The cloning, expression and crystallisation of a thermostable arginase

FEBS 17070

FEBS Letters 386 (1996) 215 218

The cloning, expression and crystallisation of a thermostable arginase Maria C. Bewley*, J. Shaun Lott, Edward N. Baker, Mark L. Patchett Department of Biochemistry, Massey University, Palmerston North, New Zealand Received 15 March 1996; revised version received 22 April 1996

Abstract The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox" has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coil using an inducible T7 RNA polymerasebased system. A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystaHised and one crystal form is isomorphous to crystals of the native protein. Key words: A r g i n a s e ; G e n e cloning; T h e r m o p h i l i c ; T h e r m o s t a b l e ; Bacillus caldovelox

1. Introduction T h e recent realisation t h a t arginases m a y play a key regulatory role in nitric oxide m e t a b o l i s m [1,2] a n d hence affect cytotoxic processes in i m m u n o l o g i c a l defence [3] has rekindled interest in this family o f enzymes. Arginases (L-arginine amidinohydrolases, E.C. 3.5.3.1) are metal ion-activated enzymes which catalyse the hydrolysis o f L-arginine to L-ornithine a n d urea. This reaction is p a r t o f the urea cycle in ureotelic animals, a n d is the initial step o f arginine c a t a b o l i s m in certain aerobic bacteria [4]. Despite considerable research o n trimeric eukaryotic arginases, n o t a b l y f r o m rat liver [5-8] a n d yeast [911], these enzymes r e m a i n very m u c h a mechanistic a n d structural mystery. T h e wide range o f transition (VO 2+, Fe 2+, Co 2+, N i 2+) a n d heavy m e t a l ions (Cd 2+) t h a t are able to substitute for the in vivo M n 2+ cofactor o f arginases also m a k e t h e m a n attractive target for the study o f m e t a l ionm e d i a t e d catalysis a n d stability [12]. W e aim to d e t e r m i n e the X - r a y crystal structure o f the h o m o - h e x a m e r i c arginase f r o m the t h e r m o p h i l i c b a c t e r i u m 'B. caldovelox'. Crystals o f the native enzyme have been obtained [13], b u t the yields o f arginase f r o m "B. caldovelox' are low. Here we r e p o r t the cloning, sequencing a n d expression in E. coli o f the 'B. caldovelox" arginase gene, a n d crystallisation o f the r e c o m b i n a n t protein, in order to facilitate further structural a n d physical studies o f this enzyme.

2. Materials and methods 'Bacillus caldovelox' (Bacillus species DSM (Deutsche Sammlung von Mikroorganism) 411) was grown with strong aeration at 70°C [12]. Enzymes for DNA manipulation were obtained from Gibco BRL and Boehringer Mannheim; Pbluescript KS(--) from Stratagene; radioisotopes from ICN; Sequenase from USB; pGEM-3Zf(--) and Taq DNA polymerase from Promega and oligonucleotides from Oligos Etc. Inc.

*Corresponding author. Fax: (64) (6) 350 5682.

