Coexpression, purification and characterization of the E and S subunitsHao-Ping Chen1, Fang-Ciao Hsui1, Li-Ying Lin1, Chien-Tai Ren2and Shih-Hsiung Wu2 1 Institute of Biotechnology and D
Trang 1Coexpression, purification and characterization of the E and S subunits
Hao-Ping Chen1, Fang-Ciao Hsui1, Li-Ying Lin1, Chien-Tai Ren2and Shih-Hsiung Wu2
1
Institute of Biotechnology and Department of Chemical Engineering, National Taipei University of Technology, Taipei, Taiwan;
2
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan
D-Ornithine aminomutase from Clostridium sticklandii
comprises two strongly associating subunits, OraS and
OraE, with molecular masses of 12 800 and 82 900 Da
Previous studies have shown that in Escherichia coli the
recombinant OraS protein is synthesized in the soluble form
and OraE as inclusion bodies Refolding experiments also
indicate that the interactions between OraS and OraE and
the binding of either pyridoxal phosphate (PLP) or
aden-osylcobalamin (AdoCbl) play important roles in the
refolding process In this study, the DNA fragment
con-taining both genes was cloned into the same expression
vector and coexpression of the oraE and oraS genes was
carried out in E coli The solubility of the coexpressed
OraS and OraE increases with decreasing isopropyl
thio-b-D-galactoside induction temperature Among substrate
analogues tested, only 2,4-diamino-n-butyric acid displays
competitive inhibition of the enzyme with a Ki of
96 ± 14 lM Lys629 is responsible for the binding of PLP The apparent Kdfor coenzyme B6binding to D-ornithine aminomutase is 224 ± 41 nMas measured by equilibrium dialysis The mutant protein, OraSE–K629M, is successfully expressed It is catalytically inactive and unable to bind PLP Because no coenzyme is involved in protein folding during
in vivotranslation of OraSE–K629M in E coli, in vitro re-folding of the enzyme employs a different re-folding mechan-ism In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation
Keywords: adenosylcobalamin; B12; D-ornithine amino-mutase
D-Ornithine aminomutase from Clostridium sticklandii
catalyzes the reversible interconversion ofD-ornithine into
2,4-diaminopentanoic acid [1] It comprises two strongly
associating subunits, OraS and OraE, with molecular
masses of 12 800 and 82 900 Da Two different coenzymes,
pyridoxal phosphate (PLP) and adenosylcobalamin
(Ado-Cbl), are involved in this enzymatic reaction The genes
encodingD-ornithine aminomutase, oraE and oraS, have
been cloned, sequenced, and expressed in Escherichia coli
[2] The recombinant OraS protein was synthesized in a
soluble homogeneous form, but the majority of OraE
protein was produced in the form of inclusion bodies The
enzymatic activity could be restored after a refolding step
However, OraE could not be properly folded in the absence
of OraS and coenzyme These observations indicate that the
binding of AdoCbl or PLP and the interactions between
OraS and OraE play important roles in the OraE refolding
process The strong interaction between the E and S
subunits of the enzyme was first reported by Barker & Stadtman [3]; Barker discovered glutamate mutase, which is also composed of weakly interacting E and S components The correlation between these interactions and protein folding is not clear
As protein refolding is labor intensive and time consu-ming, an efficient expression system to produce large amounts of soluble proteins in a short time is required Instead of expressing the oraE and oraS genes separately, the DNA fragment containing both genes was cloned into the same expression vector under the control of the T7 promoter, and coexpression of oraE and oraS genes was carried out in E coli Meanwhile, the extent of the involvement of AdoCbl or PLP in the in vivo folding process was also investigated We now describe the construction, coexpression, and purification of the apo-enzyme of D-ornithine aminomutase, together with the temperature effect on protein expression and characteriza-tion of the recombinant proteins
Materials and methods
Materials AdoCbl was obtained from Sigma Micro Dialysis tube, Q-Sepharose High Performance anion-exchange medium and Phenyl-Sepharose High Performance hydrophobic interaction gel medium were from Amersham Biosciences Restriction endonucleases, BamHI, SpeI, and NcoI,
Correspondence to H.-P Chen, Institute of Biotechnology and
Department of Chemical Engineering, National Taipei University of
Technology, 1, Sec 3, Chung-Hsiao East Road, Taipei 106, Taiwan.
