From the ¶ Department of Pharmaceutical Sciences and the
** Molecular Biology Program, University of Colorado Health Sciences
Center, Denver, Colorado 80262 and the Department of
Microbiology and the
Molecular Biology Program,
University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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The terminase enzyme from bacteriophage Terminase enzymes are found in all of the large, tailed
double-stranded DNA bacteriophages and are responsible, at least in part, for the insertion of a viral genome into an empty, pre-formed shell or procapsid (1-3). In bacteriophage The preferred packaging substrate in phage is
composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is
responsible for packaging viral DNA into the confines of an empty
procapsid. We are interested in the genetic, biochemical, and
biophysical properties of DNA packaging in phage
and, in
particular, the nucleoprotein complexes involved in these processes.
These studies require the routine purification of large quantities of
wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1
and gpA subunits. We present a simple, one-step purification scheme for
the purification of large quantities of the holoenzyme and the
individual subunits directly from the crude cell lysate. Importantly,
we have developed a method to purify the highly insoluble gpNu1 subunit
from inclusion bodies in a single step. Hexa-His terminase holoenzyme
is functional in vivo and possesses steady-state and
single-turnover ATPase activity that is indistinguishable from
wild-type enzyme. The nuclease activity of the modified holoenzyme is
near wild type, but the reaction exhibits a greater dependence on
Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the
hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is
masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a
holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is
impaired in its interactions with the gpNu1 subunit of the enzyme. The
results reported here underscore that a complete biochemical
characterization of the effects of purification tags on enzyme function
must be performed prior to their use in mechanistic studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, the enzyme is composed of large (gpA,1 73.2 kDa) and
small (gpNu1,20.4 kDa) subunits that are isolated as a
gpA1·gpNu12 holoenzyme complex (4-6). These
proteins are an integral part of a series of nucleoprotein
intermediates involved in DNA packaging, however, and the subunit
stoichiometry in each of these intermediates likely differs (7-9).
consists of a
linear concatemer of viral genomes, linked head-to-tail and up to 10 genomes in length (10). The cohesive end site
(cos) of the viral genome represents the junction between
successive genomes in the concatemer and is the site where the
terminase subunits assemble to initiate DNA packaging (11-14). A model
for genome packaging has been proposed as follows (7-9) (see Fig.
1): 1) the terminase gpA subunit
assembles as a symmetric dimer onto the cosN subsite of
cos; 2) cooperative binding of gpNu1 to three repeating
R-elements found within the cosB subsite is required for
efficient gpA assembly at cosN and the stability of the
resulting pre-nicking complex; 3) the endonuclease activity of the gpA
subunit nicks the duplex at cosN and, after
terminase-mediated strand separation, yields the mature 12-base
single-stranded left end of
DNA bound and protected by the
terminase subunits; 4) this stable nucleoprotein intermediate, known as
complex I, next binds to an empty procapsid that triggers an
ATP-dependent translocation across the duplex and initiates
active DNA packaging; 5) upon encountering the next downstream
cos in the concatemer (the end of the viral genome),
terminase again nicks the duplex at cosN and strand
separation simultaneously releases the DNA-filled capsid and
regenerates complex I, which again captures an empty procapsid; and 6)
attachment of the tail to the DNA-filled capsid completes the assembly
process and yields a fully infectious virus.
View larger version (25K):
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Fig. 1.
Model for terminase assembly at
cos. The cos region of the genome
is shown at top. The three subsites, cosQ,
cosN, and cosB, are indicated, as are the three
gpNu1-binding elements (R1-R3) and the IHF binding element
(I1) found within cosB. The terminase subunits
and IHF assemble at cos forming a pre-nicking complex which,
in the presence of Mg2+, nicks the duplex ultimately
yielding the stable packaging intermediate complex I. Complex I binds
an empty procapsid which initiates an ATP-dependent
insertion of viral DNA into the capsid (Active DNA
Packaging). We note that the stoichiometry of the terminase
subunits in each of the nucleoprotein intermediates remains
speculative. Details are presented in the text.
Genetic experiments have identified functional domains within the terminase subunits. An N-terminal domain of gpNu1 contains a putative helix-turn-helix DNA binding motif identified by sequence homology ((15) A. Becker, cited in Ref. 7), and site-specific DNA binding has been localized to this region of the protein (16, 17). Terminase-procapsid interactions have been localized to the extreme C-terminal amino acids of the gpA subunit (18, 19), and it has been postulated that gpA·gpA dimer formation occurs through the putative leucine zipper motif identified within the primary sequence of the large subunit (20). Protein-protein interactions in the holoenzyme are mediated through the association of an N-terminal domain of gpA with the C terminus of gpNu1 (16, 21). Finally, biochemical experiments have identified two ATPase catalytic sites in the holoenzyme, a high affinity site in gpA and a low affinity, DNA-stimulated site in gpNu1 (22, 23).
Our laboratories are interested in the genetic, biochemical, and
biophysical properties of DNA packaging in phage and, in particular, the nucleoprotein complexes involved in these processes. These studies require a quick, simple, and efficient method to purify
large quantities of protein, especially for the biophysical studies of
the holoenzyme and its subunits. Toward this end, we have constructed
hexahistidine (hexa-His)-tagged protein subunits and have developed
simple, one-step protocols for the purification of phage
terminase
holoenzyme and the isolated enzyme subunits. Whereas these types of
constructs are extremely useful for the rapid purification of protein,
the purification tags unavoidably alter the primary sequence of the
protein, even with "cleavable" purification tags where one or two
amino acids remain after removal of the tag. It is thus imperative that
the functional significance of these "minor" changes in protein
structure be clearly defined prior to utilization of the constructs for
detailed structural and mechanistic studies. We have therefore examined
the effect of these hexahistidine tags on the catalytic competence of
terminase holoenzyme and the isolated enzyme subunits.
