Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Overexpression and Purification of Asparagine Synthetase from Escherichia coli Akio Sugiyama, Hiroaki Kato, Takaaki Nishioka & Jun’ichi Oda To cite this article: Akio Sugiyama, Hiroaki Kato, Takaaki Nishioka & Jun’ichi Oda (1992) Overexpression and Purification of Asparagine Synthetase from Escherichia coli, Bioscience, Biotechnology, and Biochemistry, 56:3, 376-379, DOI: 10.1271/bbb.56.376 To link to this article: http://dx.doi.org/10.1271/bbb.56.376
Published online: 12 Jun 2014.
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Date: 05 November 2015, At: 23:16
Biosci. Biotech. Biochem., 56 (3), 376-379, 1992
Overexpression and Purification of Asparagine Synthetase from Escherichia coli Akio
SUGIYAMA,
Hiroaki
KATO,
Takaaki
NISHIOKA,
and Jun'ichi
ODA*
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 Received June 10, 1991
Downloaded by [117.255.242.234] at 23:16 05 November 2015
Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUCI8, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15°,10 of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The aminoterminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.
L-Asparagine synthetase [L-aspartate: ammonia ligase (AMP-forming) EC 6.3.1.1J catalyzes the synthesis of Lasparagine from L-aspartate and ammonia, while hydrolyzing ATP to AMP and PPi. 1) These enzymes are distributed in prokaryotes as Escherichia coli 2 ) and Klebsiella aerogenes 3 ) and encoded by asnA genes. On the other hand, asparagine synthetases from eukaryotes 4 - 7) use glutamine as the preferred amido nitrogen donor, although they can also use ammonia. These glutamine-dependent enzymes [L-aspartate: L-glutamine amido-ligase (AMPforming) EC 6.3.5.4J are also found in prokaryotes 3 ,8) and encoded by asnB genes. The steady state kinetic mechanisms of asparagine synthetase have been reported for both the ammonia-dependent and glutamine-dependent enzymes. 9 - 11) In the ammonia-using reaction, the mechanism of the ammonia-dependent enzymes was different from that of the glutamine-dependent enzymes. 9 ,11) Both types of asparagine synthetase genes have been cloned and sequenced from E. COli 12 ,13) and humans. 4 ) The asnA gene from E. coli coded for a polypeptide of 330 amino acid residues with a molecular weight of 36,700. A high degree of amino acid sequence homology was shown between the glutamine-dependent enzymes (asnB enzyme from E. coli and the human enzyme), however, no significant homology was found between the asnA and the asnB sequences although derived from the same origin, E. coli. 13) To elucidate these functional differences based on the protein structure, it is essential to obtain a large amount of the purified protein. We describe the construction of overexpression plasmids of the asnA gene from E. coli K-12 and a simple method for purifying the enzyme from transformed cells. This is the first time that asparagine synthetase from the asnA gene has been purified to apparent homogeneity.
Materials and Methods Bacterial strains, phages, and plasmids. Plasmid pMYlll is a pBR322 derivative containing a 2.1-kilobase pair Pstl fragment of the asparagine
synthetase gene (asnA) from Escherichia coli K_12. 12 ) The overexpression plasmid pKK-223-3 was obtained from Pharmacia (Uppsala). Plasmid pUCl8 and E. coli JMI09, 14) a host ofpUC18 and pKK223-3 derivatives, were purchased from Takara Shuzo Co., Ltd. (Kyoto). Restriction enzymes and chemicals. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, exonuclease III, mung bean nuclease, and Klenow fragment of DNA polymerase I were purchased from Takara Shuzo Co., Ltd. and Toyobo (Osaka). L-Aspartate was from Wako Pure Chemicals Co., Ltd. ATP, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase were from Oriental Yeast Co. (Tokyo). Adenylate kinase was from Unichika Co. (Osaka). DEAE-Cellulofine A-800, Blue-Cellulofine, and Cellulofine GCL-I 000 sf were gifts from Chisso Co., (Minamata). All other chemicals used in this experiment were of the purest grades commercially available. SDS-polyacrylamide gel electrophoresis. Protein samples were electrophoresed in 10% polyacrylamide gels in the presence of SDS. 15 ) Gels were then stained with Coomassie Brilliant Blue R-250. To measure the relative amount of asparagine synthetase protein, the stained gels were scanned at 570 nm using a model CS91 0 chromatoscanner (Shimadzu Co.). Assay of asparagine synthetase activity. Protein concentration was measured by the method of Bradford 16 ) with Protein Assay Reagent (Pierce Chemical Co.), and bovine serum albumin as a standard. The protein concentration of the asparagine synthetase in the purified preparation was determined from A ir'b = 13.9 cm -1 which was calculated from multiplication of the numbers of Tyr and Trp residues by their molar absorption coefficients. 