2.1. Isolation and sequencing of the arginase gene Genomic DNA from "B. caldovelox' was isolated as described for Thermus aquaticus [14]. Degenerate oligonucleotides, oligo 1 and oligo 2, were designed, on the basis of N-terminal protein sequence [12] and the conserved internal amino acid sequence -GGDHS-, respectively. Oligo 1 : 5'-ATG AAA CCG ATY TCG ATY ATY GGI GTS CCG ATG GA-3'; oligo 2: 5'-RTG RTC SCC SCC MA-3' (M=A or C; R=A or G; S=C or G and Y=C or T). A 30 cycle PCR was performed using oligos 1 and 2, with a 45°C annealing temperature (first cycle 35°C, second cycle 40°C) and a 15 s extension at 72°C in the presence of 2 mM MgCI2 with 'B. caldovelox' genomic DNA as a template. The PCR product was radiolabelled with a2p (Random Primers DNA Labelling System, Gibco BRL) and used to probe Southern blots of genomic DNA digested with a panel of restriction enzymes (Fig. 2). Hybridisation was carried out in 5 × Denhardt's reagent, 5 × SSC and 0.5% SDS at 68°C, and the filters were washed in 1 ×SSC, 0.1% SDS at 68°C. A hybridising fragment containing the arginase gene was subcloned into the vector pGEM3Zf(--), and the coding region of the arginase gene sequenced on both strands using Sequenase. Standard techniques were used for DNA manipulation and analysis [15]. 2.2. Subcloning and expression of arginase The expression vector pKS-rbs is a derivative of pBluescript K S ( - ) in which the XbaI-Ndel fragment of pT7-7 [16] containing the ribosome binding sequence has been subcloned downstream of the T7 promoter. The coding region of the arginase gene was amplified from the genomic clone in a 30 cycle PCR using Pfu polymerase (Stratagene) at an annealing temperature of 52°C with oligonucleotides 3 and 4, which contain the recognition sites for NdeI and HindllI, respectively (underlined). Oligo 3: 5'-TGG GAG ACC ATA TGA AGC CA-3'; oligo 4: 5'-CCC ACA AGC TTTACA-TGA-AGT-3'. The PCR product was digested with NdeI and HindlII and ligated into NdeI/HindlII digested pKS-rbs to yield the arginase expression plasmid pKS-Arg, in which the initiation codon of the arginase gene is 7 bp downstream of the ribosome binding site. Cells containing pKSArg were grown aerobically at 37°C in M1 medium [17], supplemented with 10 mM glucose, 8 gM MnCI2 and 100 gg/ml ampicillin, to a culture OD600 of 2.0 and induced by adding IPTG to a final concentration of 0.25 raM. Cultures were grown for a further 4 h and cells harvested by centrifugation. 2.3. Protein purification The cell pellet from a 650 ml culture (3.7 g) was resuspended in 4.5 volumes of lysis buffer (100 mM MOPS/NaOH, 50 mM MnC12, pH 7.5) and lysed by sonication on ice. Cell debris was removed by ultracentrifugation at 220000×g for 1 h at 5°C. The supernatant was rapidly heated to and incubated at 70°C for 15 min. After rapid cooling on ice, insoluble material was removed by centrifugation at 31000×g for 15 min at 5°C. Solid ammonium sulphate (enzyme grade~ BRL) was added to the supernatant to 30% saturation and the solution was centrifuged at 31000×g for 10 min at 20°C. The supernatant was made 50% saturated by further addition of ammonium sulphate and the solution was centrifuged as before. The pellet was solubilised in three volumes of buffer (200 mM MOPS/NaOH, pH 7.5) and centrifuged as before. The supernatant was dialysed against 200 volumes of 20 mM MOPS/NaOH, pH 7.5, overnight at 4°C. After dialysis, any insoluble material was removed by further centrifugation at 31 000 × g. 2.4. Characterisation of recombinant protein The purified recombinant protein was subjected to N-terminal sequencing. Arginase activity and protein concentration were determined as previously described [12].

0014-5793/96/$12.00 © 1996 Federation of European Biochemical Societies. All rights reserved. PH S 0 0 1 4 - 5 7 9 3 ( 9 6 ) 0 0 4 5 9 - 0

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M.C. Bewley et al.IFEBS Letters 386 (1996) 215-218

2.5. Crystallisation and data collection The hanging drop method was used for an initial PEG-based screening procedure, which was carried out at 20°C using 64 trial conditions in the pH range 4.9~.1 [18]. Reservoirs contained 1 ml of screening solutions and the drop contained 5 ~tl of reservoir solution and 5 ~tl of protein solution (27 ~tg/~tl protein in 20 mM MOPS/ NaOH, pH 7.5). Crystals were mounted and sealed in Lindemann tubes and X-ray data collected using the rotation method [19] with a Rigaku R-AXIS IIC image plate mounted on a Rigaku RU200 rotating anode operating at 50 kV, 100 mA. Data were processed using the program DENZO [20] and reduced using programs from the CCP4 crystallographic package [21]. Self-rotation searches were calculated using the program GLFR [22].

kb

12.0=_

3.0-The amino acid sequences of arginases from a variety of species were retrieved from version 26.2 of the O W L nonredundant protein sequence database [23] and aligned using the program C L U S T A L W [24] (see Fig. 1). This identified the amino acid sequence - G G D H S - as the longest contiguous sequence perfectly conserved in arginases from a diverse selecB. c a l d o v e l o x BACSU

.................