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EXPERIMENTAL PROCEDURES |
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Materials and Methods--
Tryptone, yeast extract, and agar
were purchased from Difco. Restriction enzymes were purchased from
Promega. Mung bean nuclease was purchased from New England Biolabs.
Guanidinium hydrochloride was purchased from Mallinckrodt.
[-32P]ATP was purchased from ICN. Unlabeled nucleoside
triphosphates and ampicillin were purchased from Sigma. Ni-NTA-agarose
was purchased from Qiagen. All other materials were of the highest
quality commercially available.
Bacterial cultures were grown in shaker flasks utilizing a New Brunswick Scientific series 25 incubator-shaker. Bacterial growth media and agar were prepared as described by Sambrook and co-workers (24). When required, kanamycin and ampicillin were added at 50 and 100 µg/ml, respectively. UV-VIS absorbance spectra were recorded on a Hewlett-Packard HP8452A spectrophotometer. Automated DNA sequence analysis was performed by the University of Colorado Cancer Center Macromolecular Resources Core facility or by the DNA sequencing facility at the University of Iowa. Both strands of the duplex were examined to verify the expected DNA sequence.
Bacterial Strains, DNA Preparation, and Protein Purification-- The viral and bacterial strains and the plasmids used in these studies are shown in Table I. Plasmids pSF1 and pAFP1 were purified from the Escherichia coli strains C600[pSF1] (25) and JM107[pAFP1] (26), respectively, using Qiagen DNA Prep® columns. Synthetic oligonucleotides used in this study were purchased from either Integrated DNA Technologies, Inc., or Life Technologies, Inc., and were used without further purification. Purification of wild-type terminase holoenzyme and the isolated wild-type gpA and gpNu1 subunits was performed as described previously (6, 27). E. coli integration host factor was purified from HN880 (generously provided by H. Nash, National Institutes of Health, Bethesda) by the method of Nash et al. (28). All of our purified proteins were homogenous as determined by SDS-PAGE and densitometric analysis using a Molecular Dynamics laser densitometer and the ImageQuant® data analysis package. Unless otherwise indicated, protein concentrations were determined spectrally using millimolar extinction coefficients (6, 27).
Construction of pQH101, a Hexa-His Terminase Holoenzyme
Overexpression Plasmid--
A vector that overexpresses terminase
holoenzyme with six histidines fused to the natural C-terminal glutamic
acid of the gpA subunit (see Fig. 2) was constructed by PCR methods
using pASY20 as a PCR template (Table I). This plasmid contains DNA extending from bp 22162
(SphI site) to bp 3522 (BssHI site) cloned into a
pIBI30 (International Biotechnologies) background (29). pASY20 thus
contains the wild-type
sequence for the A gene (3' end),
except for an XbaI site introduced at bp 2628 (Fig. 2) (29).
The forward PCR primer was complementary to bp 2206-2229 in the
A gene sequence and contained the SphI site. The
reverse PCR primer was complementary to bp 2615-2638 in the
A gene sequence, except that the TCC Ser codon at bp
2619-2621 (Ser-637, Fig. 2) was changed to an AGC. This introduced an
AflII site at bp 2615 in the A sequence but
maintained a wild-type serine codon. PCR amplification yielded the
expected 412-bp fragment that was isolated, digested with
SphI and XbaI, and ligated into similarly
digested pASY20. This afforded the plasmid pASY30 which was identical
to pASY20 except for the AflII site introduced at bp 2615. pASY30 was cut with AflII, and the cohesive ends were removed by digestion with mung bean nuclease. The linearized plasmid was next digested with BspEI (bp 3329) which deleted a
714-bp AflII-BspEI fragment (Fig. 2). The
following synthetic duplex 1 was ligated into doubly digested
pASY30.
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Construction of Phage -P1 Ahexa-His--
A
lysogen that expresses hexa-His terminase (
-P1
Ahexa-His) in place of the wild-type holoenzyme
terminase was constructed as follows. The Sse83871
restriction segment extending from
bp 2561 to 2824 (see Fig. 2) and
containing the modified segment of A gene (see above) was
cloned into the corresponding Sse83871 sites of pJM1 yielding pJM1-hexa-His. pJM1 is a derivative of pBR322 containing
DNA extending from bp 44141 through cos to 5505 (Table I). This 9-kilobase sequence includes the late promotor pR', the lysis genes S, R, and Rz, cos, the terminase genes
Nu1 and A, and genes W and
B. To cross the hexa-His modification of the A
gene into phage
, pJM1-hexa-His was crossed with
-P1
Aam42. Aam42 is an amber mutation in the
fifth-to-last codon of the A gene which renders the
terminase enzyme inactive (19). Plasmids carrying wild-type (pJM1) and
hexa-His-modified (pJM1-hexa-His) A genes were transformed
into MF1427 (
-P1 Aam42) yielding the lysogens MF1427
(
-P1 Awild-type) and MF1427 (
-P1
Ahexa-His), respectively. Selection for
transformed lysogens was accomplished by plating at 31 °C on L agar
plates containing kanamycin and ampicillin. The transformed lysogens
were grown overnight in L broth plus antibiotics at 31 °C. Prophages
were induced to undergo lytic growth by incubation at 42 °C for 20 min followed by incubation at 37 °C for 60 min. The lysates were
treated with CHCl3 and plated on MF1427 to titer the
-P1 Awild-type and
-P1
Ahexa-His recombinant phages.
