Advances in Bioscience and Biotechnology, 2010, 1, 417-425 ABB
doi:10.4236/abb.2010.15055 Published Online December 2010 (
Published Online December 2010 in SciRes.
Cloning, expression, purification and characterization of
replication protein from plasmid pGP2 from Acetobacter
Peter Grones, Jozef Grones
Comenius University, Department of Molecular Biology, Mlynska dolina B2, 842 15 Bratislava 4, Slovak Republic.
Received 10 September 2010; revised 22 October 2010; accepted 8 November 2010.
The Acetobacter estunensis Rep34 protein participates
in the replication of bacterial plasmid pGP2. The
Rep34 protein of the A. estunensis, was cloned to the
expression vector, that ensure fusion with a His-tag
sequence (Rep34 His-tagged), over-expressed in Es-
cherichia coli and purified by metal-affinity chroma-
tography to yield a highly purified and active protein.
On this purified protein number different activities
and motifs were detected. DNA band-shift assays
showed that the Rep34 His-tagged protein bound to
the regulation region for replication on the linear
double-stranded DNA. In the protein was determined
phosphatase activity, ATPase activity and protein is
possible to unwind double strand DNA.
Keywords: Acetobacter Estunensis; Rep34 Protein;
DNA-Binding Activity; ATPase Activity; Phosphatase
Activity; Unwinding Activity
The replication of eukaryotic and prokaryotic chromo-
somes, bacteriophages and bacterial plasmids DNA in-
volves several analogous events and similarities in
replisome architecture. Many systems have specific ini-
tiation proteins, including bacterial DnaA protein, phage
lambda O protein, plasmid replication initiation proteins
(Rep) and the eukaryotic origin recognition complex
(ORC). The specific mechanism for replication initiation
of a given replicon is dependent on both the structure of
the replication origin and the nature of the replication
initiation protein. The replication of bacterial ex-
tra-chromosomal replicons, such as plasmids or phages
is generally limited to a single host or a few closely re-
lated host´s proteins [1]. Replication proteins generally
initiates and regulates bacterial chromosome or plasmid
replication and serves as a transcription factor [2,3].
The origins of prokaryotic and some eukaryotic repli-
cons possess characteristic functional elements, include-
ing specific binding sites for the appropriate initiation
protein and an AT-rich region where DNA duplex desta-
bilization occurs. Plasmid origins usually contain multi-
ple binding sites (iterons) for the plasmid-specific repli-
cation initiation protein as well as one or more binding
sites for the host replication initiation protein, DnaA
(DnaA boxes) [1]. These proteins interact with repeated
regulation sequences. They also interact with Rep pro-
tein or DnaA protein on regulation boxes, which are lo-
cated within the ori regions or the promoter regions or
intergenic regions of many bacterial chromosomes and
plasmids [4].
The structural elements of the origin are employed for
broad-host-range plasmid replication and maintenance in
different host bacteria species. For example, the minimal
origin of the broad-host-range plasmid RK2 possesses
five iterons and is functional in E. coli. However, the
presence of three iterons stabilizes RK2 plasmid main-
tenance in Pseudomonas putida [5]. In addition, the re-
gion with four DnaA boxes is essential for RK2 replica-
tion in E. coli, but is dispensable for replication of the
plasmid in Pseudomonas aeruginosa [6,7].
In the E. coli chromo s o me, the replication origin (oriC)
contains five DnaA box sequences. The binding of mul-
tiple DnaA molecules in the presence of the histone-like
HU protein and the site-specific DNA-binding protein
IHF (integration host factor) results in destabilization of
the duplex DNA within the nearby AT-rich sequences of
the oriC of E. coli [3]. Origin opening of the narrow-
host-range plasmids pSC101, F, P1, and R6K requires, in
addition to E. coli DnaA, HU and/or IHF proteins, the
binding of plasmid-encoded replication initiation pro-
teins [8-12]. Similarly, the formation of an open complex
at the replication origin of the broad-host-range plasmid
RK2 by the plasmid encoded TrfA initiation protein re-
quires E. coli HU, and is stabilized by E. coli DnaA [13].
In contrast to the chromosomal oriC, plasmid origins
P. Grones et al. / Advances in Bioscience and Biotechnology 1 (2010) 417-425
Copyright © 2010 SciRes. ABB
do not require ATP for open complex formation [8-10].