17) Asparagine synthetase activity was assayed spectrophotometrically by coupling the formation of AMP to the adenylate kinase, pyruvate kinase, and lactate dehydrogenase reactions. The enzyme solution (50 pI) was added to an assay medium composed of 2 U adenylate kinase, IOU pyruvate kinase, 25 U lactate dehydrogenase, 0.24 mM NADH, I mM phosphoenolpyruvate, 1.5 mM L-aspartic acid, 20mM NH 4C1, 3 mM ATP, 100mM KCl, IOmM (CH3COO)2Mg, 5mM 2-mercaptoethanol, and 100mM Tris-HCI buffer (pH 7.8) in a total volume of 1ml, and the course of the decrease of NADH was monitored at 340 nm. One unit of enzyme was defined as the amount that catalyzed the formation of 1pmol of asparagine per min at 73°e. Plasmid construction and DNA sequencing. The recombinant DNA techniques were done essentially as described by Maniatis et al. 18 ) Deletion of DNA was carried out by the method of Henikoff et al. with exonuclease III and mung bean nuclease. 19 ) DNA sequences were determined by the dideoxy method. 20 )
* To whom correspondence should be addressed. AbbreViations: DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl {3,D-thiogalactopyranoside; asnA, asparagine synthetase gene from Escherichia coli K-12; SD sequence, Shine-Dalgarno sequence; Tris, tris(hydroxymethyl)aminomethane; SDS, sodium dodecyl sulfate; LB medium, Luria-Bertani medium; PVDF, polyvinylidene difluoride; lacZ, structural gene of {3-galactosidase. NII-Electronic Library Service
Overexpression of E. coli Asparagine Synthetase Amino acid composition and sequence analyses. Amino acid analysis was performed by the method of Spackman et al.2l) with a Hitachi model 835 amino-acid analyzer. Samples of 2.4 nmol were hydrolyzed in 6 N HCl in vacuo at 110°C for 22, 48, and 72 hr. Cysteine was determined as cysteinic acid after oxidation with performic acid. 22 ) The amino-terminal sequence analysis was done with a ABI model 477A gas-phase sequenator. After separation by SDS-polyacrylamide gel electrophoresis, proteins were electroblotted onto PVDF membrane by the method of Matsudaira. 23 ) Then the appropriate band was cut out and sequenced. Measurement of native molecular weight. The native molecular weight of asparagine synthetase was measured by gelfiltration on a Cellulofine GCL-IOOO sf column (2.6 x 85 cm) equilibrated with 50 mM Tris-HCI buffer containing 0.1 M KCl and 5 mM 2-mercaptoethanol, pH 7.5. The following proteins were used as standards (numbers in parentheses indicate the assumed M r values): ferritin (450,000), beef liver catalase (240,000), rabbit muscle aldolase (158,000), bovine serum albumin (68,000), hen egg albumin (45,000), chymotrypsinogen A (25,000) and cytochrome c (12,500).
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Results and Discussion Construction of overexpression vector for asparagine synthetase Expression plasmids for asparagine synthetase were constructed by subcloning asnA gene into a polylinker site of plasmid pKK223-3 or pUC18. Plasmid pKK223-3 has the powerful tac promoter, while pUC18 is a high copy number plasmid with the lac promoter. Both promoters can -50
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-30
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be induced by adding isopropyl P,D-thiogalactopyranoside (IPTG). Plasmid pMYlll, containing the wild-type entire asnA gene, was cleaved at the Nsp(7524)I site 43 bases downstream of the Pribnow box of the asnA gene, and treated with Klenow fragment to repair the end of the terminal. Following the PstI cleavage, a 1.2-kbp fragment containing the asnA gene was isolated from a 1.5% agarose gel and ligated into the SmaI-PstI site of plasmid pKK223-3. This derivative of pKK223-3 was designated pKNAOO. Moreover, the EcoRI-PstI fragment containing the asnA gene was transferred from the plasmid pKNAOO to the EcoRI-PstI site ofpUC18 to yield plasmid pUNAOO (Fig. 1). Expression of the asnA gene in E. coli JM 109 transformed with pKNAOO or pUNAOO was examined after induction of the cells with IPTG. SDS polyacrylamide gel electrophoresis of the cells and the asparagine synthetase activity in the cell-free extracts demonstrated that no detectable amounts of asparagine synthetase were being produced. In the DNA sequence of the inserted fragment in pKNAOO and pUNAOO, there is an inverted repeat between the Nsp(7524)I site and the putative SD sequence (Fig. 1), originating from the wild-type asnA gene. It was considered -20
-10
-1
GCATG(GTGTCGGTTTTTGTTGC;JAATCAT~GCAACAGGACGcIAGGAGITATAAAAAIATG... ... ... ... ... ...
inverted repeat
SD
~~~,
- . ORF
~~~~
~,~
........
Nsp (7524)1 ,r-
Pst 1
...--
~~~~~-
I
Dra 1
Nsp (7524)1, Blunting Pst I
Isolation of 1.2 kbp fragment
Sma I Pst 1
Sma I Pst 1
Pst I EeoRI Pst 1
Fig. 1.
EeoRI Pst I
Construction of the Overexpression Vector of asnA Gene.
pMYll1 is a pBR322 derivative containing a 2.1 kbp PsI} fragment of the asparagine synthetase gene (asnA).12) Solid bars represent the open reading frame of the asnA gene. Dotted bar arrowheads represent the lac promoter and indicate the direction of transcription. Dotted bars represent the rmB terminators. Hatched bars represent a part of the structural gene of lacZ.
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Fig. 2.
A.
SUGIYAMA
et al.
Deletion Size and Nucleotide Sequence of Each Constructed Plasmid.