Construction of gpA-Hexa-His and Hexa-His-gpNu1 Overexpression Plasmids-- A vector that overexpresses the terminase gpA subunit with six histidines fused to the natural C-terminal glutamic acid (see Fig. 2) was constructed by PCR methods previously described (27). The plasmid pSF1 contains wild-type Nu1 and A genes cloned into a pBR322 background (Table I) and was used as a PCR template. The forward primer contained an EcoRI site and was complementary to the first 21 nucleotides of the A gene, except that the GUG initiation codon in A was changed to an AUG codon with the use of the PCR primer. The reverse primer contained a BamHI site, six histidine codons, and the penultimate 18 nucleotides of the A gene. A vector that overexpresses the terminase gpNu1 subunit containing a methionine initiation codon and six histidines fused to the natural N-terminal (initiation) methionine (see Fig. 2) was similarly constructed. The primer sequences used were as follows: gpA forward primer, 5'-GAA TTC ATG AAT ATA TCG AAC AGT CAG-3'; gpA-hexa-His reverse primer, 5'-GGA TCC TCA ATG GTG ATG GTG ATG GTG TTC ATC CTC TCC GAA TAA-3'; hexa-His-gpNu1 forward primer, 5'-GAA TTC ATG CAC CAT CAC CAT CAC CAT ATG GAA GTC AAC AAA AAG-3'; gpNu1 reverse primer, 5'-GGA TCC TTA ACC TGA CTG TTC GAT ATA-3'. The EcoRI and BamHI restriction sequences are indicated in italics, and the f-MET (forward primers) and stop (reverse primers) codons are shown in bold type. Sequences complementary to the terminase genes are underlined, and the hexa-His codons are double underlined. PCR amplification of the A and Nu1 genes and construction of BL21(DE3)[pH6-A] and BL21(DE3)[pH6-Nu1], the gpA-hexa-His and hexa-His-gpNu1 expressing cell lines, respectively, were performed as described by Meyer et al. (27). We note that protein expression in these cell lines is inducible with IPTG.
Expression and Purification of Hexa-His Terminase
Holoenzyme--
One liter of 2× YT media containing 25 mM
NaH2PO4, pH 7.2, 1% glucose, and 50 µg/ml
ampicillin was inoculated with a 10-ml overnight culture of E. coli OR1265[pQH101] and maintained at 30 °C until an optical
density of 0.6 (600 nm) was obtained. The cells were then heat-induced
and harvested as described previously for wild-type holoenzyme (6). The
cell pellet was resuspended in 100 ml of 20 mM Tris-HCl
buffer, pH 8.0, containing 500 mM NaCl, 1 mM
PMSF, 0.4 mg/ml lysozyme, and 10 µg/ml aprotinin, and placed on ice
for 20 min. The cells were then lysed by sonication, and the insoluble
cellular debris was removed by centrifugation (12,000 × g for 15 min, followed by 12,000 × g for 30 min). The clarified supernatant was mixed with 5 ml of Ni-NTA agarose
followed by gentle shaking on ice for 1 h. The mixture was loaded
into an empty column and unbound protein was eluted with 2 × 4-ml
aliquots of 20 mM Tris-HCl buffer, pH 8, containing 500 mM NaCl. Bound protein was eluted in a stepwise fashion
with 20 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl and increasing concentrations of imidazole (4 × 0.5 ml each, 100, 150, and 250 mM imidazole). Hexa-His terminase holoenzyme eluted in the 250 mM imidazole
fractions that were pooled and dialyzed against 20 mM
Tris-HCl buffer, pH 8.0, containing 2 mM EDTA, 7 mM -ME, and 50% glycerol. The purified protein sample
was stored at
70 °C.
Expression and Purification of gpA-Hexa-His--
One liter of
2× YT media containing 25 mM
NaH2PO4, pH 7.2, 1% glucose, and 50 µg/ml
ampicillin was inoculated with a 10-ml overnight culture of E. coli BL21(DE3)[pH6-A] cells, and the culture was maintained at
37 °C until an optical density of 0.6 (600 nm) was obtained. The
cells were then induced with the addition of IPTG to 1.2 mM, and the culture was maintained at 37 °C for an additional 2 h. The cells were harvested by centrifugation, and the cell pellet was resuspended in 100 ml of 25 mM
Tris-HCl, pH 8.0, buffer containing 100 mM NaCl, 2 mM EDTA, and 7 mM -ME. Cell lysis was
affected as described above for hexa-His terminase holoenzyme, and the
clarified supernatant was dialyzed against 50 mM
NaH2PO4 buffer, pH 8.0, containing 500 mM NaCl and 25 mM imidazole. Five milliliters
of Ni-NTA agarose was added to the protein solution, and the mixture
was shaken gently on ice for 1 h. The mixture was then loaded into
an empty column, and the unbound protein was eluted with 2 × 4-ml
aliquots of 50 mM NaH2PO4 buffer,
pH 8.0, containing 500 mM NaCl and 50 mM
imidazole. gpA-hexa-His was finally eluted with 7 × 0.5-ml
aliquots of 50 mM NaH2PO4 buffer, pH 8.0, containing 500 mM NaCl and 250 mM
imidazole. The elution fractions were examined by SDS-PAGE, and the
appropriate fractions were pooled and dialyzed against 20 mM Tris-HCl buffer, pH 8.0, containing 100 mM
NaCl, 2 mM EDTA, 7 mM
-ME, and 50%
glycerol. The purified protein sample was stored at
70 °C.