MKP I S I I GVPMDLCQTRRGVDMGPSAMRYAGVI

................

M]DKT I S V I G M P M D L G Q A R R G V D M G P S A I

I~qGAGE I N A S R H R K E N E L K T C Q

ARGI

COCIM

- - -MTS P ST I KQKF IAKGHQLGV'VAVGFSDGQ PNQGVD- - PSGL I EAGLLDQLRDD

ARGI

YEAST

...... METGPHYNYYKNRELS

ARGI

XENLA

.............

MAKER}{SVGVI GAP FSKC-QPRRGIrEI~GPKYLREAGL I EKL RE .....

ARGI HUMAN

.............

MSAKS RT I G I I GAP FS KGQPRGGVEEGPTVLRKAGLLEKL

IL G A P V Q S G A S Q P G C L M G P D A F R T A G L T Q V L T E IVLAP FSGGQGKLGVEKGPKYMLI~GLQTS

h

~I

B. c a l d o v e l o x

.... LHY DI EDLGD I P- IGKA~R-LHEQGDS .... MGyTVEDLGD

.

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ARGI

BACSU

I

P~LRNLKA- -VA~EKI~kAAVDQWQ

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I P - I N R E K I - K N - - - D E E L K N L N S - - V L A G N E K L A Q E Y N } ( V I EE}CK

.... LG~AVTDLGDAT

PTArE P E L S H P N - - - S A V E I r L D A - - L V G W T R S L S Q K A L E M A R S FVP - EHDPNHRGMKKPRT

CD

/~RGI C O C I M

.... LEYD IRHDGQVHTYAE

ARGI_YEAST

E LE P SI~ EAQ FVG~

ARGI

XENLA

. . . . FC-NDVP, D C G D L D - F P D V P N - D T P - - F N N V K N P R T - - V G K A T E I L A N A V T A V K K A D K

ARGI

HUMAN

.... QEC DVI(DYGDLP - FAD I PN- DS P -- FQ [VT~P RS - -VGKAS EQLAGKVAQV~GR

FPLVLGGDHS

- -VSAATQQL S RQVYEHAREGR

K I ~ K D S T T G G S S V M I D G V } ( A K R A D L V G E A T KLV~" N S V S K V V Q A N R

II B. c a l d o v e l o x

IAI GTLAGVA~YER-LG

i

I

,'

. . . . V I W Y DA/{GDV]NTA~ T S P S G N I HGIVH?- - -

A2,GI B A C S U

FPLVLGGDHS IAI GTLAGTA/~Y DN-LG- - - -VIWY DAHGDLNTL ETS P S GN IHGMP- - -

ARGI_AGRT5

L PVFL GGDHSIWSAGTVS GVAQ RTAE - L GKEQ FVLWLDAHT D LHT L H TTAS GNLHGT PVA Y

ARGI_COCIM

LVLTLGGDHS

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I A I G T I S G T A K A I R E R L G R E I ~ V I W V D A H A D I N R P E D S V S G N I HGI~IPI~%F

FPLTLGGDHS IAI GTVSAVLDKY

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TCQS IGGDHSLAVGT

ARGI_HUF~

ISLVLGGDHSLAI

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P D - A G . . . . L L W I DAHA.D I N T I E S T P S G N L H G C P V S F

IAGHAAVHPN-LC

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GS ISGHARVHPD-LG-

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-- -VIWVDAHTDI NTPLTTT SGNLHGQ PVS F

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IN*

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IGGYS PKI KPEHVVLI GVRSLDEGEKKF