Fax: +886 2 27317117, Tel.: +886 2 27712171 ext 2528,
E-mail: hpchen@ntut.edu.tw
Abbreviations: AdoCbl, adenosylcobalamin; IPTG, isopropyl
thio-b-D -galactoside; PLP, pyridoxal phosphate.
(Received 30 June 2004, revised 26 August 2004,
accepted 17 September 2004)
Trang 2DNA-modifying enzymes, and Ex Taq DNA polymerase
were purchased from TaKaRa (Otsu, Japan) The E coli
strain BL21(DE3) codon plus was from Stratagene
1,4-Diaminobutane and (R,S)-2,4-diamino-n-butyric acid
were from Sigma 4-Aminopentanoic acid, and
2,5-diamino-pentanol were the kind gift from T.-L Shih (Department of
Chemistry, Tamkang University, Taiwan) All chemicals
used were of molecular biology grade or higher
Construction of expression vector poraSE
A pair of oligonucleotides, 75 and 44 (Table 1), was
designed using the nucleotide sequence of the ora genes in
order to facilitate the amplication by PCR An NcoI site was
introduced into the start of the oraS gene and a BamHI site
into the end of the oraE gene Genomic DNA was purified
from C sticklandii by phenol/chloroform extraction
meth-ods [4] The coding regions for the S and E subunits of
D-ornithine aminomutase were then amplified by PCR
using clostridial genomic DNA as template Amplification
was achieved using 30 cycles at the following temperatures:
95C for 30 sec, 50 C for 1 min, and 72 C for 4 min
Finally, the reaction was maintained at 72C for 5 min
The PCR products were gel-purified, restricted with NcoI
and BamHI, and ligated with NcoI/BamHI restricted
pET-28a vector The ligation mixture was used to transform
E coliDH5a The plasmid that carried the oraS and oraE
genes in the correct orientation was designated poraSE
Isopropyl thio-b-D-galactoside induction temperature
and small-scale expression
To facilitate the over-expression of the ora genes, poraSE
was used to transform E coli BL21(DE3) codon plus
Cultures were first grown at various temperatures in
500 mL Luria–Bertani medium containing kanamycin
(30 mgÆL)1) After isopropyl thio-b-D-galactoside (IPTG)
induction and expression, the cells were harvested by
centrifugation and resuspended in 15 mL of 50 mM
phos-phate buffer, pH 7.0 The cells were ruptured by sonication
and cell debris was removed by centrifugation at 25 000 g
for 15 min at 4C To examine the solubility of the
over-expressed proteins, 20 lL of supernatant was taken for
analysis by SDS/PAGE The insoluble fraction and cell
debris from 1 mL overnight culture were collected by
microcentrifugation at 13 000 rpm for 5 min and dissolved
in 100 lL SDS/PAGE loading buffer; 10 lL was taken to
analyse by SDS/PAGE
Large-scale protein expression and purification Cultures were grown at 25C by inoculating a 5 mL overnight culture into 4 L of Luria–Bertani medium containing kanamycin (30 mgÆL)1) Incubation was contin-ued until the culture reached an attenuance of 0.6–0.8 at
600 nm, at which point the temperature was lowered to
20C and expression was induced by the addition of
200 mgÆL)1 IPTG After overnight incubation, cells were harvested by centrifugation at 4000 g for 10 min The cells were then stored at)20 C
All purification steps were performed on ice or at 4C
In a typical purification, 15 g of cells (wet weight) were resuspended in 30 mL of 50 mMTris/Cl buffer, pH 9.0 The cells were ruptured in a volume of 30 mL by sonication Cell debris was removed by centrifugation at 25 000 g for
15 min The supernatant was applied directly to a 2.6· 20 cm Q-Sepharose Fast Flow anion-exchange col-umn equilibrated with 10 mM Tris/Cl buffer, pH 9.0 Protein was eluted with a 600 mL gradient from 0 to 0.5MKCl The flow rate was 1 mLÆmin)1; 5 mL fractions were collected Active fractions were pooled and brought to 25% saturation in ammonium sulfate by slow addition of solid The precipitate was removed by centrifugation at
25 000 g for 30 min and the supernatant was applied directly to a Phenyl-Sepharose High Performance hydro-phobic interaction column (2.6· 25 cm) equilibrated with
10 mMTris/Cl buffer, pH 9.0, containing 1M(NH4)2SO4 After washing the column with 100 mL of the same buffer, the enzyme was eluted with a linear, descending gradient of ammonium sulfate in 1000 mL of buffer The flow rate was
1 mLÆmin)1; 10 mL fractions were collected Active frac-tions were pooled and concentrated to 15 mL by ultrafil-tration in a stirred cell fitted with a YM-3 membrane The protein solution was stored at)80 C in the presence of 50% (v/v) glycerol