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The extents of the deletions are indicated by arrows. SD ,ac and SD asnA were the SD sequences derived from the lacZ gene and from the leader region of the asnA gene, respectively. The translational initiation codon, ATG, of the lacZ gene is underlined. A nucleotide sequence derived from pKK223-3 was underlined with a double line.
that this inverted repeat made a hairpin structure within Table I. Expression Level of the asnA Gene in Each Plasmid the mRNA and reduced the efficiency of transcription by Expression levela acting as a terminator structure or prevented ribosome Plasmid (%) 12 binding to the mRNA. ) To remove this inverted repeat, deletion from the Nsp(7524)I site was done. By this method ND b pUNAOO it was also expected that plasmids that have various 6.2 pUNAd12 distances betw~en the SD sequence and the ATG start codon 9.6 pUNAd15 7.6 pUNAd27 could be obtained. The changes of this distance also could 14.9 pUNAd37 affect the secondary structure of the ribosome binding region within the mRNA. Plasmid pMYlli was cleaved at the a Relative amount of asparagine synthetase protein to the total cell Nsp(7524)I and the DraI sites, and treated with T4 DNA protein measured from the intensities of protein bands. polymerase to make blunt ends. A 1.5-kbp fragment b Not detectable. containing the asnA gene was isolated from 1.5% agarose gel and treated with exonuclease III and mung bean nuclease. Following the PstI digestion, the resulting (CH 3 COOhMg, 1mM EDTA, 30mM KCl, and 5mM 2fragments were ligated into the SmaI-PstI sites of plasmid mercaptoethanol) and re-collected by centrifugation. The pKK223-3 (Fig. 1). After these were introduced into E. coli cells were then suspended in 50 ml of buffer A and disJM 109, the plasmids expressing the asnA gene were selected rupted by sonication at O°C. The extract was centrifuged by SDS polyacrylamide gel electrophoresis of the cellular at 25,000 x g for 20 min to remove cellular debris. Extraction proteins from these transformants. Four transformants was repeated after suspending the cellular debris in 50 ml producing a new protein band were obtained. Although of buffer A. All procedures below were done at 0--4°C. A cell-free this protein appeared to be asparagine synthetase judging from molecular weight, the amount was very little (slightly extract was diluted to protein concentration of about visible on SDS polyacrylamide gel). Four plasmids were 5mg/ml with buffer B (20mM Tris-HCl buffer, pH 7.4 isolated from each transformant and designated as containing 10% (w/v) glycerol, 5 mM 2-mercaptoethanol), pKNAdI2, 15,27, and 37. Next, each EcoRI-PstI fragment and applied to a DEAE-Cellulofine A-800 column (5 x 18 containing .the \ asnA gene was transferred from these cm) equilibrated with the same buffer. After washing the pKNAd series to the EcoRI-PstI site of pUCI8 to yield column with 0.12 MKCl in buffer B, elution was done with the plasmids pUNAdI2, 15, 27, and 37. Expression of the a linear gradient from 0.12 to 0.4 M KCl in buffer B. The asnA by the pUNAd series was also examined by SDS active fraction was eluted at a KCl concentration of polyacrylamide gel electrophoresis of the cell extracts approximately 0.25 M, and concentrated to about 10 ml on prepared from these transformants. In addition, each of the a Amicon UM-10 membrane. The concentrated enzyme was EcoRI-PstI fragments was subcloned into M13mpI9, and dialyzed against buffer C (the buffer B containing 10mM the nucleotide sequence of the region between 5' end and (CH 3 COOhMg), and then applied to a Blue-Cellulofine the translational start codon ATG of the inserted fragment column (2.5 x 20 cm) equilibrated with buffer C. The.column was determined by the dideoxy method (Fig. 2). The protein was washed with 0.1 M KCl in buffer C, and the enzyme presumed to be asparagine synthetase was produced in the was then eluted with a linear gradient from 0.1 MKCl, 10 mM soluble form at levels ranging from 6.2% to 14.9% of the (CH 3 COOhMg to 1.0 M KCl, 0 mM (CH 3 COOhMg in total cellular protein (Table I). The highest expression was buffer C. The active fraction was concentrated on the observed by the use of the plasmid pUNAd37 in which the Amicon membrane and dialyzed against buffer B. The inverted repeat sequence was excised. puriffed enzyme could be stored at - 70°C without loss of activity for at least one month. A typical purification Purification of asparagine synthetase procedure is summarized in Table II and analysis of the E. coli JMI09 cells transformed with pUNAd37 were enzyme by SDS polyacrylamide gel electrophoresis at grown in 2 liters of LB medium supplemented with 50,ug/ml various stages in the purification is shown in Fig. 3. The ampicillin at 37°C with reciprocal shaking. At log phase purified enzyme has a specific activity of 22.0 unit/mg which (about OD 600 =0.7), IPTG was added to a final con- is 49 fold higher than that previously reported by Ceder centration of 1mM. After 14 hr, the cells were harvest- and Schwartz,2) although they assayed the activity by ed by centrifugation at 8000 x g for 5 min, and washed measuring the radioactive asparagine produced. The factor in 50ml of buffer A (20mM Tris-HCl buffer pH 7.8, 10mM of purification after Blue-Cellulofine was 6.9. It is consistent
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Overexpression of E. coli Asparagine Synthetase Table II. Purification of Asparagine Synthetase Produced by E. coli JM 109/pUNAd37 Total Specific Total protein activity activity (mg) (unita/mg) (unin
Recovery (%)
Table III. Amino Acid Composition of Asparagine Synthetase Produced by E. coli JMI09/pUNAd37 Amino acid
Purity (-fold)
a
926 200
3.2 14.1
2963 2811
100 94.9
1.0 4.4
68
22.0
1496
50.6
6.9
One unit of enzyme was defined as the amount producing Illmol of Asn per min under the standard assay conditions.
