New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii.
ABSTRACT Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to form oxalacetate. It is composed of three subunits, the central cylindrical hexameric 12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here report the cloning, sequencing, expression, and purification of the 5S subunit. The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database. For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a protein of 505 amino acids and of M(r) 55,700.
- SourceAvailable from: Harry James Flint[Show abstract] [Hide abstract]
ABSTRACT: Propionate is produced in the human large intestine by microbial fermentation and may help maintain human health. We have examined the distribution of three different pathways used by bacteria for propionate formation using genomic and metagenomic analysis of the human gut microbiota and by designing degenerate primer sets for the detection of diagnostic genes for these pathways. Degenerate primers for the acrylate pathway (detecting the lcdA gene, encoding lactoyl-CoA dehydratase) together with metagenomic mining revealed that this pathway is restricted to only a few human colonic species within the Lachnospiraceae and Negativicutes. The operation of this pathway for lactate utilisation in Coprococcus catus (Lachnospiraceae) was confirmed using stable isotope labelling. The propanediol pathway that processes deoxy sugars such as fucose and rhamnose was more abundant within the Lachnospiraceae (based on the pduP gene, which encodes propionaldehyde dehydrogenase), occurring in relatives of Ruminococcus obeum and in Roseburia inulinivorans. The dominant source of propionate from hexose sugars, however, was concluded to be the succinate pathway, as indicated by the widespread distribution of the mmdA gene that encodes methylmalonyl-CoA decarboxylase in the Bacteroidetes and in many Negativicutes. In general, the capacity to produce propionate or butyrate from hexose sugars resided in different species, although two species of Lachnospiraceae (C. catus and R. inulinivorans) are now known to be able to switch from butyrate to propionate production on different substrates. A better understanding of the microbial ecology of short-chain fatty acid formation may allow modulation of propionate formation by the human gut microbiota.The ISME Journal 02/2014; · 8.95 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.Biotechnology Letters 05/2011; 33(5):869-81. · 1.74 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The 5S subunit of transcarboxylase was expressed and purified. Recent methods of NMR spectroscopy as transferred NOESY, INPHARMA and Saturation Transfer Difference (STD) NMR were used to investigate ligand binding of free biotin to the 5S protein. The binding epitope for biotin is very similar to that obtained at the 12S subunit of transcarboxylase, however no common binding site for pyruvate and biotin exists.Protein and Peptide Letters 02/2008; 15(6):624-9. · 1.74 Impact Factor
New and Easy Strategy for Cloning,
and Characterization of the 5S
Subunit of Transcarboxylase
from Propionibacterium f. shermanii
Rakesh Kumar Bhat and Stefan Berger
Institute of Analytical Chemistry, University of Leipzig,
Abstract: Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from
Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2
from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to
form oxalacetate. It is composed of three subunits, the central cylindrical hexameric
12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here
report the cloning, sequencing, expression, and purification of the 5S subunit. The
gene was identified by matching the amino acid sequence with that of deposited in
the NCBI database. For cloned 5S subunit sequence shows regions of high
homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The
gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S
subunit was purified to apparent homogeneity by a single step process by using
Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a
protein of 505 amino acids and of Mr55,700.