A basis for this lack of dependence on ATP, induced in
an ATP-independent mode, by the complex of the plas-
mid-encoded Rep protein and the host HU or IHF
Plasmids encoded systems that control their replica-
tion such that fairly precise, steady-state copy numbers
are maintained [16]. Plasmid replicons from Gram-
negative bacteria always seem to encode a negative
feedback control system. Two basic mechanisms for the
regulation of plasmid replication have been recognized
so far: one operates via an antisense RNA transcript that
negatively regulates the replication; the other operates
via iterons, a series of direct repeat sequence located
within ori that intereract with iteron-binding Rep pro-
teins are responsible for both the initiation of replication
and its control [16,17].
From acetic acid bacteria were purified and character-
ised several DNA plasmids which encoded rep gene
which product is able to regulate replication process.
The first identified cryptic plasmid from Acetobacter
encoding Rep protein had been used for the construction
of cloning vectors [18,19]. Later from Acetobacter pas-
teurianus was identified large plasmid pAC1 [20], and
pAP12875 [21]. From Gluconobacter was isolated
plasmid pJK2-1 [22], and from A. aceti plasmid pAG20
In this paper we presented replication protein of plas-
mid pGP2 isolated from Acetobacter estunensis GP2
strain and characterise main activities that belong to the
bacterial replication proteins.
1.1. Bacterial Strains and Cultivation Media
Escherichia coli strain XL1 Blue (tetracyclineR) [24]
was used for plasmid isolation and for cloning DNA
fragments and strain BL21 (DE3) [F ompT gal dcm lon
- mB
-) λ(DE3 [lacI lacUV5-T7 gene1 ind1 sam7
nin5]) was used as a host for protein expression. The
pGP2 plasmid isolated from Acetobacter estunensis GP2
used as template for rep34 gene. Acetobacter strain was
cultivated in YPG medium (5% yeast extract, 3% pep-
tone, and 1% manitol) and E. coli strain on LB medium
(10% Tryptone, 5% yeast extract and 5% NaCl pH 7.4)
supplemented with 50 µg/ml kanamycin and 100 µg/ml
1.2. Biochemical Material
The reagents for PCR and oligonucleotide primers were
obtained from Invitrogen Life Technologies (Carlsbad,
CA, USA). The bacterial vector pGEM-T Easy (ampicil-
linR; Promega, Madison, WI, USA) expression pET-
28a+ vector (T7 promoter, kanamycinR; Novagen, Madi-
son, WI, USA), was used for cloning and expression rep
gene. Restriction endonucleases, isopropylthio-β-D-
galactopyranoside (IPTG), and T4 DNA ligase were ob-
tained from BioLabs. p-nitrophenyl phosphate (pNPP)
and protein standard used as SDS–PAGE marker were
from Sigma Chemical (St. Louis, MO, USA).
1.3. Cloning and Expression Vector
Recombinant DNA techniques were performed using
conventional protocols. The rep34 gene of Acetobacter
estunensis (ORF2) was amplified using: forward
and reverse primer 5´-AAG CTT TTA TTC AGA TGG
CGG CTT G–3´. The amplified DNA encoding the rep34
gene produced a fragment of around 627 bp was cloned
into pGEM-T Easy vector and transformed in E. coli
XL1. Selected construct pGEM-rep34 was sequenced by
dideoxy chain termination method [25] using an ABI
Prism 3200 automated DNA sequencer (Applied Bio-
systems, Foster City, CA, USA). Sequenced data were
analyzed using BLAST-Basic local alignment search
tools program [26] through the network service of the
National Center for Biotechnology Information (http://
The DNA encoding the rep34 was then sub-cloned into
vector pET-28a- in BamHI and HindIII sites which was
used to transform E. coli XL1. The new vector construct
was named pET–rep34. Sequencing of the cloned vectors
revealed the open reading frame (ORF2) of the rep34
gene plus the expected 32 additional amino acid residues
derived from pET-28a- vector at its amino terminus
including the cluster of six histidine residues for protein
purification by metal affinity chromatography. The ex-
pression vector pET-rep34 was extracted from the trans-
formants using JETQuick Plasmid kit (Genomed), and
used to transform to E. coli BL21 (DE3) competent cells.
Selected transformant pET-28-rep34 was used for protein
expression and purification.