He
1
2
3
Cys Met Phe Tyr Arg Lys His Pro
4 ...... Origin
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Deduced from asnA
(residues/subunit) Asx Glx Thr Ser Gly Ala Val Leu
-_._~------.----'-._.--
Crude extract OEAE-Cellulofine A-800 B1ue-Cellulofine
Analyzed
26.7 41.9 11.8 21.4 31.0 27.3 24.2 41.5 14.1 2.3 6.1 10.2 5.1 19.0 15.3 12.8 13.6
27 43 12 22 29 27 24 40 14 2 6 10
5 19 15 13 14
...... 94kDa
..... 67
of a study of the enzyme by X-ray crystallography and protein engineering.
..... 43
Acknowledgments. We are indebted to Dr. M. Takanami for providing us the original E. coli strain carrying plasmid pMY III.
E
References I)
..... 30 2) 3)
...... BPB 4) Fig. 3.
SOS-Polyacrylamide Gel Analysis of Enzyme Purification.
Lane 1, cell-free extract of E. coli JM 109 not transformed; lane 2, cell-free extract of JM 109 transformed with pUNAd37; lane 3, DEAE-Cellulofine A-SOO fraction; lane 4, Blue-Cellulofine fraction.
5) 6) 7) 8) 9)
with the fact that the asparagine synthetase protein is about 14% of the total cellular protein based on the intensities of protein bands.
10)
Protein characterization
11)
The N-terminal amino acid sequence of the asparagine synthetase produced by E. coli JMI09/pUNAd37 was analyzed by a gas-phase sequenator. The first 5 residues were Met-Lys-Thr-Ala-Tyr. This sequence agreed exactly with that predicted from the DNA sequence of the asnA gene. An amino acid composition analysis of the purified enzyme was also done, and the results are compared with those expected from the DNA sequence (Table III). The disagreement is within the usual limit of variation, at most 5%. Furthermore, the native molecular weight was measured by gel filtration as 71,600. This also agreed with the fact that E. coli asparagine synthetase exists as a dimer of identical subunits. 12 ) In conclusion, we have constructed an overexpression plasmid for E. coli asparagine synthetase that produces the enzyme in the form expected. This has enabled us to prepare a large amount of the enzyme by a simple purification procedure. This work can now form the basis
12) 13) 14) 15) 16) 17)
18)
19) 20) 21) 22) 23)
A. Meister, in "The Enzymes," Vol. X, ed. by P. O. Boyer, Academic Press, New York, 1974, pp. 561~580. H. Ceder and J. H. Schwartz, 1. Bioi. Chern., 244, 4112-4121 (1969). L. J. Reitzer and B. Magasanik, J. Bacteriol., 151, 1299-1313 (1982). I. L. Andrulis, J. Chen, and P. N. Ray, Mol. Cell. Bioi., 7, 24352443 (1987). J. S. Gantt and S. M. Arfin,J. Bioi. Chern., 256, 7311-7315 (1981). C. A. Luehr and S. M. Schuster, Arch. Biochem. Biophys., 237, 335-346 (1985). S. Hongo and T. Sato, Anal. Biochem., 114, 163-166 (1981). R. Humbert and R. O. Simoni, J. Bacteriol., 142, 212-220 (1980). H. Ceder and J. H. Schwartz, 1. Bio!. Chern., 244, 4122-4127 (1969). S. Hongo and T. Sato, Arch. Biochem. Biophys., 238, 410-417 (1985). P. M. Mehlhaff, C. A. Luehr, and S. M. Schuster, Biochemistry, 24, 1104-1110 (1985). M. Nakamura, M. Yamada, Y. Hirota, K. Sugimoto, A. aka, and M. Takanami, Nucl. Acids Res., 9, 4669-4676 (1981). M. A. Scofield, W. S. Lewis, and S. M. Schuster, J. BioI. Chern., 265, 12895-12902 (1990). C. Yanisch-Perron, J. Vieira, and J. Messing, Gene, 33, 103-119 (1985). U. K. Laemmli, Nature, 227, 680-685 (1970). M. M. Bradford, Anal. Biochem., 72, 248-254 (1976). O. B. Wetlaufer, in "Advances in Protein Chemistry," Vol. 17, ed. by C. B. Anfinsen, M. L. Anson, K. Bailey, and J. T. Edsall, Academic Press, New York, 1962, pp. 303-390. T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, New York, 1989. S. Henikoff, Gene, 28, 351-359 (1984). F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74,5463-5467 (1977). O. H. Spackman, W. H. Stein, and S. Moore, Anal. Chern., 30, 1190-1206 (1958). S. Moore, J. Bioi. Chern., 238, 235-237 (1963). P. Matsudaira, 1. Bioi. Chern., 262, 10035-10038 (1987).