Biotin dependent carboxylases constitute a diverse group of enzymes,
Address correspondence to Stefan Berger, Institute of Analytical Chemistry,
Preparative Biochemistry & Biotechnology, 37: 13–26, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 1082-6068 print/1532-2297 online
methylmalonyl-CoA carboxylase, pyruvate carboxylase, geranoyl-CoA
carboxylase, oxaloacetatedecarboxylase, methylmalonyl-CoAdecarboxylase,
transcarboxylase, and urea amidolyase. Although these enzymes are found in
different biosynthetic pathways of both eukaryotes and prokaryotes[1,2]and
involve a variety of substrates, the carboxylases share common features and
mechanisms. Overall biotin dependent enzymes are organized into three
classes.[3,4]Class I enzymes comprise carboxylases, which require ATP and
Mg2þfor the transfer of CO2from hydrogen carbonate to metabolites. Class
I enzymes play an important role in metabolic pathways, such as gluconeogen-
esis, oxidation of odd chain fatty acids, and catabolism of branched amino
acids. Class II enzymes couple two carboxylation reactions and the transcar-
boxylase (TC) (EC 184.108.40.206) from Propionibacterium shermanii is the only
known enzyme of this class.[3–5]The sodium ion transport decarboxylases
form Class III. These enzymes utilize the free energy of decarboxylation to
build up a Naþgradient. Till now, only four mammalian biotin dependent car-
boxylases have been reported: acetyl-CoA carboxylase (ACC), methylcroto-
nyl-CoA carboxylase (MCC), propionyl-CoA carboxylase (PCC), and
cant sequence homology with other important biotin dependent carboxylases,
and have, therefore, long been functioning as a powerful model system for
the study of biotin dependent carboxylases.
Transcarboxylase (TC), from Propionibacterium freudenreichii subsp.
shermanii, is a multienzyme complex, made up of three different subunits.
The different subunits, i.e., 12S, 5S, and 1.3S subunits, catalyze the transfer
of a carboxyl group from methylmalonyl-CoA to pyruvate via two partial
reactions.[7–9]This reaction from Propionibacterium shermanii is unique
among biotin-containing enzymes, as it catalyzes the transfer of a carboxyl
group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and
oxalacetate without the involvement of free CO2, HCO3
first half of the reaction, the carboxyl group from the methylmalonyl-CoA,
bound to the 12S subunit, is transferred to the biotin bound to the 1.3S
subunit to form the 1.3S-carboxylated-biotin carrier which is also called —
tcc. In the second half of the reaction, pyruvate accepts the carboxyl
group from the carboxylated biotin carrier. Biotin dependent carboxylases
play an important role in mammalian metabolism. For example, Human
acetyl-CoA carboxylase (ACC) catalyses the essential step in the biosynthesis
of fatty acids.
Previous studies have shown that the transcarboxylase (EC 220.127.116.11) multi-
enzyme complex consists of 30 polypeptide chains and is formed of a central
hexameric subunit core of molecular weight 3.6 ? 105with an s20,w¼ 12S.
This subunit is surrounded by two sets of three subunits, which are dimers
with molecular weight 1.2 ? 105with s20,w¼ 1.3S, and are attached at its
opposite faces (Figure 1). The complete enzyme has a molecular weight of
12 ? 105and s20,w¼ 26S, and is, therefore, also referred to as 26S
enzyme.[7,11]The 12S central subunit is active in the transcarboxylation
2, or ATP. In the
R. K. Bhat and S. Berger14
with methylmalonyl-CoA, but is inactive with oxalacetate and the peripheral
metallo 5S subunit, is active in the transcarboxylation with oxalacetate but
inactive with methylmalonyl-CoA. These subunits, likewise, are specific for
the reverse partial reactions; the central 12S subunit catalyzing the transfer
of CO2from the carboxylated biotinyl group to propionyl-CoA to yield
methylmalonyl-CoA, and the peripheral 5S subunit to pyruvate to yield
oxalacetate. Thus, the central subunit contains the sites for the CoA esters
(methylmalonyl-CoA and propionyl-CoA) and the peripheral metallo
subunits for the keto acids (oxalacetate and pyruvate). In the overall
reaction, the biotinyl carboxyl carrier protein acts as a shuttle to carry the
carboxyl groups between the two subunits. The 5S structure serves as a
scaffold for the homology modeling of the human PC carboxyltransferase
domain, which supports the expectation of conserved fold, active site, and
The 5S subunit of TC is a homodimer containing Co2þand Zn2þand is
composed of 505 amino acid residues. The deduced sequence shows regions
of extensive homology with that of pyruvate carboxylase and oxalacetate dec-
arboxylases, which catalyze the same or the reverse reactions. The 5S subunit
of TC is functionally and sequentially homologous (27% identity) with the
C-terminal of the carboxyltransferase region of human pyruvate carboxylase
[EC 18.104.22.168].[12,13]Co2þand Zn2þhave been shown to be present and cataly-
tically important in the 5S subunit. Also, both the metals have been shown to
be located at the keto acid binding sites.[14,15]The function of the metal ions
has remained controversial. Besides Co2þand Zn2þ, Cu2þwas also found to
be present in the 5S subunits. Although shown to be catalytically inactive,
Cu2þwas thought to be important for the assembly of TC.Despite
intensive study of transcarboxylase multienzyme complex subunits in
bacteria by various groups, binding of different ligands to the 5S subunit is
still under progress. Diversity in the primary structures of the 5S subunit
can be observed. Earlier studies have shown that the primary structure of
the 5S subunit of transcarboxylase from Propionibacterium shermanii
Schematic representation of transcarboxylase multienzyme complex.