I I**I**

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B.caldovelox

LAASLGFGHPALTQ

ARGI_BACSU

LAVSLGI GHESLVNLEGYAPKI

ARGI_AGRT5

YTGQSG ..... FE GL P PLAAPVN P RNVS ~4G I RSVD P EERRRVAE I GVQVAD~VL

D EQ G

ARGI

COCIM

LTGLAKDDNEDMFGWLQPDNL

DKY G

ARGI

YEAST

LMGLNKDVP HC p E SL KWVP GNL S P KK LAY IGL RDVDAGE KKI L KDL G IAAF SMY HVDKY G

KPENVVI IGARSLDEGERKY

ARGI_XENLA

LIV~x'ELKAK~AVPGFE~PCLRSKD LLKELKGKI PDVPGFSWVTPC

BACSU

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GGLTY RE S HLAMEMLA

S LDL DGL DPND.kPGVGTPWGG

ARGI_AGRT5

V % / R P L E A F L D R V $ k'VS - - - G R L H V S L D V D F L D P A I A P A V G T T V P G G A T

ARGI

IGRVVEMALAH

COCIM

KAFS~DI

I V Y I G L R D V D P G E H Y I L K T L G I K~fL S M I EVDYL}(

I SAKD IVY IGLRDVDPGEHY

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ARGI_HUMA~

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i S YRESHLA~Y FREAHL IMEMLH

IGQD .... T P IHL S FDVDALD PQWAP STGT PVRGGLTLREGDF

INAVI EI~VHPETNGEGP

ARGI_XENLA

DD~ETLEYLVGI~H-

- KRPIHL S FD IDGLDPS ][APATGTPC PGGRTYREGR

ARGI_HU~

IGKVMEETLSYLLGRK-

- KRP IHL S FDVDGLD PS FTPATGT PVVGGLTY REGLY I TEE IY

B, c a l d o v e l o x

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I*

b

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*

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E A Q I IT S A ~ F V E V N . . . . . P I L D E R . . . . N K T A S V A V A / 2 4 G S L F G E ~ M

FLVERI~k ILHEQLH

J **

*

I

.............

ARGI _BACSU

D A G I I T S A E F V E V N . . . . . P I L D H K . . . . Nk'T G K T A V E L V E S L L GICKL L . . . . . . . . . . . . .

ARGI

DSGLVTSLDLAELN

AGRT5

..... PFLDER .... GRTARL ITDLAS SLFGRRVFDRVTTAF

......

ARGI_COCIM A2,GI Y E A S T A2,GI _ X E N L A

ETGSLVAMDLVEVN ..... PTLETLGA- - SET IRAGCSLVRSALGDTLL ............. E SGNL IALDVVECN PDLAIHD IHVSNT I SAC-CAIARCALGETLL K T G L L S C-VDT I ~ S T S R G E T } ( R D V .... EVTVKTAL DMT L S C FGKAR EG FHAS T

ARGI _HUMAN

KTGLLS GLDIMEVN ..... PSLGKTPEEVTRTVNTAVAI

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TLAC FGLAREGNHKP

I

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2.0--

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1.6--Fig. 2. Autoradiograph (3 min exposure) of a Southem blot of "B. caldovelox' genomic DNA fragments separated on a 0.7% agarose gel probed with the 32P-labelled 300 bp PCR product.

ERL ER- -

RYAHL I ERL S D .....

A~RG I _ A G R T 5

ARGI_AGRT5

~B U

9.0-7.0-6.0-5.0-4.0---

3. Results and discussion

ARGI

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+

Fig. 1. Alignment of the deduced protein sequence of 'B. caldovelox' arginase with arginase sequences from a range of species. The alignment was performed using the program CLUSTAL W 1.5 [22]. Positions showing absolute conservation of amino acid residue are marked with an asterisk; positions showing conservation of amino acid type are marked with a vertical line. Protein sequence codes are in SwissProt format: BACSU=Bacillus subtilis; AGRT=Agrobacterium tumefaciens; COCIM=Coccidioides immitis; YEAST=Saccharomyces cerevisiae; XENLA=Xenopus laevis; HUMAN=Homo sapiens. The sequence of 'B. caldovelox" arginase has been submitted to the GenBanldEMBL database; the accession number is U48226.