Expression and Purification of Hexa-His-gpNu1--
Growth and
induction of E. coli BL21(DE3)[pH6-Nu1] cells was
performed as described above for gpA-hexa-His; however, similar to the
wild-type gpNu1 (27, 31, 32), all of the expressed hexa-His-gpNu1
protein was found in the crude cell lysis pellet (data not shown). The
protein pellet was resuspended in 50 ml of 25 mM Tris-HCl,
pH 8.0, buffer containing 100 mM NaCl, 2 mM EDTA, and 7 mM -ME and re-pelleted by centrifugation
(11,500 × g for 30 min). The washed pellet was next
solubilized with 20 ml of 6 M guanidinium HCl (GdmHCl), pH
8.0, gently shaken on ice for 1 h, and insoluble material was
removed by centrifugation (10,000 × g for 30 min).
Five milliliters of Ni-NTA-agarose was added to the clarified
supernatant which was gently shaken on ice for 1 h. The mixture
was then loaded into an empty column, and unbound protein was eluted
with 2 × 4-ml aliquots of wash buffer (10 mM
Tris-HCl, 100 mM NaH2PO4 buffer
containing 6 M GdmHCl) at pH 5.9. Nonspecifically bound
protein was eluted with 2 × 4-ml aliquots of wash buffer at pH
5.5, followed by 1 ml of wash buffer at pH 5.0. Hexa-His-gpNu1 protein
was finally eluted with 6 × 0.5-ml aliquots of the same buffer at
pH 4.5. The fractions were examined by SDS-PAGE, and the appropriate
fractions were pooled, dialyzed against 25 mM Tris-HCl
buffer, pH 8.0, containing 2 mM EDTA, 7 mM
-ME, 2.5 M GdmHCl, and 20% glycerol. The concentrated protein samples showed no signs of aggregation upon prolonged storage
at
70 °C in the presence of 2.5 M GdmHCl.
Reconstitution of Terminase Holoenzyme-- Reconstitution of terminase holoenzyme from the individually purified subunits was accomplished by mixing gpA and gpNu1 (wild-type or hexa-His-tagged) in a 1:2 molar ratio and incubating on ice for 5 min. We note that gpNu1 is stored as a concentrated stock in 2.5 M GdmHCl. Dilution of the protein sample during reconstitution simultaneously diluted the GdmHCl thus allowing re-folding of gpNu1 into an active enzyme complex. Control experiments confirmed that GdmHCl concentrations as high as 50 mM had no effect on the catalytic activities of the enzyme (not shown).
In Vitro DNA Packaging Assay--
The in vitro
packaging assay was performed as described by Chow et al.
(30). The reaction mixture (20 µl) contained 10 µl of a sonic
extract of an induced culture of MF2517 (Table I) in 30 mM
Tris-HCl buffer, pH 9.0, containing 10 mM
MgCl2, 3 mM spermidine, 6 mM
putrescine, 7 mM -mercaptoethanol, 1.5 mM
EDTA, 1.5 mM ATP, 1.5 nM of mature
cI857 Sam7 DNA, and the indicated concentration
of either wild-type or hexa-His-tagged terminase holoenzyme. The sonic
extract was prepared as described previously (33) and provides
proheads, tails, and assembly proteins required for virus assembly. The
reaction samples were incubated at room temperature for 30 min to allow
the assembly of infectious virus in vitro, and appropriate
dilutions were plated on the supF strain MF1968 to determine
virus yield.
In Vivo Virus Development (Virus Yield Assay)--
MF1427
(IHF+) or MF1972 (IHF), lysogenized with
-P1 carrying either a wild-type A gene (
-P1
Awild-type) or with
-P1 carrying the
hexa-His-A gene (
-P1 Ahexa-His),
were grown overnight with aeration in L broth plus kanamycin at
31 °C. To determine the number of viable lysogens in each culture, the cultures were diluted into L broth (1:100 dilution) and grown to
approximately 2 × 107 cells/ml. Then portions of each
culture were removed, diluted, and spread on L plates plus kanamycin;
the plates were incubated overnight at 31 °C. Lysogens were induced
by thermal induction at 42 °C for 20 min and then incubated at
37 °C for 60 min. The lysates were treated with chloroform,
clarified, and plated for phage yield on the IHF+ strain MF1427.
Terminase Activity Assays-- The cos cleavage assay was performed as described previously using pAFP1 as a nuclease substrate (6, 34). ATPase catalytic activity was examined as described previously (22). Where indicated, DNA (ScaI-linearized pAFP1) was added to the ATPase assay mixtures at a concentration of 25-50 nM. The concentration of protein used in these assays is indicated in each individual experiment.
Kinetic Analysis-- Steady-state ATPase activity was analyzed using linear regression techniques as described previously (6, 34). Only data within the linear portion of the reaction time course were used in the analysis. Data from single turnover experiments were analyzed according to both Equations 1 and 2, which describe monophasic and biphasic reaction time courses, respectively.
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(Eq. 1) |
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(Eq. 2) |
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RESULTS |
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Construction, Expression, and Purification of Hexa-His-gpNu1,
gpA-Hexa-His, and Hexa-His Terminase Holoenzyme--
Previous studies
have demonstrated that the C-terminal 38 amino acids of gpA define a
functional domain that is required for interaction of complex I with
the procapsid (18, 19). Interestingly, whereas the penultimate 5 amino
acids are strictly required for procapsid binding (19), addition of up
to 4 random amino acids to the C terminus of the gpA subunit did not
appear to significantly affect the phage yield in E. coli
(29). Based upon these data, we reasoned that the addition of six
histidines to the C terminus of the gpA subunit might provide a
convenient and efficient purification tag with little effect on enzyme
function. Vectors that overexpress the hexa-His-tagged gpA subunit,
alone and co-expressed with the wild-type gpNu1 subunit, were thus
constructed as described under "Experimental Procedures." We
further constructed a vector for the expression of an isolated gpNu1
subunit that contains a hexa-His tag at the N
terminus.3 The C-terminal
amino acid sequence of gpA-hexa-His and the N-terminal amino acid
sequence of hexa-His-gpNu1 are shown in Fig.