5S Subunit of Transcarboxylase 15
varies from one strain of bacteria to another;[12,17]this has led us to clone and
purify the 5S subunit protein for the future NMR studies in our laboratory. The
present study describes the DNA cloning, expression, and purification of
the 5S subunit of transcarboxylase.
The main aim of the present research is to find out the protein ligand
interaction by NMR. Here, in this paper, we present a new and easy
strategy for the cloning and purification of the 5S subunit of TC. It was
difficult to obtain the protein in the quantity and quality required for NMR
studies by using the normal protein purification procedures.We tried to
optimize the cloning, sequencing, and initial expression of 5S subunit in
Escherichia coli (E. coli). To improve the expression, the 5S subunit gene
was subcloned into several vectors and later transformed into different
E. coli strains to determine the optimal conditions for the expression of the
recombinant subunit (Figure 2).
Bacterial Strains and Plasmids
The Propionibacterium f. shermanii DSMZ 4902 strain was obtained from the
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany, and was used as the source for the 5S subunit
gene. For the amplification of the newly constructed plasmid DNA, ptc5S,
E. coli DH5a (ATCC 53868) was used as a host strain for subcloning and
marker (6H-SDS); 2) Cells after harvest; 3) Cells before induction; 4) Clear cell lysate.
SDS-PAGE gel analysis of the 5STC of TC expression in E. coli. 1) HMW
R. K. Bhat and S. Berger 16
E. coli BL21(DE3) (Novagen) was used for protein expression. Vector pTXB1
(New England Biolabs, GmbH) was used for cloning, sequencing, and
expression. Restriction enzymes and shrimp alkaline phosphatase were from
New England Biolabs, GmbH. Reagents for ligation were obtained from
MBI Fermentas, GmbH. Plasmid purification was done using the Qiagen
plasmid purification kit (GmbH).
Media and Growth Conditions
Propionibacteria strain DSMZ 4902 was cultivated anaerobically at 308C in
PYG modified medium, as described by the supplier. E. coli DH5a was culti-
vated at 378C in Luria and Bertani (LB) medium supplemented with 50 mg of
ampicillin per ml if necessary. Transformants were grown at 378C in LB
medium containing 0.1 mg/mL ampicillin.
Isolation of Genomic DNA from Propionibacteria
Propionibacteria were cultivated in 5 mL of medium for 48 h. Genomic DNA
was extracted from the bacteria by using the Wizard Genomic DNA Purifi-
cation System, from Promega, GmbH.
Construction of Cloning Vector Containing the Gene of 5S Subunit
DNA manipulations, including amplification, ligation, cloning, and transform-
ation, followed the methods described by Sambrook et al.Restriction endo-
nucleases and T4 DNA Ligase were purchased from New England Biolabs.
Taq polymerase and pfu high fidelity DNA polymerases were purchased
from New England Biolabs, GmbH and Stratagene, respectively. All the
enzymes were used according to the manufacturers’ instructions.