Mutant construction The construction of mutant poraSE-K629M was carried out using recombinant PCR [5] Two overlapping, comple-mentary oligonucleotides were designed to introduce the mutagenic sequence A 1.8 kb and 700 base pair region of the oraE gene were PCR amplified using poraEX as template and oligonucleotide pairs 40/66 and 41/67 as primers Both PCR products were gel-purified and assem-bled in a second round of PCR using oligonucleotides 40 and 41 as primers and cotemplates The PCR product was purified, restricted with SpeI and BamHI, and ligated with SpeI/BamHI-restricted poraSE vector The resulting plas-mid was designated poraSE-K629M The DNA fragment amplified by PCR was resequenced by automated methods (Mission Biotech Co Ltd., Nankang, Taipei, Taiwan; ABI
3730 XL DNA Analyzer, Applied Biosystems, CA, USA)
to confirm that no unwanted mutation had been introduced The procedures for production and purification of the mutant protein were the same as those of the wild-type
Protein determination and enzyme assay Protein concentrations were determined by the method of Bradford using bovine serum albumin as standard [6] The
Table 1 PCR primer names and sequences.
Primer
name Sequence
21 GGGTCTAGAATGGAAAAAGATCTACAGTTAAGA
33 CCGGAATTCTTATTTCCCTTCTCTCATCTC
40 GCGCGCCATGGAAAAAGATCTACAGTTAAGA
41 GGGGGATCCCCATAATCCACTCCACCTGCTAAA
44 GGGGGGGATCCT CATTATTTCCCTTCT
66 AATACCGCCATGTATAATATCTATTACTTC
67 GTAATAGATATTATACATGGCGGTATTGAA
75 GGGGGGGCCATGGAAAGAGCAGACGATTT
Trang 3assay kit was obtained from Bio-Rad, Hercules, CA, USA.
A rapid spectrophotometric method was used to assay
D-ornithine aminomutase activity [7] The assay couples
the reduction of NADP+ to form 2,4-diaminopentanoic
acid through the action of NAD+/NADP+-dependent
2,4-diaminopentanoic acid dehydrogenase The Ki value
of the competitive inhibitor, 2,4-diamino-n-butyric acid,
was determined by measuring the apparent Kmvalue of
D-ornithine at 25, 50, 100, 200 and 400 lMof the inhibitor
For the measurement of the activity of the substrate
analogues, an HPLC and NMR-based method was
devel-oped A 1.0 mL solution in a septum-sealed vial containing
6 lM D-ornithine aminomutase, 0.4 mMPLP, and 0.14 mM
AdoCbl in 100 mM potassium phosphate buffer, pH 7.8,
was made anaerobic by purging with argon A concentrated
anaerobic 0.1 mL solution of substrate analog (0.5M) in the
same buffer was introduced into the vial by syringe to
initiate the reaction After overnight incubation at room
temperature in the dark, the reactions were stopped by
freeze-drying The reaction products were separated by
HPLC on a C18reverse phase column with a linear gradient
of acetonitrile containing 0.1% (v/v) trifluoroacetic acid
The substrate analogues, presumed products, and
phos-phate ion were eluted at the beginning of the run and
collected by hand The mixture was dried by evaporation
under vacuum and redissolved in 0.4 mL D2O three times
The solution was transferred to an NMR tube and the
spectra were recorded at 400 MHz
Measurement of the binding of PLP to proteins
The binding of coenzyme B6toD-ornithine aminomutase
was measured by equilibrium dialysis About 250 lL of
30 lMpurified proteins were loaded into the Micro Dialysis
tube The protein solutions were dialyzed against 400 mL of
10 mMTris buffer, pH 9.0, in the presence of 6000, 1500,
960, 480, 300 and 150 nM coenzyme B6 at 4C for 4 h
Absorbance was recorded at 420 nm using an Amersham
Bioscience Ultrospec 2100 spectrophotometer; a sample of
the corresponding dialysis buffer was used to subtract out
the contribution of unbound PLP from the absorbance of
the enzyme A computer program (KALEIDA GRAPH, Synergy
Software, Reading, PA, USA) was used to fit the data in
order to estimate the dissociation constant
Ultraviolet–visible protein spectrum
About 16 mgÆmL)1proteins (wild-type or mutant
OraSE-K629M) and 3 lMPLP in 10 mMTris/Cl buffer, pH 9.0,
were dialyzed in the dark at 4C, against 10 mMTris/Cl
buffer, pH 9.0, containing 3 lM PLP for 24 h, by which
time equilibrium had been reached Sepctra were recorded
using an Amersham Bioscience U2100 spectrophotometer;
a sample of the dialysis buffer was used to subtract out the
contribution of unbound PLP from the spectra of proteins
Results
The expression of poraSE was first carried out at 37C with
a shaking speed of 180 r.p.m It is worth noting that, alone,
OraS protein can be expressed in a soluble form However,
the OraE and OraS proteins were coexpressed in an
insoluble form under the same conditions The codon usage difference between C sticklandii and E coli does not seem
to be responsible for this result, because the E coli strain, Epicarian Coli-Codon PlusTM(DE3)-RIL, contains extra copies of the argU, ileY, and leuW tRNA genes