1.4. Expression and Purification of Recombinant
A total of 100 µl of an overnight culture of E. coli BL21
(DE3) with pET-28-rep34 was diluted into 100 ml of LB
medium containing kanamycin (50 µg/ml). The culture
was grown at 37 until optical density at 590 nm
reached 0.6 and was induced with 0.5 mM IPTG. The
incubation continued for an additional 2 h in the same
temperature. After incubation, the culture was harvested
by centrifugation at 8,000 g for 10 min at 4. The pellet
cells were suspended in 25 mM Tris.HCl, pH 8.0 buffer
P. Grones et al. / Advances in Bioscience and Biotechnology 1 (2010) 417-425
Copyright © 2010 SciRes. ABB
containing 20 mM NaCl, and 5% glycerol and disrupted
for 10 min at 4 followed by sonication. The suspend-
sion was then centrifuged at 16,000 g for 10 min at 4
to separate the cell debris, and the solubility of the re-
combinant fusion protein was analyzed by 12% SDS–
PAGE. The soluble fraction containing recombinant
pET-rep34 was loaded onto a Ni–NTA column (Qiagen,
Hilden, Germany) pre-equilibrated with 50 mM sodium
phosphate, pH 8.0 buffer containing 300 mM NaCl and
10 mM imidasole. The unbound proteins were washed
out with the same buffer used for equilibration of the
column. Subsequently, the recombinant proteins were
eluted with the same buffer containing 50 to 250 mM
imidazole. The resulting pET-rep34 was exhaustively
dialyzed in 50 mM Tris.HCl, pH 8.0 buffer containing
50 mM NaCl for elimination of imidazole. The protein
purity was confirmed by the presence of a single band on
SDS–PAGE 12% of molecular weight predicted for the
Rep34 (about 25.5 kDa, with His-tag). For protein visu-
alization, the gels were stained with Coomassie Brilliant
Blue G-250 (Bio-Rad Laboratories) and distained with
10% acetic acid and 20% methanol. The purified protein
was frozen and stored at –80. Protein concentration
recombinant soluble protein concentration was deter-
mined by UV absorbance at 280 nm (Spectrophotometer
ND-1000 UV-Vis, NanoDrop Technologies).
1.5. Computational Methods for Secondary
Structure Prediction
The DNA nucleotide sequence was translated into a pro-
tein sequence and the deduced amino acid sequence was
analyzed using the Expasy SwissProt Web server
( The sequence of amino acids
from Rep34 was aligned with 45 sequences of GST en-
zymes using ClustalW [27]. The methods used for
general secondary- structure prediction were Jpred [28],
PHD [29], PSIPRED [30], and SSpro [31]. The predict-
tion of secondary structure and analysion of protein mo-
tifs were made using program CLC Main Workbench
1.6. Assay of A TPase Activity
The ATPase activity was assayed by using non radioac-
tive modified method [32]. With interaction of free
anorganic phosphate with fresh prepared 0.045% (w/v)
malachit green and 4.2% (w/v) molibden ammonium in
4 M HCl in the rate of 3:1. Reaction mixture contained
in 50 µl (1 µg protein, 50 mM Tris-HCl pH 7.9, 5 mM
MgCl2 and 1 mM ATP) at 37 in times volume 0, 5, 10,
15, 20 min. After reference times was added 800 µl
malachit-molybdene solution and after 1 min added 100
µl of 34% (v/v) sodium citrate and the absorbance
change was measured spectrophotometrically at 660 nm.
As a standard was used reaction with different con-
centration of Na3PO4.
1.7. Helicase Assay
Helicase activity was detected by the release of a Fam3-
labeled oligonucleotide annealed to M13mp18 single-
stranded (ss) DNA [33]. The oligonucleotide, which
consisted of 20 bases (5´ – fam3-GTT GTA AAA CGA
CGG CCA GT – 3´) was complementary to the M13
DNA. Labeled oligonucleotide was annealed with M13
ss DNA (New England BioLabs) in 10 mM Tris.HCl (pH
8.0), 1 mM EDTA, and 100 mM NaCl, heat for 5 min at
65 and slowly cooling down for 30 min to room tem-
perature. The double-strand (ds) substrate was purified
by ethanol precipitation to remove unannealed oligonu-
cleotide. The helicase reaction mixture contained 5 nM
substrate in 20 mM Tris.HCl, 2 mM DTT, 5 mM MgCl2,
5% glycerol, 5 mM ATP, 0.1 mg/ml BSA, and the solu-
tion was adjusted to pH 7.5. The reactions were incu-
bated at 37 for 30 min and stopped adding 1/10 vol-
ume of 3 M sodium acetate and ethanol precipitate. After
precipitate pellet was suspended in 100 µl of sterile
water and unwound ds DNA substrate was quantified by
fluorometric analysis at 492 nm on fluorescence ana-
lyzer (Tecan Safire 2 Microplate Fluorescence Reader).