Construction of Expression Vector
The ‘in-frame’ 1.5-kbp fragment of the transcarboxylase of the 5S subunit
gene (referred as TC5S gene) was amplified from genomic DNA by a poly-
merase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) with
the following primers: sense primer (50- TTT CGT CCA TCA TAT GAG
TCC GCG AGA AAT TGA GGT TTC CG-30) and anti-sense primer (50-
TTT CCA CGC TCT TCC GCA CGC CTG AAC GGT GAC TTG-30).
The two PCR primers contain Nde I and Sap I restriction sites (in bold),
respectively, to allow subcloning into the multiple cloning sites of the
pTXB1 vector. After initial denaturation of DNA for 5 minutes at 958C,
5S Subunit of Transcarboxylase 17
amplification was performed for 25 cycles, with denaturation at 948C for
1 min, 10 s, annealing at 658C for 55 s, and extension for 2 min 30 s at
728C, with a final extension step for 10 min at 958C.The PCR products
were double digested with Nde I and Sap I, gel-purified and inserted into
the similarly digested pTXB1 vector. The recombinant construct containing
the 5S subunit gene of TC from P. shermanii (TC5S), inserted between the
Nde I and Sap I restriction sites of pTXB1 vector, was referred to as
pTXBTC5S. Transformation of Escherichia coli DH5a with pTXBTC5S
was carried out using the standard chemical transformation method.
The recombinant colonies grown on LB plates containing ampicillin
(0.1 mg/mL) were initially screened for the TC5S gene insert by colony
PCR using a forward vector specific primer and a reverse insert specific
primer or vice versa to confirm the insert and its orientation.The
colonies which gave the positive PCR results were then selected for
digestion with Nde I and BamH I. After confirming the presence of the
TC5S gene and determining its orientation, the recombinant construct was
sequenced. The nucleotide sequence of plasmid pTXBTC5S was determined
by the primer walking strategy, starting from both ends. Overall, 3 primers
were designed to cover the complete 1.5 kb, and each nucleotide was read
at least two times in each direction. Once the integrity of the plasmid
sequence was confirmed by DNA sequencing, competent E. coli BL21
(DE3) cells were prepared and transformed with pTXBTC5S plasmid
according to the Hanahan protocol.The 5S subunit producer clone was
named E. coli BL21pTXBTC5S and was used to express the TC5S gene in
E. coli BL21(DE3) under control of the T7 promoter.
Cell Culture and Protein Overexpression
A single transformed BL21(DE3) colony, containing BL21pTXB5S, was used
to inoculate 10 mL Luria-Bertani (LB) medium supplemented with ampicillin
(100 mg/mL) grown with 190 rpm shaking overnight at 378C. The 10 mL
culture was diluted 100-fold into 1 L of medium in baffled culture flasks
(200 ml/L) with continuous shaking at 378C until an OD600 ¼ 0.8 was
reached. The culture was then chilled to approximately 238C by swirling
the flasks in an ice-water bath for 6 min. Sterile IPTG was added to a final con-
centration of 1 mM and shaking resumed at 308C for 6 h.The cells were
harvested by centrifugation and the cell pellets were rapidly frozen in liquid
nitrogen and stored at 2208C.
Cell Disruption and Purification of Recombinant 5STC Protein
All purification procedures were performed at 48C. About 5 g of wet cell mass
was suspended in 20 mL of buffer A (20 mM HEPES pH 8.0, 500 mM NaCl,
R. K. Bhat and S. Berger18
0.1 mM EDTA) containing 20 mg/mL lysozyme, 0.1 mM tris- (2-carbox-
yethyl) phosphine (TCEP), 20 mM Phenylmethylsulfonyl fluoride (PMSF),
10 mg/mL Aprotitin, 10 mg/mL pepstatin. This solution was incubated at
48C for 2 hrs and then sonicated at 48C with Sonifier Cell Disruptor B-30
(20 s per time, six times). 10 mg/mL protease free Dnase I in presence of
5 mM MgCl2 was added to reduce the viscosity of the cell lysate. The
lysate was kept at room temperature for 1 hour. The supernatant from the cen-
trifugation of the cell lysate at 12,000-x g for 30 minutes at 48C was referred to
as clear cell lysate. The expressed protein was purified using a chitin-bead
system, following the protocol provided by the manufacturer. Briefly, clear
cell lysate from E. coli, containing fusion protein, was passed over a 20 mL
pre-equilibrated chitin bead column at 48C. The column was washed with
more than seven column volumes of washing buffer (20 mM HEPES pH
8.0, 500 mM NaCl, 0.1 mM EDTA) and then quickly flushed with two
column volumes of cleavage buffer (20 mM HEPES pH 8.0, 50 mM NaCl,
0.1 mM EDTA, and 30 mM freshly diluted dithiothreitol (DTT)) (Figure 3).