2. For the purpose of clarity, we use the
term terminase holoenzyme to describe enzyme that was directly purified
as a gpA1·gpNu12 holoenzyme complex from
cells simultaneously expressing both enzyme subunits and the term
reconstituted terminase holoenzyme to describe enzyme that was prepared
by mixing the individually purified subunits.
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Both hexa-His terminase holoenzyme and the isolated gpA-hexa-His
subunit were efficiently expressed in E. coli (Fig.
3) and were found in the soluble fraction
of the crude cell lysate (not shown). The proteins were purified from
the clarified cell lysate in a single step as described under
"Experimental Procedures" and yielded hexa-His terminase holoenzyme
(10 mg/liter cells) and gpA-hexa-His (12 mg/liter cells) preparations
that were >95% homogenous as determined by SDS-PAGE. The isolated
hexa-His-gpNu1 subunit was similarly efficiently expressed in E. coli (Fig. 3). However, as is observed with the wild-type protein
(31, 32, 35), hexa-His-gpNu1 was found exclusively in the insoluble
cell lysis pellet (data not shown). The protein was solubilized from these inclusion bodies using 6 M guanidinium hydrochloride
and purified to homogeneity in a single step using the nickel-chelate column as described under "Experimental Procedures" (29 mg/liter cells, >95% pure). Hexa-His-gpNu1 has been stored as a concentrated protein solution in 2.5 M GdmHCl for up to 12 months with
no evidence of aggregation and with no loss of catalytic activity.
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In Vitro Packaging Activity of Wild-type and Hexa-His Terminase
Holoenzyme--
An in vitro packaging assay was used to
examine the biological activity of the purified hexa-His-tagged
terminase holoenzyme. This assay utilizes extracts of induced cultures
of E. coli MF2517 (Table I) as
a source of viral procapsids, tails, and all the necessary assembly
proteins, except for a functional terminase enzyme (30). Addition of
viral DNA and terminase holoenzyme allows virus assembly in
vitro and yields fully infectious phage that are quantitated by
their ability to form plaques (pfu). Fig. 4 shows that both wild-type and hexa-His
terminase holoenzymes are biologically active and may be used to
assemble infectious virus in vitro. We note, however, that
the concentration dependence of pfu formation is slightly greater for
hexa-His terminase than for the wild-type enzyme, with 10-fold more
enzyme required for similar phage yields. This difference disappears at
enzyme concentrations greater than 10 nM, however,
presumably because terminase is no longer limiting in the assay
mixture.
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ATPase Activity of Wild-type and Hexa-His Terminase
Holoenzyme--
We have previously identified two ATPase catalytic
sites in terminase holoenzyme, a high affinity site in gpA
(Km 5 µM) and a low affinity,
DNA-stimulated site in gpNu1 (Km
1,300 and 500 µM, minus and plus DNA, respectively) (22, 23). Fig.
5A shows the steady-state rate
of ATP hydrolysis using an ATP concentration of 1 mM and
thus examines ATPase activity of both catalytic sites in the
holoenzyme. The figure shows that the ATPase activity of both wild-type
and hexa-His-tagged terminase holoenzymes are essentially identical.
Moreover, the ATPase activity of both the wild-type and mutant
holoenzymes is significantly stimulated by DNA, and the degree of
stimulation is virtually identical for each (Fig. 5A, Table
II). Furthermore, the steady-state rate
of ATP hydrolysis by both enzymes using an ATP concentration of 20 µM, a concentration where catalytic activity is
predominantly localized within the gpA subunit, is similarly identical
(Fig. 5B and Table II).
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In order to characterize more fully the effect of the hexa-His purification tag on ATP hydrolysis activity, we next examined the rate of ATP hydrolysis in a single-turnover experiment. In this experiment, the concentration of enzyme and ATP were both 25 nM, and a single catalytic turnover by the enzyme is observed. Furthermore, the low concentration of ATP used ensures that hydrolysis is observed at the high affinity gpA subunit of the enzyme only. Fig. 5C shows that under these experimental conditions, both wild-type and hexa-His terminase holoenzymes hydrolyze ATP with a similar time course and that both proteins are similarly stimulated by DNA. These data were analyzed as described under "Experimental Procedures" yielding the observed rate constants presented in Table III.
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Endonuclease Activity of Wild-type and Hexa-His Terminase
Holoenzyme--
Terminase holoenzyme possesses a site-specific
nuclease activity that is required for excision of a single genone from
a concatemeric precursor. The site at which terminase assembles and
cleaves the duplex is known as cos, an abbreviation for the
cohesive end site of the viral genome (see Fig.
1). Fig. 6A shows the results of a
cos-cleavage activity assay for wild-type and hexa-His
terminase holoenzymes. Whereas both enzymes exhibit significant
activity in this assay, the time course for the hexa-His-tagged enzyme lags slightly behind that of wild-type terminase. We have suggested that assembly of the terminase subunits onto DNA is the rate-limiting step in the cos-cleavage reaction (6, 34), and these data suggested that the hexa-His-tagged holoenzyme might have impaired DNA
binding interactions. In order to explore more fully this possibility,
we examined the cos-cleavage activity of these enzymes in
the absence of E. coli integration host factor (IHF). IHF, although not strictly required for cos-cleavage activity
in vitro or virus assembly in vivo, stimulates
these reactions (6, 36, 37); however, IHF becomes essential for plaque
formation when terminase assembly at cos is impaired by
mutations in the cos sequence of the genome (26, 38).