DNA unwinding was calculated as a percentage of the
total counts in each reaction.
1.8. Gel mobility Shift Assays
The DNA probe for the electrophoretic mobility shift
assay (EMSA) was the plasmid pGP2 transcription and
replication region in position 1671-2761 bp. The probe
was synthesized by PCR, using the primers 5’ – GAG
plification, the DNA probe was cleaved by PvuII which
created two fragments 539 and 475 bp. Mobility shifts
were performed as previously described [34], with some
modifications. The reaction mixtures (15 µl) contained
50 mM Tris.HCl (pH 8.0), 50 mM NaCl, 4.8 mM di-
thiothreitol, 1.0 mg/ml bovine serum albumin per ml and
20% (w/v) glycerol. DNA probe, reaction mixture and
recombinant Rep34 protein (0.4 pmol) were incubated at
30 for 15 min. The separation take place on a 4%
polyacrylamide gel containing 0.09% bis-acrylamide,
2.5% glycerol, and 1x TBE at 5-10 V/cm. The complexes
were visualized after colored ethidium bromide [35].
1.9. Phosphatase Activity
The enzymatic activity of Rep34 towards p-nitrophenyl
phosphate (pNPP) substrate was assayed at 37 by
spectrophotometric detection of the absorbance at 405
nm due to the release of p-nitrophenol (pNP) [36].
P. Grones et al. / Advances in Bioscience and Biotechnology 1 (2010) 417-425
Copyright © 2010 SciRes. ABB
Kinetic measurements assigned to determine substrate
kinetic parameters were performed in reaction mixtures
of a total volume of 400 μl. The mixtures contained 50
mM Tris–acetate, pH 5.5, 50-100 nM Rep34, in the
presence or absence of 10 mM MgCl2, and different
concentrations of pNPP. The reactions were initiated by
the addition of the enzyme, and quenched after 10 min
by the addition of 100 μl of 2 M NaOH followed by
centrifugation at 14,000g for 5 min. The absorbance at
405 nm was read for supernatants of reaction mixtures
(containing enzyme) and controls (the same substrate
concentration omitting the enzyme), and the difference
between these measurements represented the real rate of
product formation.
1.10. Protein Analysis
Proteins were analyzed by SDS–PAGE [37] stained ei-
ther by Coomassie Brilliant Blue R-250 method. Protein
concentrations were determined by the Bradford proce-
dure [38] or by densitometric analyses of Coomassie
Brilliant Blue-stained SDS–PAGE gels. Bovine serum
albumin (BSA) was used as the standard.
Bacterial plasmid pGP2 purified from Acetobacter es-
tunensis GP2 (2 797 bp) encoded three proteins. ORF2
encoded 209 aa large protein belonging to group of rep-
lication protein designed as Rep34 (Figure 1). By the
analysis of amino acid sequence were determined the
number of alpha helixes (4 larger than 4 aa) and beta
structures (4 larger than 4 aa) and two domains: Helicase
conserved C-terminal domain (137-175 aa) and HTH
motive (169-203 aa). Isoelectric point of this protein,
determined at pI 9.25, is similar to the isoelectric point
determined in replication proteins of plasmid pAG20
from A. aceti 3620 (pI 8.2) [23], but a bit distinct to
isoelectric point of protein from pAP12875 plasmid
from A. pasteurianus (pI 12.1) [21].
The rep gene was amplified using PCR amplification
and this product was cloned into pGEM-T easy vector
(pGEM-rep34 recombinant). Re-cloning gene in pET28a-
expression vector was in E. coli BL21 (DE3) cells am-
plified protein under T7 promoter and was constructed
2.1. Protein Purification
The expression vector containing the encoding rep34
gene was used to transform to E. coli BL21 (DE3) cells,
which over expressed a Rep34 protein after IPTG induc-
tion. An over-expressed band on SDS–PAGE corre-
sponding to a protein of approximately 25.55 kDa (with
His-tag) was observed in the crude bacterial lysates,
consistent with that expected for the recombinant protein
(Figure 2(a)). The greater part of Rep34 was found in the
supernatant after lysis and after using affinity chroma-
tography, the recombinant protein was quickly purified
to apparent homogeneity (Figure 2(b)). More detailed
data and purification steps were presented in Table 1.