The column was then left at 48C for 36 hrs. The TC5S protein was eluted
with three column volumes of cleavage buffer without DTT. The purified
protein was dialyzed four times with Millipore 30,000 against 100 mM
potassium phosphate buffer, pH 7.4, SDS/PAGE was carried out with 12%
Protein concentration was determined in accordance with Lowry et al. using
BSA as standard.
purification. 1) HMW marker (6H-SDS); 2) Column flow through; 3) Column washing;
4) 30 mM DTT washing; 5) Chitin beads after elution (36 h); 6) Column elution frac-
tion No. 1.
SDS-PAGE gel analysis of the expressed 5STC of TC in E. coli and its
5S Subunit of Transcarboxylase 19
Mass Spectrometric Analysis
The molecular weight of the intact purified recombinant 5STC protein was
measured by nanoelectrospray ionization (ESI), using a Q-TOF (Quadrupole
Time-of-Flight) mass spectrometer (Qstar Applied Biosystems, Germany),
which is accurate to within 1 Da for intact protein samples (Figure 4). The
control Software AnalystQS within the instrument control was used to recon-
struct the multiple charged ion mass spectra to the singly charged ion mass
spectra (Figure 5). The sequence analysis of the peptides was done after
tryptic digestion by nano LC-ESI-MS (MS/MS) with a collision energy
varying from 20 to 40 eV applied in the hexapole collision cell with N2as
the collision gas.
Approximately 50 nmol of TC5S was buffer exchanged with by Millipore cen-
tricons with a molecular cut-off of 10 kDa. TPCK treated trypsin was added to
the TC5S protein in the ratio of 25:1 and incubated at 378C for 4 hours. The
reaction was stopped by the addition of TFA. Peptides were eluted using a 35
minute gradient from 97% solvent A (Water: formic acid, 100:0.1 v/v) to
70% Solvent B (acetronitrile: formic acid, 100:0.1 v/v). The peptides were
subsequently separated on a C18reverse phase column. The sequence was
confirmed by ESI MS/MS, which gives a sequence coverage of 59% with
protein transcarboxylase 5S subunit,geninfo identifier (gi) ¼ 38304072,
and 31% sequence coverage with methylmalonyl-CoA carboxyltransferase
5S subunit monomer,gi ¼ 150929.
Primary Structure of the 5S Subunit of TC
The primary structure of the 5S subunit of transcarboxylase was deduced from
the nucleotide sequence of cloned PCR fragment from the genomic DNA from
Propionibacterium shermanii. Cleavage at the predicted intein sequence of
the cloned 5STC would result in a mature protein with a predicted
spectrum of the tryptic digested 5STC protein mass showing the location of fragment
ions used in the de novo sequence interpretation.
Mass spectra of charged ion series of the protein. A Q-ToF ESI MS/MS
R. K. Bhat and S. Berger20
molecular mass of 55,620 kDa, a value in excellent agreement with the
molecular weight of ? 55,500 estimated by SDS-PAGE for the expressed
5S subunit. The deduced nucleotide sequence of the 5S subunit gene, which
contains 1518 nucleotides, from P. shermanii strain used here differs from
the sequence originally reported (Figure 6).[1,12]The corresponding DNA
sequence was sequenced by the Interdisciplinary Centre for Clinical
Research (IZKF), Leipzig, and has been deposited in the GenBank
databases (under accession number DQ231350).
