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 ofazole compounds causes accumulation of methylated sterol precursors in fungi, leading to an imbalance in cell-wall stability andreduced 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 celllysis 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 tono 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-1020

Smith et al.  The Protein Maker

glucopyranoside (BOG) with 500 mM salt at the lowest pH tested(Fig. 3). CHAPS and BOG are nonionic detergents that are favoredin membrane-protein extraction, partly owing to the relative ease of
their removal further downstream in the puriﬁcation. BOG is especially 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, thedetergents 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 tothe 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 conditions for further work on scale-up and puriﬁcation of CYP51A1 andhave led to further work in gene optimization.

4. Conclusions
As more genomes are sequenced, the range of uncharacterizedbiological targets of interest expands, increasing the need to rapidly
clone, express and purify targets for structural and functional genomics. 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 crystallizationgrade protein. A single person using a single-channel FPLC system inour facility can fully process up to four protein targets in a week fromlysis 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 increasein 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 tohave taken two weeks of effort, fully occupying the instrument and asingle researcher for that duration. In such a scenario, constructdesign may have been limited to residue lengths which previously
worked for CBP2 from H1N1 and H5N1, constructs which to datehave not generated crystals for H3N2. Although only two conditions
appeared to increase the yield of our CYP51A1 protein target, thisinformation was sufﬁcient to improve lysis conditions for milligramscale puriﬁcation. The identiﬁcation of key components in the celllysis buffer also prompted a more thorough investigation of theconstruct and has led to second-generation deletion mutants toobtain the P450 domain of this important antifungal drug target.
Whether for method development or high-throughput processing, theparallel puriﬁcation options afforded through the Protein Maker has
made it a valuable asset for the SSGCID pipeline and the determination 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 andfunctionality 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.

electronic reprint

Acta Cryst. (2011). F67, 1015–1021

laboratory communications
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