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ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Overexpression and Purification of Asparagine Synthetase from Escherichia coli Akio Sugiyama, Hiroaki Kato, Takaaki Nishioka & Jun’ichi Oda To cite this article: Akio Sugiyama, Hiroaki Kato, Takaaki Nishioka & Jun’ichi Oda (1992) Overexpression and Purification of Asparagine Synthetase from Escherichia coli, Bioscience, Biotechnology, and Biochemistry, 56:3, 376-379, DOI: 10.1271/bbb.56.376 To link to this article: http://dx.doi.org/10.1271/bbb.56.376
Published online: 12 Jun 2014.
Submit your article to this journal
Article views: 34
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Download by: [117.255.242.234]
Date: 05 November 2015, At: 23:16
Biosci. Biotech. Biochem., 56 (3), 376-379, 1992
Overexpression and Purification of Asparagine Synthetase from Escherichia coli Akio
SUGIYAMA,
Hiroaki
KATO,
Takaaki
NISHIOKA,
and Jun'ichi
ODA*
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 Received June 10, 1991
Downloaded by [117.255.242.234] at 23:16 05 November 2015
Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUCI8, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15°,10 of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The aminoterminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.
L-Asparagine synthetase [L-aspartate: ammonia ligase (AMP-forming) EC 6.3.1.1J catalyzes the synthesis of Lasparagine from L-aspartate and ammonia, while hydrolyzing ATP to AMP and PPi. 1) These enzymes are distributed in prokaryotes as Escherichia coli 2 ) and Klebsiella aerogenes 3 ) and encoded by asnA genes. On the other hand, asparagine synthetases from eukaryotes 4 - 7) use glutamine as the preferred amido nitrogen donor, although they can also use ammonia. These glutamine-dependent enzymes [L-aspartate: L-glutamine amido-ligase (AMPforming) EC 6.3.5.4J are also found in prokaryotes 3 ,8) and encoded by asnB genes. The steady state kinetic mechanisms of asparagine synthetase have been reported for both the ammonia-dependent and glutamine-dependent enzymes. 9 - 11) In the ammonia-using reaction, the mechanism of the ammonia-dependent enzymes was different from that of the glutamine-dependent enzymes. 9 ,11) Both types of asparagine synthetase genes have been cloned and sequenced from E. COli 12 ,13) and humans. 4 ) The asnA gene from E. coli coded for a polypeptide of 330 amino acid residues with a molecular weight of 36,700. A high degree of amino acid sequence homology was shown between the glutamine-dependent enzymes (asnB enzyme from E. coli and the human enzyme), however, no significant homology was found between the asnA and the asnB sequences although derived from the same origin, E. coli. 13) To elucidate these functional differences based on the protein structure, it is essential to obtain a large amount of the purified protein. We describe the construction of overexpression plasmids of the asnA gene from E. coli K-12 and a simple method for purifying the enzyme from transformed cells. This is the first time that asparagine synthetase from the asnA gene has been purified to apparent homogeneity.
Materials and Methods Bacterial strains, phages, and plasmids. Plasmid pMYlll is a pBR322 derivative containing a 2.1-kilobase pair Pstl fragment of the asparagine
synthetase gene (asnA) from Escherichia coli K_12. 12 ) The overexpression plasmid pKK-223-3 was obtained from Pharmacia (Uppsala). Plasmid pUCl8 and E. coli JMI09, 14) a host ofpUC18 and pKK223-3 derivatives, were purchased from Takara Shuzo Co., Ltd. (Kyoto). Restriction enzymes and chemicals. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, exonuclease III, mung bean nuclease, and Klenow fragment of DNA polymerase I were purchased from Takara Shuzo Co., Ltd. and Toyobo (Osaka). L-Aspartate was from Wako Pure Chemicals Co., Ltd. ATP, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase were from Oriental Yeast Co. (Tokyo). Adenylate kinase was from Unichika Co. (Osaka). DEAE-Cellulofine A-800, Blue-Cellulofine, and Cellulofine GCL-I 000 sf were gifts from Chisso Co., (Minamata). All other chemicals used in this experiment were of the purest grades commercially available. SDS-polyacrylamide gel electrophoresis. Protein samples were electrophoresed in 10% polyacrylamide gels in the presence of SDS. 15 ) Gels were then stained with Coomassie Brilliant Blue R-250. To measure the relative amount of asparagine synthetase protein, the stained gels were scanned at 570 nm using a model CS91 0 chromatoscanner (Shimadzu Co.). Assay of asparagine synthetase activity. Protein concentration was measured by the method of Bradford 16 ) with Protein Assay Reagent (Pierce Chemical Co.), and bovine serum albumin as a standard. The protein concentration of the asparagine synthetase in the purified preparation was determined from A ir'b = 13.9 cm -1 which was calculated from multiplication of the numbers of Tyr and Trp residues by their molar absorption coefficients. 