RESULTS AND DISCUSSION
Wefirst clonedthe 5Ssubunit intothepUC18 vectorsuchthatthe 5Sgenewas
downstream of the lac promoter in the vector, and expressed in E. coli JM109
cells. This system takes much time for purification and the yields are very low,
as there is loss of protein at each step of the purification. Our second method of
choice was the pET28 a (þ) vector system, which keeps the protein under the
tight control of the T7 promoter, and expresses the protein as a fusion protein
with a His- tag at N/C-terminal end. The 5S subunit protein was expressed in
Reconstruction of the charged ion series to singly charged ion peak.
5S Subunit of Transcarboxylase 21
TC to related sequences from databases. ORF-derived amino acid sequences of P. sher-
manii 5STC monomer of TC clones 5STC (shown as 5STC in the figure) 15STC/A/B
(EMBL accession numbers A ¼ DQ231350; B ¼ AJ606310; and C ¼ L06488). The
amino acid residues, which differ, are greyed and the residues, which are matched in
ESI MS/MS, are underlined.
Alignment of amino acid sequence of purified 5STC subunit monomer of
R. K. Bhat and S. Berger22
E. coli strain BL21(DE3) and purified by using His-Bind Fractogel resin
charged with Cu2þ. In this case, the yields of the protein were good enough
for NMR studies, but the problem was the removal of the His-tag. Using the
thrombin as the cleavage protease in the presence of Tween-20 detergent,
we were able to get enough His-tag cleaved, but then the problem was
removal of Tween-20 which poses problems in our NMR measurements. We
tried to remove the Tween-20, first, by using a Sephadex G-200 gel filtration
column and, later, by using Extracti-Gel D Detergent Removal Gel (Pierce
Biotechnology). We were not able to remove the Tween-20, but loss of the
protein during these processes was observed. It seems that Tween-20 binds
strongly to the protein (Figure 7).
Then, we tried the cleavage of His-tag in absence of a detergent. In this
case, the cleavage reaction takes a longer time and cleavage was not sufficient.
One more problem which arises after removing the His-tag from the protein,
was that, now we were not able to concentrate the protein solution; it was
being precipitated when the concentration reached around 25 mg/mL.
By incubating the protein for longer times with thrombin for the cleavage
of His-tag, thrombin was showing some secondary protease activity.
Finally, we tried the Intein mediated protein ligation (IPL) system.
temperature in potassium phosphate buffer pH?7.3 in D2O (?not corrected for H2O):
(A) tween-20 (B) 5S subunit protein after Sephadex G-200 and Extracti-Gel D Deter-
gent gel filtration column chromatography.
Comparison of two1H spectra. The spectra were recorded at 108C system
5S Subunit of Transcarboxylase 23
The efficiency of protein synthesis by means of the IPL method depends
greatly on the proper choice of the ligation sites used. The ligation reaction
occurs exclusively between the C-terminal thioester and the N-terminal
cysteine, thus requiring a cysteine in the sequence of a target protein.
An intein expressed plasmid, pTXB1, containing the 5S gene of transcar-
boxylase was generated and overexpressed in E. coli. First, different induction
times and the amount of IPTG for induction and temperatures were tested to
achieve a successful, high yield, and soluble expression of the fusion protein.
When the BL21(DE3) cells transformed with pTXBTC5S were induced with
0.1 mM IPTG at 308C, or at room temperature (23–278C), practically all of
the expressed fusion protein was found in the soluble fraction. The protein
was purified to homogeneity by a one-step purification using chitin beads.
