IRJET- Subcellular Localization of Transmembrane E-cadherin-GFP Fusion Pr...
SHIAffinityPurificationPEP
1. High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a
catalytically-active nickel-free intermediate form
Sanjeev K. Chandrayan, Chang-Hao Wu, Patrick M. McTernan, Michael W.W. Adams ⇑
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
Article history:
Received 25 August 2014
and in revised form 28 October 2014
Available online 21 November 2014
Keywords:
Hydrogen
Hydrogenase
Enzyme maturation
Pyrococcus
Affinity purification
a b s t r a c t
The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen
gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture
of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times
greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large
scale biohydrogen production. Further development of this process at an industrial scale is limited by the
availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and matura-
tion pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in
P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase
in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also
identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity
similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the
proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit
assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.
Ó 2014 Elsevier Inc. All rights reserved.
Introduction
Hydrogen is one of the most efficient energy carriers and is car-
bon neutral [22]. A major hurdle for its widespread utilization is
the economic and efficient production of hydrogen from renewable
sources [10]. Currently, most hydrogen comes from steam-reform-
ing of non-renewable natural gas, which is not economical nor
environmentally-friendly because greenhouse gases are produced
[10]. Biohydrogen production from renewable sources has the
capability to replace the current method of hydrogen production
[10,26]. Amid current methodologies for biohydrogen production
from biomass sugars, an in vitro cell-free synthetic pathway holds
great promise as the yield (12 mol H2/mol glucose) is three times
the theoretical limit for biohydrogen from microbial fermentation
(4 mol H2/mol glucose [25]). In the synthetic pathway, all of the
reductant generated from glucose oxidation is channeled to
NADPH. The key enzyme is a NADPH-dependent hydrogenase
termed SHI (for soluble hydrogenase I), which oxidizes NADPH
and produces hydrogen gas. SHI is obtained from the hypertherm-
ophilic archaeon, Pyrococcus furiosus, which grows optimally near
100 °C [2,1,21,18]. In addition to biohydrogen production, the
reversible nature of the SHI reaction also makes it an attractive
catalyst for the regeneration of the cofactor NADPH in in vitro sys-
tems using hydrogen as the reductant [13,14].
In order to scale up the in vitro system for hydrogen production,
sufficient quantities of purified SHI will be needed. In addition to
its use in biofuel-related applications, the availability of pure SHI
would also stimulate attempts to structurally characterize the
enzyme. Crystal structures are not available for this particular type
of hydrogenase, which is part of the group 3b type [23]), nor for a
hydrogenase from a hyperthermophile, which are microorganisms
that grow optimally above 80 °C. Hence the primary goal of the
present work was to overproduce P. furiosus SHI and devise a sim-
ple purification procedure that would yield enzyme for both
applied and basic studies. SHI has been studied in detail
[2,1,21,18]. It is a heterotetramer encoded by a single operon
(PF0891-PF0894). The four subunits are predicted to contain the
[NiFe] active site (a), two iron sulfur clusters (b), a flavin adenine
dinucleotide with one iron sulfur cluster (c), and three iron sulfur
clusters (d) [1].
Obtaining a [NiFe]-hydrogenase in large quantity from any
source by heterologous expression is difficult because of the com-
plex maturation and assembly of active NiFe catalytic site, which
involves eight or more maturation proteins and enzymes [6,9].
Efforts to overexpression [NiFe]-hydrogenases in Escherichia coli
have typically met with limited or no success [24,8]. Although
the active form of the enzyme from Cupriavidus necator H16
http://dx.doi.org/10.1016/j.pep.2014.10.018
1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
⇑ Corresponding author. Tel.: +1 (706) 542 2060; fax: +1 (706) 542 0229.
E-mail address: adams@bmb.uga.edu (M.W.W. Adams).
Protein Expression and Purification 107 (2015) 90–94
Contents lists available at ScienceDirect
Protein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep
2. (Ralstonia eutropha H16) was very recently obtained in E. coli, the
yield of the pure protein was still much lower than that obtained
from the native host [17]. This was also the case in our attempt
to heterologously express P. furiosus SHI in E. coli [20].