The total protein yield at the last purification step was
approximately 71 mg of Rep34 per liter of bacterial cul-
Upon SDS–PAGE under reducing conditions, the iso-
lated protein migrated as a large homogenous band with
a molecular weight between 20 and 30 kDa, consistent
with that expected for a Rep34 monomer. A monomer of
the recombinant protein has a predicted molecular
weight of approximately 23.31 kDa. The molecular mass
of Rep34 is lower than the molecular mass of RepA
monomer protein described in plasmid pRSF1010 that
was determined on 31 kDa [39]. The results of the sec-
ondary-structure prediction indicate that overall Rep34 is
composed of the same amount of α-helical and β-sheet
2.2. In Vitro DNA-Binding Activity
DNA band-shift assays showed that the purified Rep34
His-tagged protein was able to bind specifically to linear
double-stranded amplified 1091 bp fragment from repli-
cation region of plasmid pGP2 in position 1670-2761 bp.
PCR product was cleaved by PvuII and afford two frag-
ments 475 bp and 539 bp which were used as substrate
for gel shift assay. Incubation of increasing concentra-
tions of Rep34 with a fixed amount of DNA progress-
sively altered the mobility of the DNA, indicating the
formation of protein–DNA complexes. Since the Rep34
apparently has sequence specificity. Furthermore, the
increase in DNA band retardation dependent on the
Rep34 protein concentration is probably due to the pro-
gressive occupation of the DNA molecule by Rep34
(Figure 3 lane 2-6).
Interaction Rep34 protein with substrate DNA was
specific on the 539 bp large fragment encoded regulation
region for transcription of replication protein as well as
ori region of plasmid pGP2. The second smaller DNA
fragment 475 bp did not change its position during
experiment and showed that it is not a specific substrate
for analyzed protein.
2.3. Assay of A TPase Activity
The most of enzymes, that catalyse biomolecular reac-
tions involving binding to the DNA and modificate its
structure, need energy from ATP dissociation for their
right functioning. ATP molecule can be dissociated by
the supporting protein or by the enzyme itself. Rep34
protein belongs to the second group and the ATPase ac-
P. Grones et al. / Advances in Bioscience and Biotechnology 1 (2010) 417-425
Copyright © 2010 SciRes. ABB
2797 bp
or i
HelixStand Helix
Stand St and
Stand He lix
209 aa
Figure 1. Genetic map of pGP2 plasmid from Acetobacter estunensis GP2. Replication protein
encoded ORF2 (1674-2301 bp; 209 aa). Gray fraim determined HTH region in protein, full
frames determined α helix and dashed frame determined β structure of protein. The gray L
represents amino acids residues leucine, that may form the structure of leucine zipper.
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 9
25.55 kDa25.55 kDa
(a) (b)
Figure 2. Rep34 expression and purification. 12% SDS–PAGE showing: (a) lane 8, molecular
weight standard; lane 1, total proteins from cells culture before induction with IPTG; lanes 2-4,
total proteins from cells after 0.5, 1, and 2 h of induction with IPTG (0.5 mM IPTG at 37),
respectively, lanes 5-7, total proteins from cells after 0.5, 1, and 2 h of induction with IPTG
(0.5 mM IPTG at 30). (b) lane 1, exprimed protein loaded to column, lanes 2, 3 washed
column with 5 mM imidazole, lane4-9 recombinant protein Rep34 eluted from Ni–NTA resin
with 20 mM, 40 mM, 60 mM, 80 mM, 100 mM and 250 mM imidazole, respectively. The
Rep34 bands are indicated by an arrow approximately 25.55 kDa).
Table 1. Purification of Rep34 protein after expression from Escherichia coli.
Procedure Volume (ml) Protein (mg)
Yield (%)Fold
sonification 5 271 54.2 100 1
sulphate 4.5 216 48 88 1.25
Ni-affi-Gel 1 7.1 7.1 13 38
Starting material was 0.5 g E. coli BL21 (DE3) cell paste
tivity was determined by standard modified method [32]
by removing anorganic phosphate from ATP. As showed
Figure 4, one microgram of protein converse about 0.58
± 0.11 μg/min ATP to ADP. This activity is higher than
the ATPase activity of SecA protein from E. coli (0.32
μg/min) [40], but similar to the products of rep genes
from E. coli [41] and bacteriophages replication proteins
P. Grones et al. / Advances in Bioscience and Biotechnology 1 (2010) 417-425
Copyright © 2010 SciRes. ABB
Figure 3. DNA-binding activity of the Rep34 His-tagged
protein. The purified Rep34 His-tagged protein was analyzed
for DNA-binding activity in band-shift assays using linear two
fragments 475 and 539 bp of regulation region of pGP2
plasmid Samples were analyzed in 4% non denaturated PAGE
in TBE buffer and visualized after ethidium bromide staining.