The cleavage of the protein from the beads was performed by using 30 mM
dithiothreitol (DTT) instead of 2-mercaptoethanesulfonic acid (MESNA) or
thiophenol, because the a-thioester derivate of the target protein is stable
enough and can be stored for several months at 2708C. The cleaved TC5S
protein eluted from the chitin column was characterized by MS-MS data
(peptide mass finger print), ESI-MS, and SDS-PAGE. The ESI-MS analysis
resulted in a molecular mass of 55,623 Da, whereas the mass of recombinant
5STC was calculated to be 55,620 Da, and ?55,500 Da estimated from the
SDS-PAGE are in good agreement with each other.
This method has advantages over the method described earlier.[12,17]The
protein is purified to homogeneity by a single purification step. Thus, in this
case, one has full recovery of the expressed protein, contrary to the conven-
tional methods where there is loss of protein in each purification step.
Another advantage of using intein-based purification is that the protein
eluted is without the tag, which is not the case with the His.tag purification
system. For example, the thrombin-based cleavage does not cleave the tag
In case of the sensitive analytical measurements such as NMR, one cannot
have two species instead of one in the solution. This additional tag on the
protein can pose some problems, i.e., a ligand may have affinity towards the
tag or the tag can interfere in the conformation of the native protein. So, in
such cases, one can obtain artifacts which are difficult to differentiate from
the desired protein. It was also found that, after two rounds of freezing and
thawing, the protein was precipitating. As our main concern at this time is
to find the binding between the 5S subunit and different ligands, we assume
that we are working with a catalytically active protein. But this has to be
verified in our future experiments.
of transcarboxylase to homogeneity by a single step purification procedure.
R. K. Bhat and S. Berger 24
The sequenced subunit was identified as the subunit of transcarboxylase by
comparison to the peptide sequence data obtained by Thornton et al.and
Hall et al.The alignment of different 5S subunit sequences shows that the
sequence differs from one bacterial strain to another. The investigation of the
protein ligand interaction is being continued in our laboratory.
ATP: Adenosine triphosphate; NMR: Nuclear magnetic resonance; DSMZ:
Deutsche Sammlung von Mikroorganismen und Zellkulturen; DNA: Deoxyr-
ibonucleic acid; RNA: Ribonucleic acid; PCR: Polymerase chain reaction;
TPCK: Tosyl phenylalanyl chloromethyl ketone; DTT: dithiothreitol; IPL:
Intein mediated protein ligation; ESI: electrospray ionization.
We gratefully acknowledge Prof. Dr. Annette Beck-Sickinger (Faculty of
Biology, Pharmacy and Psychology, institute of Biochemistry, University of
Leipzig) for providing the laboratory for performing the molecular biology
work and DNA sequencing; Prof. Dr. Ralf. Hoffmann (Faculty of Chemistry
and Mineralogy, University of Leipzig) for MALDI-TOF MS determination
of the molecular mass and related discussions. Support of this project by The
Sonderforschungsbereich 610 Fund (SFB 610) is gratefully acknowledged.
1. Hall, P.R.; Wang, Y.-F.; Rivera-Hainaj, R.E.; Zheng, X.; Pusztai-Carey, M.;
Carey, P.R.; Yee, V.C. Transcarboxylase 12S crystal structure: hexamer assembly
and substrate binding to a multienzyme core. EMBO J. 2003, 22 (10), 2334–2347.
2. Toh, H.; Kondo, H.; Tanabe, T. Molecular evolution of biotin-dependent carboxy-
lases. Eur. J. Biochem. 1993, 215, 687–696.
3. Samols, D.; Thornton, C.G.; Murtif, V.L.; Kumar, G.K.; Haase, F.C.; Wood, H.G.
Evolutionary conservation among biotin enzymes. J. Biol. Chem. 1988, 263,
4. Knowles, J.R. Ann. Rev. Biochem. 1989, 58, 195–221.
5. Wood, H.G.; Barden, R.E. Biotin enzymes. Ann. Rev. Biochem. 1977, 46,
6. Moss, J.; Lane, M.D. The biotin-dependent enzymes. Adv. Enzymol. Rel. Areas
Molec. Biol. 1971, 35, 321–442.