SHI purification from P. furiosus is complicated and time-con-
suming involving four chromatography steps [2]. However, genetic
tools were recently developed for P. furious [11] and we were suc-
cessful in our initial attempt to homologously overexpress SHI
with a Strep-tag II inserted at the N-terminus of the b-subunit
(PF0891) [5]. However, while the Strep-tag II has been successfully
used as an affinity tag for E. coli hydrogenase [12], the affinity puri-
fication step for P. furiosus SHI gave unsatisfactory results. The
yield of SHI from the cytoplasmic extract was at best 20%, the pro-
cedure was difficult to scale-up and the results were not reproduc-
ible. We had been reluctant to use a poly-histidine tag for SHI
affinity purification as it might interfere with the assembly of the
nickel-containing catalytic site. Nevertheless, we show here that
that is not the case. Moreover, this new procedure is so efficient
that an unprocessed or immature form of SHI was also purified that
lacked the nickel-containing catalytic subunit while retaining
NADPH-oxidizing activity.
Materials and methods
Genetic manipulation of COM1
The knock-in cassette for constructing the 9x-His tagged ver-
sions of SHI was generated by combining four different PCR frag-
ments: (1) 1 kbp upstream flanking region (UFR)1
, (2) the marker
cassette (Pgdh-pyrF), (3) the S-Layer protein promoter (Pslp) and a
9X-Histidine tag, and (4) 1 kbp downstream flanking regions (DFR),
into one cassette by overlapping PCR [3]. UFR, DFR and Pslp were
amplified using Pfu-genomic DNA as a template and for amplifica-
tion of Pgdh-pyrF, plasmid pGLW021 was used as template for the
PCR reaction [11]. For routine PCR and overlapping PCR, the high
fidelity enzyme Prime Star HS premix (Clonetech, Takara) was used.
The genetic manipulation using the knock in cassette was performed
on the P. furiosus COM1 strain as described previously [11]. The
knock in cassette was placed at the N-terminus of SHIb (PF0891)
in MW0430 and at the N-terminus of SHIa (PF0894) in MW0434
(Fig. 1). Both MW0430 and MW0434 were confirmed by PCR and
DNA sequencing (Macrogen sequencing services, Maryland, USA).
Large-scale cell growth, affinity protein purification and SDS PAGE gel
electrophoresis
The P. furiosus strains were grown in a rich medium in a 20-liter
fermenter as described previously [5]. Cells were harvested and
frozen in liquid nitrogen and stored at À80 °C. For purification, fro-
zen cells (100 g) were suspended in 50 mM Tris, pH 8.0, containing
2 mM dithiothreitol (DTT) and 50 lg/ml DNase with a cell to buffer
ratio of 1:5 and lysed by stirring for 4 h at 23 °C anaerobically in an
anaerobic glove box (Coy Lab products, Michigan, USA). The cyto-
plasmic extract (S100) was obtained by centrifuging the lysed cells
for 1 h at 100,000Âg. The S100 fraction was loaded onto a His Trap
FF 5 ml column (GE Healthcare, Piscataway, NJ, USA) equilibrated
with 50 mM Tris, pH 8.0, containing 400 mM NaCl and 2 mM
DTT. The column was washed with twenty column volumes of
the equilibration buffer and bound protein was eluted using a gra-
dient of imidazole (0–100%) where 100% is the equilibration buffer
containing 500 mM imidazole. Imidazole was removed from frac-
tions by buffer exchange with 50 mM Tris pH 8.0, containing
300 mM NaCl and 2 mM dithionite using a Amicon Ultracentrifu-
gation filter with a 30 kDa cutoff (Millipore, Massachusetts, USA).
For SDS PAGE analysis, samples analyzed using precast Criterion
TGX (4–15%) gels and a Bio-Rad gel electrophoresis system.
Gel filtration chromatography
A HiLoad 26/60 Superdex-200 preparatory grade column was
used for purification of the trimeric intermediate from tetrameric
SHI. The Ni–NTA fractions containing tetrameric SHI (F3–F6 in
Fig. S1A) were applied on HiLoad 26/60 Superdex-200 and eluted
at 0.3 ml/minute using 50 mM Tris pH 8.0, containing 300 mM
NaCl and 2 mM dithionite. The fractions were analyzed by SDS
PAGE and those containing the trimeric form (F9–F11 in Fig. S2)
were further purified on a Superdex-200 10/300 GL analytical col-
umn. The column was elucted at 0.3 ml/minute using the same
buffer for HiLoad 26/60 Superdex-200. The samples were analyzed
and those containing the trimeric form were pooled and used for
enzyme assays.