Fig. 6B demonstrates that the cos-cleavage
activity of hexa-His terminase is significantly reduced in the absence
of IHF, whereas that of wild-type enzyme is only modestly affected.
These data support the posit that the hexa-His-tagged holoenzyme is
slightly impaired in its interactions with cos-containing
DNA.
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Catalytic Activity of the Isolated Wild-Type and Hexa-His-tagged Terminase Subunits-- We (17) and others (39) have previously demonstrated that whereas gpNu1 is devoid of cos-cleavage activity, the isolated gpA subunit possesses a weak nuclease activity that is strongly stimulated in the holoenzyme complex. Table IV demonstrates that the hexa-His-tagged subunits behave similarly to the wild-type subunits. Moreover, the mutant proteins may be reconstituted into catalytically competent holoenzyme complexes that possess cos-cleavage activity. This is particularly true for the hexa-His-tagged gpNu1 subunit that may be reconstituted into an enzyme complex that is fully active compared with wild-type reconstituted enzyme (Table IV). Consistent with the cos-cleavage activity of wild-type and hexa-His terminase holoenzymes, terminase reconstituted with a hexa-His-tagged gpA subunit possesses nuclease activity that is slightly impaired when compared with wild-type reconstituted enzyme (see Table IV and Fig. 6A).
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Studies on the ATPase activity of the isolated terminase subunits have similarly revealed that the isolated gpNu1 subunit possesses weak ATPase activity while gpA efficiently hydrolyzes ATP (17, 34, 40). Consistently, the isolated hexa-His gpNu1 subunit possesses modest ATP hydrolysis activity, whereas hexa-His gpA hydrolyzes ATP at a rate similar to that of the wild-type subunit (Table V). Reconstitution of terminase with wild-type gpA and hexa-His gpNu1 subunits affords a holoenzyme with ATPase activity that is virtually identical to that obtained with a fully wild-type enzyme (Table V). Interestingly, however, reconstitution of the isolated gpA-hexa-His subunit with either wild-type or hexa-His-gpNu1 yields an enzyme complex that does not possess ATP hydrolysis activity beyond that of the isolated gpA-hexa-His subunit alone (Table V).
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In order to characterize more fully the ATPase activity of the isolated
gpA-hexa-His subunit, a single-turnover kinetic analysis of ATP
hydrolysis was performed, and the results are presented in Fig.
7 and Table
VI. The wild-type gpA subunit hydrolyzes
ATP with a time course that is well described by a single exponential curve function (Fig. 7A) and with an observed rate that is
similar to that observed for terminase holoenzyme in the absence of DNA (compare Tables III and VI). This confirms that at this concentration of ATP, hydrolysis in the holoenzyme is limited to the gpA subunit of
the enzyme. Addition of DNA to the reaction mixture strongly stimulates
the ATPase activity of the wild-type gpA subunit and the rate of ATP
hydrolysis is more appropriately described by a double-exponential
curve function. Analysis of these data yield the fast and slow rate
constants presented in Table VI. Interestingly, the rate constant for
the fast phase of the reaction is similar to that observed for
terminase holoenzyme in the presence of DNA (compare Tables III and
VI), suggesting that at this concentration of ATP, the observed
stimulation of ATP hydrolysis in holoenzyme is mediated by DNA
interactions with the gpA subunit.
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Unlike wild-type protein, single-turnover ATP hydrolysis by the isolated gpA-hexa-His subunit exhibits biphasic behavior, even in the absence of DNA (Fig. 7B). Moreover, the rate constants obtained from analysis of these data are quite similar to the kslow and kfast rate constants obtained for wild-type gpA in the presence of DNA (Table VI). Only a small proportion of the time course (14-22%) is attributable to kfast for the hexa-His-tagged protein, however, and unlike the wild-type protein, gpA-hexa-His is unresponsive to DNA (Fig. 7B and Table VI).
The Effect of Hexa-His Terminase on in Vivo Virus
Development--
-Pl Ahexa-His was
constructed by crossing phage
-P1 Aam42 (Table I) and
plasmid pJM1-hexa-His as described under "Experimental Procedures."
The Aam42 mutation is a lethal amber mutation located in the
5th-to-last codon of the A gene. Since there are only 4 codons between the Aam42 mutation in the phage and the six
His codons of the modified A gene in the plasmid, it was
expected that virtually all plaque-forming
A+ recombinant phage would contain
the hexa-His modification.4
This was confirmed by sequencing studies on the
-Pl
Ahexa-His recombinants, which directly
demonstrated the presence of the hexa-His modification. The yield of
plaque-forming (A+) recombinants was 1.43 × 107 and 1.62 × 107 pfu/ml in crosses
with pJMI-Awild-type and
pJM1-Ahexa-His plasmids, respectively. A control
cross with no plasmid yielded less than 1 × 104
pfu/ml, indicating that revertants of the
-P1 Aam42 phage
were not contributing significantly to the titers of the cross-lysates. Since the frequencies of plaque-forming recombinants were essentially the same for each plasmid, we conclude that terminase with the hexa-His
modification is functional in virus development.