17) Asparagine synthetase activity was assayed spectrophotometrically by coupling the formation of AMP to the adenylate kinase, pyruvate kinase, and lactate dehydrogenase reactions. The enzyme solution (50 pI) was added to an assay medium composed of 2 U adenylate kinase, IOU pyruvate kinase, 25 U lactate dehydrogenase, 0.24 mM NADH, I mM phosphoenolpyruvate, 1.5 mM L-aspartic acid, 20mM NH 4C1, 3 mM ATP, 100mM KCl, IOmM (CH3COO)2Mg, 5mM 2-mercaptoethanol, and 100mM Tris-HCI buffer (pH 7.8) in a total volume of 1ml, and the course of the decrease of NADH was monitored at 340 nm. One unit of enzyme was defined as the amount that catalyzed the formation of 1pmol of asparagine per min at 73°e. Plasmid construction and DNA sequencing. The recombinant DNA techniques were done essentially as described by Maniatis et al. 18 ) Deletion of DNA was carried out by the method of Henikoff et al. with exonuclease III and mung bean nuclease. 19 ) DNA sequences were determined by the dideoxy method. 20 )
* To whom correspondence should be addressed. AbbreViations: DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl {3,D-thiogalactopyranoside; asnA, asparagine synthetase gene from Escherichia coli K-12; SD sequence, Shine-Dalgarno sequence; Tris, tris(hydroxymethyl)aminomethane; SDS, sodium dodecyl sulfate; LB medium, Luria-Bertani medium; PVDF, polyvinylidene difluoride; lacZ, structural gene of {3-galactosidase. NII-Electronic Library Service
Overexpression of E. coli Asparagine Synthetase Amino acid composition and sequence analyses. Amino acid analysis was performed by the method of Spackman et al.2l) with a Hitachi model 835 amino-acid analyzer. Samples of 2.4 nmol were hydrolyzed in 6 N HCl in vacuo at 110°C for 22, 48, and 72 hr. Cysteine was determined as cysteinic acid after oxidation with performic acid. 22 ) The amino-terminal sequence analysis was done with a ABI model 477A gas-phase sequenator. After separation by SDS-polyacrylamide gel electrophoresis, proteins were electroblotted onto PVDF membrane by the method of Matsudaira. 23 ) Then the appropriate band was cut out and sequenced. Measurement of native molecular weight. The native molecular weight of asparagine synthetase was measured by gelfiltration on a Cellulofine GCL-IOOO sf column (2.6 x 85 cm) equilibrated with 50 mM Tris-HCI buffer containing 0.1 M KCl and 5 mM 2-mercaptoethanol, pH 7.5. The following proteins were used as standards (numbers in parentheses indicate the assumed M r values): ferritin (450,000), beef liver catalase (240,000), rabbit muscle aldolase (158,000), bovine serum albumin (68,000), hen egg albumin (45,000), chymotrypsinogen A (25,000) and cytochrome c (12,500).
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Results and Discussion Construction of overexpression vector for asparagine synthetase Expression plasmids for asparagine synthetase were constructed by subcloning asnA gene into a polylinker site of plasmid pKK223-3 or pUC18. Plasmid pKK223-3 has the powerful tac promoter, while pUC18 is a high copy number plasmid with the lac promoter. Both promoters can -50
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be induced by adding isopropyl P,D-thiogalactopyranoside (IPTG). Plasmid pMYlll, containing the wild-type entire asnA gene, was cleaved at the Nsp(7524)I site 43 bases downstream of the Pribnow box of the asnA gene, and treated with Klenow fragment to repair the end of the terminal. Following the PstI cleavage, a 1.2-kbp fragment containing the asnA gene was isolated from a 1.5% agarose gel and ligated into the SmaI-PstI site of plasmid pKK223-3. This derivative of pKK223-3 was designated pKNAOO. Moreover, the EcoRI-PstI fragment containing the asnA gene was transferred from the plasmid pKNAOO to the EcoRI-PstI site ofpUC18 to yield plasmid pUNAOO (Fig. 1). Expression of the asnA gene in E. coli JM 109 transformed with pKNAOO or pUNAOO was examined after induction of the cells with IPTG. SDS polyacrylamide gel electrophoresis of the cells and the asparagine synthetase activity in the cell-free extracts demonstrated that no detectable amounts of asparagine synthetase were being produced. In the DNA sequence of the inserted fragment in pKNAOO and pUNAOO, there is an inverted repeat between the Nsp(7524)I site and the putative SD sequence (Fig. 1), originating from the wild-type asnA gene. It was considered -20
-10
-1
GCATG(GTGTCGGTTTTTGTTGC;JAATCAT~GCAACAGGACGcIAGGAGITATAAAAAIATG... ... ... ... ... ...
inverted repeat
SD
~~~,
- . ORF
~~~~
~,~
........
Nsp (7524)1 ,r-
Pst 1
...--
~~~~~-
I
Dra 1
Nsp (7524)1, Blunting Pst I
Isolation of 1.2 kbp fragment
Sma I Pst 1
Sma I Pst 1
Pst I EeoRI Pst 1
Fig. 1.
EeoRI Pst I
Construction of the Overexpression Vector of asnA Gene.
pMYll1 is a pBR322 derivative containing a 2.1 kbp PsI} fragment of the asparagine synthetase gene (asnA).12) Solid bars represent the open reading frame of the asnA gene. Dotted bar arrowheads represent the lac promoter and indicate the direction of transcription. Dotted bars represent the rmB terminators. Hatched bars represent a part of the structural gene of lacZ.
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Fig. 2.
A.
SUGIYAMA
et al.
Deletion Size and Nucleotide Sequence of Each Constructed Plasmid.