Enzyme assays
All enzyme assays were performed at 80 °C under anaerobic
conditions. Purified protein ($0.1 mg/ml) was used to assay for
both the trimeric and tetrameric forms of SHI. Hydrogen evolution
assays were carried out using dithionite-reduced methyl viologen
(MV) as the electron donor [2]. An anaerobically sealed assay vial
containing 2 ml of 10 mM dithionite, 1 mM MV and 100 mM EPPS,
pH 8.4, was preheated for 1 min and purified protein was added to
start the reaction. The vial was incubated at 80 °C for 6 min and
100 ll of the gaseous headspace was removed and injected into a
6850 Network GC system from Agilent Technologies (Santa Clara,
CA, USA). One unit (U) of activity is equal to the production of
1 lmol of H2 per minute. For the hydrogen uptake assay, the head-
space of a sealed assay vial containing 2.0 ml of 100 mM EPPS, pH
8.4, and 1.0 mM benzyl viologen was replaced by 100% hydrogen
gas and incubated at 80 °C. The purified protein was added to start
the reaction and hydrogenase activity was measured by the
increase of absorbance at 580 nm using a 100 Cary UV–Vis spectro-
photometer with a peltier-based temperature controller from Agi-
lent technologies (Santa Clara, CA, USA) as previously described
(see [20]. Benzyl viologen oxidoreductase (BVOR) activity was
measured in a similar manner except that NADH or NADPH
(2 mM) were used as the electron donor rather than hydrogen
gas. In both cases, an extinction coefficient of 8.8 mMÀ1
cmÀ1
was used for reduced benzyl viologen (BV).
Results
Construct of overexpression strain MW0430 and MW0434
P. furiosus strain MW0430 was constructed using the parent
COM1 in which the four-gene SHI operon (PF0891-PF0894) was
placed under control of the constitutive and highly expressed pro-
moter, Pslp, of the gene encoding the S-layer protein (PF1399)
(Fig. 1A). In addition, strain MW0434 was constructed in which
the four-gene SHI operon was split to two operons that SHIbcd
(PF0891-PF0893) were controlled by PshI and SHIa was controlled
by Pslp (Fig. 1B). The strains were obtained by homologous recom-
bination. The genotypes of the parent and daughter strains are
given in Table 1. The 9x-Histidine affinity tag was placed at the
N-terminus of the b subunit (PF0891) in MW0430 and at the N-ter-
minus of the a subunit (PF0894) in MW0434, as shown in Fig. 2.
These strains did not exhibit any phenotype during growth on
1
Abbreviations used: UFR, upstream flanking region; DFR, downstream flanking
regions; MV, methyl viologen; BVOR, Benzyl viologen oxidoreductase.
S.K. Chandrayan et al. / Protein Expression and Purification 107 (2015) 90–94 91
3. sugar (the disaccharide, maltose) compared to the parent COM1
strain.
Purification of affinity-tagged SHI
SHI could be purified from the cytoplasmic fraction of P. furiosus
strain MW0430 cells by a single affinity chromatography step.
Moreover, as shown in Table 2, a total of 76% of the hydrogenase
activity was absorbed to and eluted from the Ni–NTA column. This
compares with a yield of only 21% with Strep-tag II-SHI [5] and
with 32% for the Strep-tag II form of the tetrameric hydrogenase
of R. eutropha [17]. In contrast, the yield of MW0434 was only
25% using the same purification procedure used for MW0430.
Hence, placing the affinity tag at the b-subunit (produced by strain
MW0430) leads to a more efficient purification procedure than at
the a-subunit (produced by strain MW0434), and strain
MW0430 was used for the characterization described below. The
hydrogen elution activities and the SDS gel profiles of the fractions
eluting from the Ni–NTA column are shown in Fig. S1. A total of
135 mg of His-SHI was obtained from 100 g of P. furiosus cells,
Fig. 1. Scheme of the construction of P. furiosus strains MW0430 and MW0434. The knock in cassette has homologous arms shown as the upstream flanking region (UFR) and
downstream flanking region (DFR). Marker cassette is indicated as Pgdh-pyrF, the strong constitutive promoter is indicated as Pslp. The native SHI promoter is indicated as PshI
and the affinity tag is indicated as 9x-His. The corresponding homologous loci of the UFR and DFR in the parent COM1 strain are depicted as PF0890 and SHIb respectively in
1A, and SHId and SHIa respectively in 1B. Arrangement of SHI locus after recombination is also shown.