The coprecipitation of OraS and OraE might imply that (a) the apoenzyme or OraE is not properly folded; and (b) the noncovalent interaction between these two subunits is strong enough to result in the coprecipitation of OraS In many instances, the folding of the desired expressed protein can be improved at lower induction temperatures [8–10] As shown in Fig 1, the solubility of the overexpressed OraS and OraE increases with decreasing IPTG induction temperature When the incubator shaking speed reduced from 180 to 50 r.p.m., no significant difference in the expressed protein solubility can be observed (data not shown)
The protocol described above gave good expression of the oraS and oraE genes Approximately 15 mg of purified protein was obtained per litre of culture A purification method based on chromatography on Q-Sepharose
ion-Fig 1 The over-expression of oraS and oraE at different temperatures (A) Supernatant fraction (B) Precipitation fraction Lane 1, marker; lane 2, 37 C; lane 3, 30 C; lane 4, 25 C; lane 5, 20 C.
Trang 4exchange and Phenyl-Sepharose hydrophobic interaction
matrixes was developed In both purification steps, OraS
and OraE eluted during the end of the run in a well-resolved
broad peak, resulting in protein that was nearly
homogen-eous (Fig 2) This method of preparation proved very
reproducible, and purified enzyme could be stored in
concentrated solution in the presence of 50% glycerol for
several months, frozen at)80 C
A lysine residue is thought to be involved in PLP-binding
through a Schiff base linkage Comparison of the deduced
amino acid sequence of oraE to those of known
PLP-dependent aminomutases reveals the presence of a
con-served PLP-binding site, a lysine residue at position 629, at
the C-terminus of the OraE protein [11] The binding of PLP
to D-ornithine aminomutase was investigated by
equilib-rium dialysis The proteins in the Micro Dialysis tube were
equilibrated in various concentrations of PLP, and the
binding of coenzyme was measured PLP was bound with
an apparent Kdof 227 ± 41 nM(Fig 3)
The production and purification methods for mutant protein, OraSE-K629M, were as described above No significant difference in protein solubility could be found between wild-type and mutant protein at various IPTG induction and expression temperatures Perhaps not sur-prisingly the mutation of the Lys629 residue to Met caused a complete loss of catalytic activity Meanwhile the binding of PLP by mutant OraE-K629M was too weak to allow binding constants to be determined with any accuracy, as shown by the equilibrium dialysis experiment The ultra-violet–visible spectrum of wild-type and mutant enzyme is shown in Fig 4 The presence of an absorption maximum
at 420 nm suggests thatD-ornithine aminomutase, as is the case with other pyridoxal 5¢-phosphate dependent enzymes, binds pyridoxal 5¢-phosphate via an azomethine link between the formyl group of the cofactor and the amino group of a protein residue In contrast, the absence of absorption maximum at 420 nm of the mutant enzyme spectrum directly demonstrates that the Lys629 residue is responsible for the binding of PLP inD-ornithine amino-mutase (Fig 4)
High substrate specificity is a common feature for most AdoCbl-dependent mutases However, alternative sub-strates exit in the case of B12-dependent glutamate mutase and lysine aminomutase [12,13] The enzymatic activity of
D-ornithine aminomutase to four substrate analogues, 1,4-diaminobutane, 2,4-diamino-n-butyric acid, 4-amino-pentanoic acid, and 2,5-diaminopentanol, was also exami-ned in this study Our results show that none of them could
be catalyzed by the enzyme Moreover, only 2,4-diamino-n-butyric acid is able to behave as a competitive inhibitor of the enzyme with a Ki of 96 ± 14 lM as measured by photometric assay The other three analogues showed neither inhibitory potential nor suggestion of cleavage of the cobalt–carbon bond of AdoCbl (H.-P Chen, unpub-lished results) These results suggest that the substrate specificity ofD-ornithine aminomutase is strict
Discussion
The genes encoding D-ornithine aminomutase, oraE and oraS, are adjacent on the clostridial chromosome They
Fig 2 SDS/PAGE results of samples taken after each step in the
purification of the recombinant enzyme Purification of OraSE (20%
gel) Lane 1, marker; lane 2, crude cell extract before IPTG induction;
lane 3, crude cell extract after IPTG induction; lane 4, supernatant
after cell disruption by sonication; lane 5, pooled fractions after
Q-Sepharose HP chromatography; lane 6, pooled fractions after
Phenyl-Sepharose HP hydrophobic interaction chromatography.