tion of species. This sequence and N-terminal sequence data [12] were used to design oligonucleotides for amplification of a 300 bp portion of the 'B. caldovelox' arginase gene by PCR. Direct sequencing confirmed that the single 300 bp P C R product coded for the N-terminal one third of an arginase, and radiolabelled P C R product was used to probe a Southern blot of ' R caldovelox" genomic D N A that had been digested with a panel of restriction enzymes (see Fig. 2). Restriction mapping of the hybridising fragments indicated that a 3.2 kb EcoRI/ BgIII fragment contained the arginase gene. Genomic D N A was digested with E e o R I and BglII and size fractionated by agarose gel electrophoresis. A mini-library of 2.9-3.5 kb EcoRI/BgllI fragments was produced by ligation into EcoRI/BamHI-digested p G E M - 3 Z f ( - ) . The ligation mix was transformed into XL1-Blue cells by electroporation and colonies with the desired genomic clone were identified by P C R on colony D N A , using oligonucleotides 1 and 2 as primers. The 3.2 kb fragment contained an open reading frame encoding a protein of 299 amino acids, with a calculated molecular weight of 32.4 kDa. Both the conserved internal sequence and the previously obtained N-terminal sequence were present in the deduced amino acid sequence, which is shown in Fig. 3. In order to subclone this coding region into an expression vector, P C R was carried out using the "B. caldovelox" genomic clone as a template and oligos 3 and 4 as primers. A single product of approximately 900 bp was amplified. This product was digested with NdeI and HindlII and subcloned into pKSrbs to form the expression plasmid pKS-Arg. E. coli BL21(DE3) cells were transformed with pKS-Arg by chemical means, and small-scale cultures of these cells were treated with IPTG. Addition of I P T G to the medium specifically induced the expression of a protein with an approximate molecular weight of 31 k D a as judged by S D S - P A G E (Fig. 4). Crude cell lysates from these cultures showed considerable arginase activity which was not present in uninduced cultures. This expression was subsequently scaled up to larger culture volumes in order to purify recombinant protein in large enough quantities to undertake crystallographic studies. The purification protocol is an elegant one which effectively utilises the thermostable character of the protein. The yield is

217

M.C. Bewley et al./FEBS Letters 386 (1996) 215~18 -221

GGGGCCTTAGCTCAGCTGGGA~GNGCCTGCTTTGCACGCAG~GGTCATCGGTTCGATC

-162

-161

CCGATAGGCTCCATC~GAGCAGACTGCTTGAC~CCAAAAGCAGCCTGCTCTTTTTTGT

-102

-i01

TATATCGGTATAAATATTCAAAATTTTGTC~GTTGAGTCATCCCCTTTCTTTCCTCTAC

-41~TAAA~CGT~CAGG~CCGGA~GGGAGACAGCATG~GCC~TTTC~TTA 1 M K P

SI

20 T C G G G G T T C C G A T G G A T T T A ~ G C A G A C A C G C C G C G G C G T T G A T A T G G G G C C ~ G C G C ~ 8 G V M D L QT RG D M G S A M 2 7 80 T G C G T T A T G C A G G C G T C A T C G ~ C G T C T G G ~ C G T C T T C A T T A C G A T A T T G ~ G A T T T G G 2 8 R Y A G V I RL RL Y D I DL

-42 19 7 79

139 47

140 G A G A T A T T C C G A T T G G A A A A G C A G A G C G G T T G C A C G A G C ~ G G A G A T T C A C G G T T G C G C A 4 8 D I GK ER HE G D S R L R N 6 7

199

200 A T T T G A A A G C G G T T G C G G ~ G C G ~ C ~ G A A A C T T G C G G C G G C G G T T G A C C ~ G T C G T T C 68 LK V A E A N E LA A V D Q V V

259 87

260 A G C G G G G G C ~ T T T C C G C T T G T G T T G G G C G G C G A C C A T A G C A T C G C C A T T G G C A C G C T C G 8 8 R G F P L V L G DH I A I G T L A I 0 7

319

320 C C G G G G T G G C G A A A C A T T A T G A G C G G C T T G G A G T G A T C T G G T A T G A C G C G C A T G G C G A C G 1 0 8 G V K H Y RL V I W Y D A H G D V I 2 7

379

380 T C ~ C A C C G C G G A / h ~ C G T C G C C G T C T G G A A A C A T T C A T G G C A T G C C G C T G G C G G C G A G C C 1 2 8 N T A E T S P S G N I H G M P L A A S L I 4 7