Effect of IHF on -Pl Awild-type and
-Pl
Ahexa-His in Vivo Virus Development--
The distinct
requirement for IHF in cos-cleavage by hexa-His terminase
holoenzyme (Fig. 6) suggested that phage development in vivo
might exhibit a similar requirement for IHF. This is especially important as the hexa-His purification tag might be expected to weaken
interactions with the procapsid and thus require increased stability of
complex I to ensure that progression toward active DNA packaging would
occur. We thus examined the requirement for IHF on in vivo
phage development by
-P1 Awild-type and
-P1 Ahexa-His, viruses that express wild-type
and hexa-His-tagged terminase holoenzymes, respectively (Table I).
Initial studies demonstrated that although
-P1
Ahexa-His formed normal plaques an IHF
+ host, the mutant phage formed minute pinpoint
plaques on an IHF-deficient host (data not shown). To quantify these
effects, virus burst studies were performed using
-P1
Awild-type and
-P1
Ahexa-His lysogens in IHF+ and
IHF
hosts. Table VII shows
that the yield of
-P1 Ahexa-His was, within
experimental error, identical to that of
-P1
Awild-type when an IHF+ strain was
used. This result is similar to that observed in the in
vitro DNA packaging (Fig. 4) and cos-cleavage (Fig. 6)
assays, where only modest effects are observed between the wild-type
and hexa-His-tagged holoenzymes in the presence of IHF. Whereas both phages showed significantly reduced yields in the IHF
host, the deficit was much more pronounced for
-P1
Ahexa-His (Table VII). In fact, the observed
decrease in burst size
-P1 Ahexa-His was
sufficient to lower virus yield to a level just above that required for
plaque formation on an IHF
host, thus yielding minute
pinpoint plaques.
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DISCUSSION |
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The terminase enzyme from bacteriophage lambda is responsible, at
least in part, for the insertion of viral DNA into a procapsid (7-9).
Our laboratories are interested in the genetic, biochemical, and
biophysical aspects of phage assembly and, specifically, the
nucleoprotein complexes required for genome packaging. These studies
require a simple, rapid, and efficient purification scheme for the
isolation of large quantities of wild-type and mutant terminase
holoenzymes, as well as the isolated enzyme subunits. The use of
terminase in the biotechnology industry further underscores the need
for simple and efficient purification protocols. Several vectors have
been developed for the expression of terminase holoenzyme and the
individual subunits in E. coli (27, 30), and several purification schemes have been published over the years (4, 6, 23, 31,
32). Even the most efficient protocols are relatively laborious and
time consuming, however, for the routine purification of large
quantities of enzyme. Purification of hexa-His-tagged proteins using
nickel-chelate chromatography has become routinely used for rapid and
efficient protein purification, and we thus constructed vectors that
express hexa-His-tagged holoenzyme and the individual terminase
subunits. All of the vectors described here express significant
quantities of protein, and our purification protocols allow their
purification in one step from the crude cell lysate. Importantly, we
have developed a method to purify the highly insoluble terminase gpNu1
subunit in a single step from the crude cell lysis pellet in the
GdmHCl-denatured state. This protein may be stored as a concentrated
stock solution that, upon dilution of the GdmHCL, refolds into an
active conformation.
Expression from the vectors described above yield proteins that possess a hexa-His purification tag directly attached to the C-terminal glutamic acid of gpA or the N-terminal methionine of gpNu1 (see Fig. 2). Most vectors that express hexa-His-tagged proteins provide a cleavage site that allows the removal of most, but not all, of the purification tag. Previous studies have suggested that addition of 4 amino acids to the C terminus of the gpA subunit did not significantly affect in vivo viral development (29), and we reasoned that the 6 histidines at the C terminus of gpA-hexa-His would similarly be tolerated. Our expression vectors were thus constructed to add a minimal purification tag that would remain part of the purified protein. This concept provides a simple method to purify the proteins and avoids a proteolysis step that would add time and significant expense to the purification procedure.
Although the addition of 4 random amino acids to the C terminus of the gpA subunit did not affect terminase function in vivo, it was necessary to confirm that addition of the hexa-His purification tag similarly did not affect the catalytic activities of the enzyme. Initial experiments demonstrated that the tag only modestly affected phage development in vivo and phage assembly in vitro. A more detailed investigation is required, however, if these proteins are to be used for mechanistic studies on the enzyme, and we next examined the ATPase activity of the modified enzyme. Both steady-state and single-turnover kinetic experiments demonstrated that ATP hydrolysis by terminase holoenzyme is little affected by introduction of the hexa-His purification tag into the gpA subunit. Importantly, both enzymes hydrolyze ATP with identical rates, and both enzymes are similarly responsive to DNA. Similarly, only modest differences were observed between the enzymes in our standard cos-cleavage activity assay; however, further investigation demonstrated that hexa-His terminase holoenzyme has a mild DNA-binding defect that is masked by IHF, an E. coli protein known to stabilize terminase-DNA interactions. This result was also apparent in experiments that similarly showed a significant requirement for IHF in the in vivo development of a hexa-His terminase-containing phage.
The mild defect in hexa-His terminase holoenzyme was significantly magnified in the isolated gpA-hexa-His subunit. Unlike wild-type protein, gpA-hexa-His exhibited biphasic ATPase kinetics in the absence of DNA and was completely unresponsive to the addition of polynucleotide. These data suggest that while gpA-hexa-His in a holoenzyme complex is relatively "normal," the isolated subunit is more severely impaired, particularly in its interactions with DNA. Moreover, the isolated gpA-hexa-His subunit does not appear to interact appropriately with gpNu1 to form a "natural" holoenzyme complex. Although the nuclease activity of terminase reconstituted from the gpA-hexa-His subunit is near wild type, this reconstituted holoenzyme does not possess ATPase activity beyond that observed with the isolated enzyme subunits. Conversely, however, terminase reconstituted from wild-type gpA and a hexa-His-tagged gpNu1 subunit yields a catalytically competent holoenzyme complex with wild-type nuclease and ATPase activities.