Table 1
Strains used in this study.
Strain designation Genotype Deleted or inserted ORF/elements References
COM1 DpyrF PF1114 [11]
MW0430 DpyrF :: Pgdh-pyrF- Pslp9X-His Pslp9x-His, Pgdh-pyrF This study
MW0434 DpyrF :: Pgdh-pyrF- Pslp9X-His Pslp9x-His, Pgdh-pyrF This study
92 S.K. Chandrayan et al. / Protein Expression and Purification 107 (2015) 90–94
4. which compares with a yield of 17 mg of Strep-tag II SHI from the
same cell quantity (Table 3). As shown in Table 3, the yield of His-
SHI is more than 50-times that of untagged SHI from native bio-
mass and almost 1000-times the yield of heterologously-expressed
SHI from E. coli.
Identification and characterization of the trimeric NADPH
oxidoreductase
As shown in Fig. S1, the hydrogenase active fractions eluting
from the Ni–NTA column contained all four SHI subunits, but when
these fractions were further analyzed by Superdex-200 chromatog-
raphy, the later eluting fractions contained much less of the largest
of the four SHI subunits, the NiFe-containing a-subunit, when ana-
lyzed by SDS–PAGE (lane 9, 10 and 11 in Fig. S2). As shown in Fig. 3,
the resulting enzyme contained only three of the SHI subunits and
this trimeric form represented approximately 1% of the total SHI
from the Ni–NTA column. The proposed model of the trimeric form
(Fig. 3) predicts that it should not possess hydrogenase activity as
it lacks the NiFe-containing a-subunit but it might still catalyze
NADPH oxidation if a suitable electron acceptor was present. As
expected, the trimeric form did not exhibit significant hydrogen
evolution or hydrogen oxidation activity compared to the holopro-
tein tetrameric form of SHI (<0.1 and 1.1 units/mg, and 121 and
211 units/mg, respectively). However, the trimeric form did reduce
the artificial dye, benzyl viologen (BV) using NADPH as the electron
donor (BV oxidoreductase or BVOR activity). Moreover, the activi-
ties of the trimeric form and the holoprotein were comparable (98
and 86 units/mg, respectively). These data suggest that the trimeric
SHI is fully folded and completely functional, except it lacks the
catalytic a-subunit to couple NADPH oxidation to hydrogen
production.
Discussion
By changing the affinity tag from a Strep-tag II to a 9x-Histidine
tag, we were able to improve the protein yield of active SHI from P.
furiosus by almost an order of magnitude compared to a previously
published procedure and by almost three orders of magnitude
compared to the recombinant form produced in E. coli (Table 3).
Clearly, the poly-histidine tag does not interfere with nickel incor-
poration into SHI during biosynthesis. Moreover, in this study, it
was shown that the N-terminus of the b-subunit was a better posi-
tion for the affinity tag than the N-terminus of the a-subunit, pre-
sumably due to its accessibility. Since, a structure of a group 3b
[NiFe] hydrogenase is not available, the relative accessibilities of
the N-termini of the a- and b-subunits could not be addressed by
homology modeling. Nevertheless, with the availability of a highly
efficient purification procedure, sufficient SHI can be generated for
crystallographic studies and these are in progress. It is not clear
why the His-tag enabled a much more efficient and reproducible
purification of SHI compared to the use of the Step-tag. For exam-
ple, in our previous study [5], three 5-ml Strep-Tactin columns
were linked together and the flow-through material was reloaded
to optimize the yield of SHI. In contrast, for the same amount of P.
furiosus cytoplasmic extract (from 100 g of cells), the His tag ver-
e-
2H+ H2
FAD
NADPH NADP+
e-
e-
Ni CO
CN
CN
Fe
S
S
S
S
[4Fe-4S]
[4Fe-4S]
[4Fe-4S]
[2Fe-2S]
[4Fe-4S]
[4Fe-4S]
9xHis Tag
SHI α
SHI δSHI β
SHI γ
9xHis Tag
MW0430
MW0434
Fig. 2. A model based on sequence analysis showing the cofactor content and likely
pathway of electron flow in the four subunits of 9x-His SHI. Modified from [20].