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The extents of the deletions are indicated by arrows. SD ,ac and SD asnA were the SD sequences derived from the lacZ gene and from the leader region of the asnA gene, respectively. The translational initiation codon, ATG, of the lacZ gene is underlined. A nucleotide sequence derived from pKK223-3 was underlined with a double line.
that this inverted repeat made a hairpin structure within Table I. Expression Level of the asnA Gene in Each Plasmid the mRNA and reduced the efficiency of transcription by Expression levela acting as a terminator structure or prevented ribosome Plasmid (%) 12 binding to the mRNA. ) To remove this inverted repeat, deletion from the Nsp(7524)I site was done. By this method ND b pUNAOO it was also expected that plasmids that have various 6.2 pUNAd12 distances betw~en the SD sequence and the ATG start codon 9.6 pUNAd15 7.6 pUNAd27 could be obtained. The changes of this distance also could 14.9 pUNAd37 affect the secondary structure of the ribosome binding region within the mRNA. Plasmid pMYlli was cleaved at the a Relative amount of asparagine synthetase protein to the total cell Nsp(7524)I and the DraI sites, and treated with T4 DNA protein measured from the intensities of protein bands. polymerase to make blunt ends. A 1.5-kbp fragment b Not detectable. containing the asnA gene was isolated from 1.5% agarose gel and treated with exonuclease III and mung bean nuclease. Following the PstI digestion, the resulting (CH 3 COOhMg, 1mM EDTA, 30mM KCl, and 5mM 2fragments were ligated into the SmaI-PstI sites of plasmid mercaptoethanol) and re-collected by centrifugation. The pKK223-3 (Fig. 1). After these were introduced into E. coli cells were then suspended in 50 ml of buffer A and disJM 109, the plasmids expressing the asnA gene were selected rupted by sonication at O°C. The extract was centrifuged by SDS polyacrylamide gel electrophoresis of the cellular at 25,000 x g for 20 min to remove cellular debris. Extraction proteins from these transformants. Four transformants was repeated after suspending the cellular debris in 50 ml producing a new protein band were obtained. Although of buffer A. All procedures below were done at 0--4°C. A cell-free this protein appeared to be asparagine synthetase judging from molecular weight, the amount was very little (slightly extract was diluted to protein concentration of about visible on SDS polyacrylamide gel). Four plasmids were 5mg/ml with buffer B (20mM Tris-HCl buffer, pH 7.4 isolated from each transformant and designated as containing 10% (w/v) glycerol, 5 mM 2-mercaptoethanol), pKNAdI2, 15,27, and 37. Next, each EcoRI-PstI fragment and applied to a DEAE-Cellulofine A-800 column (5 x 18 containing .the \ asnA gene was transferred from these cm) equilibrated with the same buffer. After washing the pKNAd series to the EcoRI-PstI site of pUCI8 to yield column with 0.12 MKCl in buffer B, elution was done with the plasmids pUNAdI2, 15, 27, and 37. Expression of the a linear gradient from 0.12 to 0.4 M KCl in buffer B. The asnA by the pUNAd series was also examined by SDS active fraction was eluted at a KCl concentration of polyacrylamide gel electrophoresis of the cell extracts approximately 0.25 M, and concentrated to about 10 ml on prepared from these transformants. In addition, each of the a Amicon UM-10 membrane. The concentrated enzyme was EcoRI-PstI fragments was subcloned into M13mpI9, and dialyzed against buffer C (the buffer B containing 10mM the nucleotide sequence of the region between 5' end and (CH 3 COOhMg), and then applied to a Blue-Cellulofine the translational start codon ATG of the inserted fragment column (2.5 x 20 cm) equilibrated with buffer C. The.column was determined by the dideoxy method (Fig. 2). The protein was washed with 0.1 M KCl in buffer C, and the enzyme presumed to be asparagine synthetase was produced in the was then eluted with a linear gradient from 0.1 MKCl, 10 mM soluble form at levels ranging from 6.2% to 14.9% of the (CH 3 COOhMg to 1.0 M KCl, 0 mM (CH 3 COOhMg in total cellular protein (Table I). The highest expression was buffer C. The active fraction was concentrated on the observed by the use of the plasmid pUNAd37 in which the Amicon membrane and dialyzed against buffer B. The inverted repeat sequence was excised. puriffed enzyme could be stored at - 70°C without loss of activity for at least one month. A typical purification Purification of asparagine synthetase procedure is summarized in Table II and analysis of the E. coli JMI09 cells transformed with pUNAd37 were enzyme by SDS polyacrylamide gel electrophoresis at grown in 2 liters of LB medium supplemented with 50,ug/ml various stages in the purification is shown in Fig. 3. The ampicillin at 37°C with reciprocal shaking. At log phase purified enzyme has a specific activity of 22.0 unit/mg which (about OD 600 =0.7), IPTG was added to a final con- is 49 fold higher than that previously reported by Ceder centration of 1mM. After 14 hr, the cells were harvest- and Schwartz,2) although they assayed the activity by ed by centrifugation at 8000 x g for 5 min, and washed measuring the radioactive asparagine produced. The factor in 50ml of buffer A (20mM Tris-HCl buffer pH 7.8, 10mM of purification after Blue-Cellulofine was 6.9. It is consistent
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Overexpression of E. coli Asparagine Synthetase Table II. Purification of Asparagine Synthetase Produced by E. coli JM 109/pUNAd37 Total Specific Total protein activity activity (mg) (unita/mg) (unin
Recovery (%)
Table III. Amino Acid Composition of Asparagine Synthetase Produced by E. coli JMI09/pUNAd37 Amino acid
Purity (-fold)
a
926 200
3.2 14.1
2963 2811
100 94.9
1.0 4.4
68
22.0
1496
50.6
6.9
One unit of enzyme was defined as the amount producing Illmol of Asn per min under the standard assay conditions.