During the course of this investigation, we have uncovered an
interesting aspect of ATP hydrolysis by terminase holoenzyme. Previous
studies have suggested that the steady-state rate of ATP hydrolysis by
the gpA subunit in terminase holoenzyme was unaffected by DNA and that
DNA-mediated stimulation of ATPase activity occurred primarily at the
low affinity, gpNu1 ATP-binding site of the enzyme (22). Contrary to
these earlier results, however, the data presented here demonstrate
that DNA directly stimulates ATP hydrolysis at the high affinity gpA
subunit of the enzyme. Steady-state kinetic experiments performed at an
ATP concentration of 20 µM, a concentration well below
the Km for ATP binding by the gpNu1 subunit (500 µM), show a 4-5-fold increase in the rate of ATP
hydrolysis with the addition of DNA to the reaction mixture. Virtually
identical results were obtained in the single-turnover experiments
under conditions where ATP hydrolysis at the low affinity gpNu1
catalytic site is expected to be minimal. The above experiments were
performed with terminase holoenzyme, however, and it was feasible that
DNA interactions with the gpNu1 subunit were responsible for the
observed increases in catalytic activity. Single turnover experiments
with the isolated gpA subunit were thus performed to provide additional
mechanistic insight. The single-turnover rate constant for ATP
hydrolysis by the wild-type gpA subunit was virtually identical to that
of the holoenzyme, confirming that ATP hydrolysis at the gpNu1 subunit under these conditions is essentially zero. Addition of DNA to gpA
resulted in the appearance of biphasic kinetics and the introduction of
a fast rate of ATP hydrolysis. Interestingly, the rate constant for the
fast phase of the reaction was quite similar to the rate constant
obtained with the holoenzyme, again suggesting that DNA stimulates ATP
hydrolysis directly at the gpA subunit. However, although a single,
fast rate was observed with the holoenzyme, only 50% of the time
course may be ascribed to the fast phase with the isolated gpA subunit.
These data suggest that protein-protein interactions with gpNu1 in the
holoenzyme complex are required for full expression of DNA stimulation
at the gpA subunit. The mechanistic implications of the biphasic
kinetics observed in these single-turnover experiments, and the
protein-protein interactions affecting these kinetic profiles is
currently under investigation in our laboratory.
In conclusion, we have constructed vectors that efficiently express
hexa-His-tagged terminase holoenzyme and the individual enzyme
subunits, and we have developed simple purification protocols that
allow their purification in one step from the crude cell lysates. The
catalytic activities of the tagged holoenzymes is, at first glance,
indistinguishable from the wild-type enzyme. Moreover, phages that
express these mutant proteins efficiently replicate in vivo,
perhaps the most stringent test of biological activity. Upon closer
examination, however, the catalytic properties of these proteins reveal
subtle defects. The primary defect appears to be in the interaction of
gpA-hexa-His with DNA. Although most apparent with the isolated
subunit, effects are also observed with the hexa-His-tagged holoenzyme.
The deficiency can be overcome with the addition of IHF to the reaction
mixture, and these proteins will find utility in a number of
experimental systems; however, the results reported here underscore
that a complete biochemical characterization of the effects of
purification tags on enzyme function must be performed prior to their
use in mechanistic studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM50328-03 (to C. E. C.) and GM51611 (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 303-315-8561;
Fax: 303-315-6281; E-mail: carlos.catalano{at}uchsc.edu.
2 DNA sequences are listed as described by Daniels et al. (41).
3 Given that gpA-gpNu1 interactions occur at the C terminus of the gpNu1 subunit, we did not attempt to construct a C-terminally hexa-His-tagged gpNu1 protein as we felt that reconstitution of terminase holoenzyme would be adversely affected.
4
An alternative outcome was that the hexa-His tag
in -P1 Ahexa-His would interfere with virus
development and be lethal. In this event, few viable
Ahexa-His recombinants would be expected.
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ABBREVIATIONS |
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The abbreviations used are:
gpA, the large
subunit of phage terminase;
gpA-hexa-His, a gpA subunit containing
a hexahistidine purification tag at the C terminus of the protein;
bp, base pair;
-ME, 2-mercaptoethanol;
cos, cohesive end site, the junction between individual genomes in immature
concatemeric
DNA;
gpNu1, the small subunit of phage
terminase;
GdmHCl, guanidinium hydrochloride;
hexa-His-gpNu1, a gpNu1 subunit
containing a hexahistidine purification tag at the N terminus of the
protein;
hexa-His terminase, phage
terminase enzyme containing a
hexahistidine purification tag at the C terminus of the gpA subunit;
IHF, E. coli integration host factor;
PAGE, polyacrylamide
gel electrophoresis;
phage
-P1 Awild-type, bacteriophage lambda that carries a wild-type terminase holoenzyme;
phage
-P1 Ahexa-His, bacteriophage lambda
virus that carries a hexa-His terminase holoenzyme construct;
reconstituted terminase holoenzyme, enzyme that was prepared by mixing
the individually purified subunits;
terminase holoenzyme, enzyme
directly purified as a gpA1·gpNu12 holoenzyme
complex from cells simultaneously expressing both enzyme subunits;
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
PCR, polymerase chain reaction;
Ni-NTA, nickel-nitrilotriacetic acid;
pfu, plaque-forming units.
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REFERENCES |
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