Table 2
Purification His-SHI from MW0430 Strain.
Step Units
(U)
Protein
(mg)
Specific activity
(U mgÀ1
)
Yield
(%)
Fold
purification
S100 18973 4490 4.4 100 1
IMAC (Ni–NTA) 14503 120 121 76 27
Table 3
Protein yields from different purification procedures for SHI.
Protein Expression host Affinity tag Purification steps Protein yield (mg)1
References
Native SHI P. furiosus – 4 2.5 [2]
Recombinant SHI E. coli – 3 0.16 [20]
Strep-tag II SHI P. furiosus Strep-tag II 1 17.0 [5]
9x-His-SHI P. furiosus 9x His tag 1 135.0 This study
1
Protein yield from 100 g of cells (wet weight).
Fig. 3. Left: SDS–PAGE gel showing the three subunits of the trimeric form (SHI(3))
in comparison of the four subunits of the holoenzyme. Right: A model showing the
three subunits of the trimeric form. The abbreviations are: MV, methyl viologen;
BV, benzyl viologen. The activities shown in the table for each form are given in
lmol.
S.K. Chandrayan et al. / Protein Expression and Purification 107 (2015) 90–94 93
5. sion of SHI required only a one-step purification using a single 5 ml
Ni–NTA column. It has been reported that the 9x-Histidine and
Strep-tag-II versions of the maltodextrin-binding protein of P.
furiosus were purified with similar efficiency, although interest-
ingly the His-tagged form was successfully crystallized but that
was not the case with the Strep-tag II version [4]. The use of the
His-tagged form of SHI may therefore increase the chances of
obtaining crystals of this protein for structure determination.
A trimeric form of SHI was also characterized in this study and
shown to be catalytically-active toward NADPH oxidation but not
to have hydrogenase activity, in agreement with it lacking the
nickel-containing subunit that catalyzes hydrogen production.
Great progress has been made in our understanding of how the
NiFe-cofactor, which has carbon monoxide and cyanide ligands,
is synthesized and inserted deep into the catalytic site of the so-
called large subunit of [NiFe]-hydrogenases (subunit a in the case
of SHI, see Fig. 2). However, it is not clear whether folding and
assembly of the FeS-containing small subunit (subunit d in the case
of SHI, see Fig. 2) is independent of processing the large a-subunit
[7,15,16,19]. The isolation of an active and functional trimeric form
SHI, containing subunits b, c and d, suggests that this is an interme-
diate in hydrogenase maturation, to which the a-subunit is added
to and further processed.
Previously, for E. coli hydrogenase, the large subunit was iden-
tified in three different forms: (1) unprocessed containing iron
but not nickel (2) processed containing both iron and nickel
but inactive and (3) processed and active and in a complex with
the small subunit, where only the latter form appeared to be
stable [15,19]. This is also supported by our attempts to purify
a monomeric form of SHI, which contains only the catalytic
a -subunit. For MW0434, surprisingly, the active tetrameric SHI
could be purified even though the four-gene operon was split
to yield two separate transcripts. Moreover, in QPCR analysis of
MW0434 (data not shown), the expression of SHIa (controlled
by Pslp) was twice SHIbcd (controlled by PshI). With a 9X-Histidine
tag at the N-terminus of a-subunit of SHI, we expected to purify
both tetrameric and monomeric forms of SHI due to this differen-
tial expression. However, when Ni–NTA fractions of MW0434
were further analyzed by size exclusion chromatography, no
trace of the monomeric form of SHI could be observed. We have
also constructed another strain expressing only the 9x-Histidine
tagged SHIa without other three subunits. Unfortunately, the a-
subunit of SHI could not be purified neither in an active nor inac-
tive form. Therefore, our results support the view that [NiFe]-cat-
alytic subunit is not catalytically active or indeed stable without
the presence of the small subunit [15].
Funding
This work was supported by a Grant (DE-FG05-95ER20175 to
MWA) from the Division of Chemical Sciences, Geosciences and
Biosciences, Office of Basic Energy Sciences of the Department of
Energy.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.pep.2014.10.018.
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