439

440 T C G G G T T T G G C C A T C C G G C G C T G A C G C A A A T C G G C G ~ T A C A G C C C C A A A A T C ~ G C C G G 1 4 8 G F G H P A L T Q G G Y S P K I K P E I 6 7

499

500 ~ C A T G T C G T G T T G A T C G G C G T C C G T T C C C T T G A T G ~ G G G G A G ~ G ~ G T T T A T T C G C G 1 6 8 H V V L I G V R S L D E G E K K F I R E 1 8 7

559

560 A A A A A G G ~ T C A A A A T T T A C A C G A T G C A T G A G G T T ~ T C G G C T C G G ~ T G A C ~ G T G A 1 8 8 K G I K I Y T M H E V D R L G M T R V M 2 0

619 7

620 T G G ~ G A A A C G A T C G C C T A T T T A A A A G ~ C G ~ C G G A T G G C G T T C A T T T G T C G C T T G A C T 2 0 8 E E T I A Y L K E R T D G V H L S L D

679 227

680 T G G A T G G C C T T G A C C C ~ G C G A C G C A C C G G G A G T C G G ~ C G C C T G T C A T T G G A G G A T T G A 228 D G L D P S D A P G V G T P V I G G L

739 247

740 C A T A C C G C G A A A G C C A T T T G G C G A T G G A G A T G C T G G C C G A G G C A C A A A T ~ T C A C T T C A G 248 Y R E S H L A M E M L A A Q I I T S

799 267

800 C G G ~ T T T G T C G ~ G T G ~ C C C G A T C T T G G A T G A G C G G ~ C A A A A C A G C A T C A G T G G C T G 2 6 8 E F V E V N IL E R N K T A S V A

859 287

860 T A G C G C T G A T G G G G T C G T T G T T T G G T G A A A A A C T C A T G T ~ T G C A T G T G G G C A A A G A G G G 288 A L M G S L G E L M

919 299

920 T T G G T T G C G G G A T T C A C G ~ T A T G A T ~ T ~ T T T G T T C C C G C T G T T T G T G A A A G A G G G G C

979

980 T G C C A T T T G T T T G G A G T C C C C T C T T T T N C C G T T G T A T G A T A C A A T A A A T A T G T T G C A T G A

1039

1040 A A C T T T T C T T G A T C G A G C G C C G T A A T A T A C A G T A A G C C G T G A G C G N G G T A T T G T A T T T T A

1099

Fig. 3. DNA sequence of the portion of the genomic arginase clone around the coding region. The deduced arginase protein sequence is shown below the DNA sequence. Potential --35 and -10 promoter regions are highlighted in bold type, and the likely transcriptional start site is underlined. The probable Shine-Dalgarno sequence is marked in bold italics. The double-underlined region contains several potential stem-loop structures, one of which may act as a transcriptional termination signal. approximately 3 0 4 0 mg of purified protein per gram wet weight of induced cells. Approximately 90% of protein contaminants are removed during heat treatment. The purified protein ran as a single band on SDS-PAGE and appeared identical to purified native protein. Fig. 4 illustrates the purification protocol. Amino-terminal amino acid sequencing of the purified recombinant protein was performed and yielded the sequence M-K-P-I-S-I-I-G-V-P-M-D-L-G-Q-, showing that the recombinant protein was correctly expressed in E. coli.

The specific activity and Km of the protein are 4.6 kU/mg (at 60°C) and 3.5 mM respectively. Both values are in close agreement with those reported for the native protein [12]. Crystals were observed under two separate conditions and were subsequently found to be distinct crystal forms: type I and type II. Type I crystals were grown in 14% methoxy-PEG 5000, 200 mM MES/KOH, pH 6.5 and were fully grown with-