The bands observed in lane 1 in the absence of added protein
and lane 2-6 in presence of different concentration of binding
protein (0.1 µg, 0.5 µg, 1 µg, 2 µg, and 5 µg of Rep34 protein).
0510 15 20
Release phosphate [g]
Time [min]
Figure 4. Determination of ATPase activity replication protein
Rep34 from plasmid pGP2.
2.4. Determination of Phosphatase Activity
Catalytic activity of this protein was studied as an acidic
phosphatase, since the highest activity was observed at
pH 5.5. Thus, we analyzed the enzymatic activity of
Rep34 protein and determined the catalytic constants of
(a) (b)
(c) (d)
Figure 5. Enzymatic phosphatase activity Rep34 protein in the presence or absence of 10 mM Mg2+. (a) Standard
Michaelis–Menten curve of Rep34 activity assayed with pNPP in the concentration range of 0.01-0.3 mM in the buffer
comprising 50 mM Tris–acetate, pH 5.5, 10 mM MgCl2. (b) Standard Michaelis–Menten curve of Rep34 activity assayed with
pNPP in the concentration range of 0.01-0.3 mM in the buffer comprising 50 mM Tris–acetate, pH 5.5. (c) Lineweaver–Burk
plot of the kinetic process presented in (a) was used to derive Km and Vmax. (d) Lineweaver–Burk plot of the kinetic process
presented in (b) was used to derive Km and Vmax.
P. Grones et al. / Advances in Bioscience and Biotechnology 1 (2010) 417-425
Copyright © 2010 SciRes. ABB
(a) (b)
Figure 6. A representative DNA helicase assay in the presence of ATP. (a) Effect of decrease fam-labeled ds DNA
substrate after inteaction with Rep34 protein associed helicase activity. Helicase reactions were incubated at 37 for
30 min and the substrate and product were quantified by fluorometric analysis. (b) Unwinding super coiled form of
plasmid pGP2 by Rep34 helicase activity. Lane 1 standard plasmid pGP2, lane 2-8 activity measured after every 10
minutes of incubation with rep34 protein in optimal reaction condition (OC – open circular, SC – super coiled form).
the protein, using the cleavage of synthetic substrate
p-nitrophenyl phosphate. The Rep34 was able to cleave
pNPP in the presence and in the absence of Mg2+ ions
(Figure 5). Enzyme activity is higher about 20% in the
presence than absence Mg2+ ions. However phosphatase
activity of this protein is about hundred times lower than
is described in bacterial acid phosphatases [43]. Assum-
ing a Michaelis–Menten model of the enzymatic activity,
we used Lineweaver–Burk plots to derive catalytic pa-
rameters Km and Vmax. The Km value calculated in the
presence of Mg2+ for Rep34 is 0.46 ± 0.01 mM and in the
absence of Mg2+ is 1.09 ± 0.01. The phospthatase active-
ity is lower than described in bacterial C acid phos-
phatase of Helicobacter pylori (1.20 ± 0.25 mM) [44].
2.5. Helicase Assay
To demonstrate DNA helicase activity, unwinding was
monitored by the release of a Fam-labeled 32-mer from
a partially double-stranded circular M13 DNA substrate.
The release labeled primer from double strand DNA was
separated by ethanol precipitation. The pellet was dis-
solved in water and used for quantification on a fluores-
cent reader (Figure 6(a)). Unwinding activity Rep34 pro-
tein showed continuously decrease labeled dsDNA after
30 min incubation at 37 with 0.5 µg of protein (Fig-
ure 6(b)). Although a concentration-dependent increase
in helicase activity was observed at lower protein con-
centrations, the amount of unwinding was not signifi-
cantly increased at greater than 0.5 µg of protein (data
not shown).
Finally, new replication protein from plasmid isolated
from Acetobacter strain was cloned and exprimed in E.
coli expression systems. Small protein with basic
isoelectric point has two domains one for interaction
with other replication protein with leucine zipper and
second HTH domain for interaction with DNA. Purified
replication protein has phosphatase activity, ATP-ase
activity and is able to unwind double strand DNA
molecule. HTH domain specific recognise boundig region
for iniciation replication and transcription of plasmid
pGP2. Fusion of this replication protein with GFP
protein was used to monitor protein expression by
fluorometric microscopy.
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