7. Wood, H.G.; Zwolinski, G.K. Transcarboxylase: role of biotin, metals, and
subunits in the reaction and its quaternary structure. CRC Crit. Rev. Biochem.
1976, 4 (1), 47–122.
8. Wood, H.G. The anatomy of transcarboxylase and the role of its subunits. CRC
Crit. Rev. Biochem. 1979, 7 (2), 143–160.
5S Subunit of Transcarboxylase25
9. Wood, H.G.; Kumar, G.K. Transcarboxylase: its quaternary structure and the role
of the biotinyl subunit in the assembly of the enzyme and in catalysis. Ann. N. Y.
Acad. Sci. 1985, 447, 1–22.
10. Abu-Elheiga, L.; Jayakumar, A.; Baldini, A.; Chirala, S.S.; Wakil, S.J. Human
acetyl-CoA carboxylase: characterization, molecular cloning and evidence for
two isoforms. Proc. Natl Acad. Sci. USA 1995, 92, 4011–4015.
11. Poto, E.M.; Wood, H.G. Association-dissociation of transcarboxylase. Biochemis-
try 1977, 16 (9), 1949–1955.
12. Thornton, C.G.; Kumar, G.K.; Shenoy, B.C.; Haase, F.C.; Phillips, N.F.;
Park, V.M.; Magner, W.J.; Hejlik, D.P.; Wood, H.G.; Samols, D. Primary
structure of the 5S subunit of transcarboxylase as deduced from the genomic
DNA sequence. FEBS Lett. 1993, 330 (2), 191–196.
13. Wexler, I.D.; Du, Y.; Lisgaris, M.V.; Manda1, S.K.; Freytag, S.O.; Yang, B.S.;
Liu, T.C.; Kwon, M.; Patel, M.S.; Kerr, D.S. Primary amino acid sequence and
structure of human pyruvate carboxylase. Biochim. Biophys. Acta 1994, 1227,
14. Ahmad, F.; Lygre, D.G.; Jacobson, B.E.; Wood, H.G. Transcarboxylase: XII.
identification of the metal-containing subunits of transcarboxylase and stability
of the binding. J. Biol. Chem. 1972, 247, 6299–6305.
15. Northrop, D.B. Transcarboxylase VI. Kinetic analysis of the reaction mechanism.
J. Biol. Chem. 1969, 244, 5808–5819.
16. Fung, C.H.; Mildvan, A.S.; Leigh, J.S., Jr. Electron and nuclear magnetic
resonance studies of the interaction of pyruvate with transcarboxylase. Biochem-
istry 1974, 13 (6), 1160–1169.
17. Hall, P.R.; Zheng, R.; Pusztai-Carey, M.; van den Akker, F.; Carey, P.R.;
Yee, V.C. Expression and crystallization of several forms of the Propionibacter-
ium shermanii transcarboxylase 5S subunit. Acta Crystallogr. D: Biol. Crystallogr.
2004, 60 (PT3), 521–523.
18. Leblanc, D.J.; Lee, L.N. Physical and genetic analysis of streptococcal plasmid
pAM1 and cloning of its replication origin. J. Bacteriol. 1984, 157, 445–453.
19. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory
Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
20. Gussow, D.; Clackson, T. Direct clone characterization from plaques and colonies
by the polymerase chain reaction. Nucleic Acids Res. 1989, 17, 4000.
21. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol.
Biol. 1983, 166, 557–580.
22. De Man, J.C.; Rogosa, M.; Sharpe, M.E. A medium for the cultivation of lactoba-
cilli. J. Appl. Bacteriol. 1960, 23, 130–135.
23. Lawry, O.H.; Rosenbrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement
with the folin–phenol reagents. J. Biol. Chem. 1951, 193, 265–275.
24. Rehm, T.; Huber, R.; Holak, T.A. Application of NMR in structural proteomics
screening for proteins amenable to structural analysis. Structure 2002, 10,
Received April 18, 2006
Accepted May 29, 2006
R. K. Bhat and S. Berger 26