in 24 h. A typical crystal grew as a long needle (10 m m x 0 . 1 m m x 0.1 mm) and diffracted to 2.2 ,~ resolution. The space group was P21212 with cell dimensions a=83.4 ,~, b=146.6 A,, c=155.1 ,%. Self-rotation functions calculated for two-fold and three-fold axes showed correlation maxima which were consistent with the arginase hexamer having 32 symmetry. Assuming six molecules in the asymmetric unit, the solvent content is ,~ 53% which is in the normal range found in protein crystals [25]. Type II crystals, grown in 28% PEG 6000, 200 mM Bis-Tris propane/KOH, pH 8.5, appeared after 48 h and were fully grown within 1 week. They grew as square prisms (0.4×0.4x0.5 mm) and diffracted to 3.0 A, with c¢=90°, 13=90° , +~=120° and unit cell dimensions of a=81.1 A, c=152.0 A. The point group symmetry is 622 and thus assuming one monomer in the asymmetric unit, the estimated solvent content is 46% [25]. Arginase undergoes a pH-driven cofactor release, and hence the difference in crystal packing at low and high pH as illustrated by type I and II crystals may reflect a conformational change caused by release of manganese ions. Comparison of the deduced amino acid sequence of 'B. caldovelox' arginase with those of other arginases from a diverse range of species (Fig. 1) shows that this is a highly conserved family of homologous proteins. Sequence identity between the 'B. caldovelox' and B. subtilis enzymes is 70% and between 'B. caldovelox' and human arginase is 43%. The latter figure seems remarkable considering the distinct metabolic roles played by arginase in the two species; clearly the mechanism of action is highly conserved. As well as the sequence GGDHS-, found in all species, a conserved triplet -DAH- is present 22 residues downstream. The two histidine residues are potential ligands to the cofactor in the active site of arginase. A number of general rules have been suggested in the literature which attempt to account for the thermostability or

1

2

I

3

4

5

6

7

Dg

O +

_

Fig. 4. SDS-PAGE analysis of 'B. caldovelox' arginase purification and expression. A 15% acrylamide gel running a Laemmli buffer system was stained with Coomassie blue R-250. Lanes: (1) cell lysate; (2) cell-free supernatant; (3) heat treatment supernatant; (4) dialysed (NH4)2SO4 pellet; (5) SDS-7 molecular weight markers (Sigma) containing a mixture of seven proteins of molecular weights 66, 45, 36, 29, 24, 20 and 14 kDa; (6) IPTG-induced BL21(DE3)/ pKS-Arg (whole cell lysate); (7) IPTG-induced BL21(DE3)/pKS-rbs (whole cell lysate).

218 otherwise of proteins. For instance, comparison of thermostable proteins with non-thermostable homologues seems to indicate that increased thermostability correlates with decreased cysteine, asparagine and glutamine residue content [26], or an increased proline residue content [27]. Cysteine, asparagine and glutamine are thought to be susceptible to covalent damage at high temperatures (e.g. deamidation) and thus tend to be replaced in thermostable proteins [26]. A comparison of the amino acid sequence of the arginase from 'B. caldovelox' with that of the arginase from the mesophile B. subtilis shows that there are fewer cysteines (none compared to one) and asparagines (6 compared to 13) in the more thermostable protein. However, the number of prolines remains unchanged at twelve, and the number of glutamines increases from two to five. Clearly, the phenomenon of protein thermostability cannot be generally explained by amino acid sequence data alone. However, the 'B. caldovelox" arginase does contain an increased number of charged amino acid residues compared to the B. subtilis protein, which would appear to give weight to the hypothesis that thermostability is achieved by an increase in the number of salt bridges buried within the protein [28]. That said, it is clear that protein thermostability is not a property which can be convincingly explained by general rules, and specific structural information is required to fully understand and analyse the interactions that are responsible for the increased thermostability of a protein relative to nonthermostable homologues. The previous structural studies on 'B. caldovelox" arginase [13] were frustrated by the low yield of native protein: even large-scale fermentations produced only 0.1 mg of purified protein per litre of culture. The expression and facile purification of recombinant enzyme presented here allow the structural characterisation to be a viable project. The type I crystals are isomorphous to those previously grown from native protein, and the search for heavy atom derivatives is under way. Acknowledgements: We gratefully acknowledge financial support from the Foundation for Research, Science and Technology. E.N.B. also receives support as an International Research Scholar of the Howard Hughes Medical Institute. References

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M.C. Bewley et al./FEBS Letters 386 (1996) 215-218

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