Fig 3 Binding of PLP to recombinant D -ornithine aminomutase
measured by equilibrium dialysis.
Fig 4 UV–visible spectrum of wild-type and mutant D -ornithine amino-mutase The maximal absorption at 420 nm of indicated that pyridoxal 5¢-phosphate is bound to the wild-type enzyme.
Trang 5share overlapping start and stop codons which might lead to
transcription coupling so as to produce equal amounts of
the two proteins [2] In the open reading frames for the oraS
and oraE genes, an E coli ribosome-binding site is located
upstream of the initiation codon of oraS and a clostridial
ribosome-binding site on oraE Although the different
prokaryotic Shine–Dalgarno sequences might have different
affinities for ribosomes, both oraS and oraE genes are
successfully overexpressed (Fig 5)
The strong interaction between OraS and OraE was
first reported by Barker & Stadtman [3] OraS shows no
significant homology to other proteins in the
SWISS-PROT database The sequence alignment results indicate
that the coenzyme-binding and catalytic domains are
located in the E subunit [2] Unfortunately, varying the
induction temperature and inducer concentration had
little effect on the solubility of OraE, and any attempt to
refold OraE by itself was not successful Although the
role of the S subunit remains obscure, it seems likely that
OraS somehow interacts with OraE to stabilize the
protein in an active conformation Moreover, the
calcu-lated isoelectric point of the E component is 9.2, whereas
the S component is 5.1 This result might provide an
explanation for the strong interaction between the S and
E components
Previous studies have shown that it is necessary to include
coenzyme B12or B6during the refolding of OraE and OraS
in vitro[2] Both B12and B6-binding motifs are located at the
C-terminal of OraE and only separated from each other by
about 10 amino acid residues It seems likely that inclusion
of AdoCbl or PLP during refolding might facilitate the
correct folding of OraE The dissociation constant, Kd, for
PLP inD-ornithine aminomutase is 224 ± 41 nM,
indica-ting that the apoenzyme can bind it with high affinity It is
not clear whether coenzyme B12or B6plays a role in protein
folding during in vivo translation To examine this, a mutant
protein, OraSE-K629M, which is unable to bind PLP, was
constructed and produced in E coli As the bacterial strain
used to express protein is unable to synthesize cobalamin by
itself, neither AdoCbl nor PLP could be involved in the
mutant protein folding process during in vivo translation
However, no significant difference in protein solubility
could be found between wild-type and mutant protein This
result indicates that (a) the recombinant protein folding
pathway during in vivo translation in E coli is different from
the in vitro refolding process, and (b) the association of the
S and E subunit is important forD-ornithine aminomutase
to maintain an active conformation in both cases In summary, we have successfully constructed, overexpressed, and purified the recombinant D-ornithine aminomutase Future work in our group will focus on the determination of the quaternary structure of the holoenzyme and the catalytic mechanism of this 1,2-rearrangement reaction
Acknowledgements
This work was supported by grant NSC 91-2320-B032-001 from the National Science Council, Taiwan, Republic of China (to H.-P Chen).
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Fig 5 The plasmid construction map of poraSE RBS, Ribosome
binding site.