He
1
2
3
Cys Met Phe Tyr Arg Lys His Pro
4 ...... Origin
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Deduced from asnA
(residues/subunit) Asx Glx Thr Ser Gly Ala Val Leu
-_._~------.----'-._.--
Crude extract OEAE-Cellulofine A-800 B1ue-Cellulofine
Analyzed
26.7 41.9 11.8 21.4 31.0 27.3 24.2 41.5 14.1 2.3 6.1 10.2 5.1 19.0 15.3 12.8 13.6
27 43 12 22 29 27 24 40 14 2 6 10
5 19 15 13 14
...... 94kDa
..... 67
of a study of the enzyme by X-ray crystallography and protein engineering.
..... 43
Acknowledgments. We are indebted to Dr. M. Takanami for providing us the original E. coli strain carrying plasmid pMY III.
E
References I)
..... 30 2) 3)
...... BPB 4) Fig. 3.
SOS-Polyacrylamide Gel Analysis of Enzyme Purification.
Lane 1, cell-free extract of E. coli JM 109 not transformed; lane 2, cell-free extract of JM 109 transformed with pUNAd37; lane 3, DEAE-Cellulofine A-SOO fraction; lane 4, Blue-Cellulofine fraction.
5) 6) 7) 8) 9)
with the fact that the asparagine synthetase protein is about 14% of the total cellular protein based on the intensities of protein bands.
10)
Protein characterization
11)
The N-terminal amino acid sequence of the asparagine synthetase produced by E. coli JMI09/pUNAd37 was analyzed by a gas-phase sequenator. The first 5 residues were Met-Lys-Thr-Ala-Tyr. This sequence agreed exactly with that predicted from the DNA sequence of the asnA gene. An amino acid composition analysis of the purified enzyme was also done, and the results are compared with those expected from the DNA sequence (Table III). The disagreement is within the usual limit of variation, at most 5%. Furthermore, the native molecular weight was measured by gel filtration as 71,600. This also agreed with the fact that E. coli asparagine synthetase exists as a dimer of identical subunits. 12 ) In conclusion, we have constructed an overexpression plasmid for E. coli asparagine synthetase that produces the enzyme in the form expected. This has enabled us to prepare a large amount of the enzyme by a simple purification procedure. This work can now form the basis
12) 13) 14) 15) 16) 17)
18)
19) 20) 21) 22) 23)
A. Meister, in "The Enzymes," Vol. X, ed. by P. O. Boyer, Academic Press, New York, 1974, pp. 561~580. H. Ceder and J. H. Schwartz, 1. Bioi. Chern., 244, 4112-4121 (1969). L. J. Reitzer and B. Magasanik, J. Bacteriol., 151, 1299-1313 (1982). I. L. Andrulis, J. Chen, and P. N. Ray, Mol. Cell. Bioi., 7, 24352443 (1987). J. S. Gantt and S. M. Arfin,J. Bioi. Chern., 256, 7311-7315 (1981). C. A. Luehr and S. M. Schuster, Arch. Biochem. Biophys., 237, 335-346 (1985). S. Hongo and T. Sato, Anal. Biochem., 114, 163-166 (1981). R. Humbert and R. O. Simoni, J. Bacteriol., 142, 212-220 (1980). H. Ceder and J. H. Schwartz, 1. Bio!. Chern., 244, 4122-4127 (1969). S. Hongo and T. Sato, Arch. Biochem. Biophys., 238, 410-417 (1985). P. M. Mehlhaff, C. A. Luehr, and S. M. Schuster, Biochemistry, 24, 1104-1110 (1985). M. Nakamura, M. Yamada, Y. Hirota, K. Sugimoto, A. aka, and M. Takanami, Nucl. Acids Res., 9, 4669-4676 (1981). M. A. Scofield, W. S. Lewis, and S. M. Schuster, J. BioI. Chern., 265, 12895-12902 (1990). C. Yanisch-Perron, J. Vieira, and J. Messing, Gene, 33, 103-119 (1985). U. K. Laemmli, Nature, 227, 680-685 (1970). M. M. Bradford, Anal. Biochem., 72, 248-254 (1976). O. B. Wetlaufer, in "Advances in Protein Chemistry," Vol. 17, ed. by C. B. Anfinsen, M. L. Anson, K. Bailey, and J. T. Edsall, Academic Press, New York, 1962, pp. 303-390. T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, New York, 1989. S. Henikoff, Gene, 28, 351-359 (1984). F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74,5463-5467 (1977). O. H. Spackman, W. H. Stein, and S. Moore, Anal. Chern., 30, 1190-1206 (1958). S. Moore, J. Bioi. Chern., 238, 235-237 (1963). P. Matsudaira, 1. Bioi. Chern., 262, 10035-10038 (1987).
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