we chose to study purine salvage by Schistosoma ..... fold purification and a 23% recovery. ... the phosphate concentration of the medium to 100 mM (data.
THE JOURNAL DF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry
Vol. 26.5, No. 23, Issue of August 15, pp. 13528-13532,199O
The Hypoxanthine-Guanine
Schistosoma FURTHER
Printed in U.S. A.
and Molecular Biology, Inc.
Phosphoribosyltransferase
of
mansoni
CHARACTERIZATION
AND
GENE
EXPRESSION
IN ESCHERICHIA
COLP
(Received for publication, Ling
Yuan,
Sydney
From the DeDartment California 94i43
P. Craig, of Pharmaceutical
James
H. McKerrowS,
Chemistrv
and
Due to the lack of de nouo purine nucleotide hiosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.
In our continuous effort to establish a model for biochemical approaches to effective chemotherapy of infectious diseases, we chose to study purine salvage by Schistosoma mansoni, a pathogenic trematode parasite in human, because it lacks de nouo synthesis of purine nucleotides (1). The salvage of purine bases depends primarily on the purine phosphoribosyltransferases which catalyze the conversion of hypoxanthine, guanine, and adenine to their respective nucleotides, IMP, GMP, and AMP, in the presence of 5phosphorylribose l-pyrophos* This investigation was supported by National Institutes of Health Grant AI-24011. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. § To whom correspondence and reprint requests should be addressed: Box 0446, Dept. of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143.
and Ching
the *Department
March
19, 1990)
C. Wang5
of Pathology,
University
of California,
San Francisco,
phate (1, 2). An earlier study showed that hypoxanthine and guanine phosphoribosyltransferase activities are associated with a single enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase),l in S. mansoni (3). Due to the lack of interconversions between adenine and guanine nucleotides in the parasite, the schistosomal HGPRTase has been proposed as a potential target for antischistosomal chemotherapy, since inhibition of this enzyme will effectively block the supply of guanine nucleotides for the parasite. To rationally design a specific inhibitor of the schistosomal HGPRTase, extensive kinetic and structural studies of the enzyme will be necessary. Dovey et al. (3) reported that the schistosomal HGPRTase could be purified from crude extracts of adult S. mansoni by either ion-exchange or isoelectrofocusing chromatography. Since sufficient quantities of the enzyme cannot be obtained from adult worms, we sought to express the schistosomal HGPRTase cDNA in bacteria. Recently, Craig et al. (4,5) isolated the cDNA and gene encoding the schistosomal HGPRTase. The cDNA was sequenced and the 231-amino acid sequence thus deduced has only 47.9% identity in a 217-amino acid overlap with human HGPRTase (4). Due to the uncertainties concerning the amino terminus of the parasite enzyme, however, a precise estimation of the molecular weight of this protein has been difficult. Also, it was impossible to make a correct genomic construct for the expression of native S. mansoni HGPRTase in bacteria. In the present paper, we purified a significant amount of S. mansoni HGPRTase. It enabled us to determine the amino terminus of the enzyme protein and to estimate the accurate size of the protein subunit, which is found to be 26 kDa, not 64 kDa as reported previously (3). We then cloned the correct size cDNA into a vector regulated by an Escherichia coli alkaline phosphatase promoter and signal peptide for secreting the expressed product into the periplasmic space. Native S. mansoni HGPRTase was found in the periplasmic space of the transformed E. coli, which signifies, to our knowledge, the first time a eucaryotic HGPRTase gene has been successfully expressed in a bacterial system. EXPERIMENTAL
PROCEDURES
Materials-UltraDure ammonium sulfate, and low molecular weight protein standards Bio-Rad. [S-%]Hypoxanthine (57.0 mCi/mM) (55.0 mCi/mM) were from ICN Radiochemicals. ’ The
abbreviations
are: HGPRTase, hypoxanthine-guanine HPLC, high-_ pressure liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacryiamide gel electrophoresis; HPRTase, hypoxanthine phosphoribosyltransferase; PRPP, 5-phosphorylribose l-pyrophosphate; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid.
.nhosnhoribosvltransferase: -
13528
used
SDS-PAGE reagents, were purchased-from and [S-%]guanine Polyethyleneimine-
Hyporanthine-Guanine
Phosphoribosyltransferase
cellulose-coated plastic sheets were from Brinkmann Instruments, Inc. Reagents for bacterial culture media, including vitamin-free casamino acids, were from Difco. Restriction endonucleases were from Bethesda Research Laboratories. Immobilon was from Millipore. All other reagents were purchased from Sigma in the highest purity available. E. coli strain GPlZOD (rlpro-gpt-lac, thi, pur E) was a generous gift from Dr. Joseph Gots of the University of Pennsylvania. Schistosomal adult worm protein extracts (SWAP (8), 86 ml, -7.3 mg/ml) were kindly provided by Sara Heiny and Dr. Alan Sher of the Laboratory of Parasitic Diseases, National Institutes of Health. To prevent enzymatic degradation, proteinase inhibitors (N-ethylmaleimide, iodoacetic acid, and trans-epoxysuccinyl-L-leucylamido-(4guanidino)butane were added to the extracts to a final concentration of 1 mM. PRPP (1 mM) was also added to stabilize the phosphoribosyltransferases (3). Enzyme Assays-HPRTase and guanine phosphoribosyltransferase activities were measured using the conditions described by Dovey et al. (3) with the following modifications. Reactions were terminated by adding an equal volume of 1 mM base and nucleotide monophosphate. Twenty ~1 of the terminated reaction mixture was chromatographed on a polyethyleneimine-cellulose-coated plastic sheet using 5 mM ammonium acetate, pH 4.5, as mobile phase. The substrates and products were located under UV light, cut out, and the radioactivity was assayed using a Beckman LS 3801 scintillation counter. Protein Analysis-Protein concentrations were determined by the Bio-Rad Bradford protein assay using IgG as standard. To determine the amount of protein on a Coomassie Blue-stained SDS-PAGE, the gel was scanned using an LKB 2202 laser densitometer. SDS-PAGE (15%) was performed as described by Laemmli (10). For electrotransfer of protein bands from the gel to Immobilon, the gel was pre-run using Laemmli running buffer containing 50 pM reduced glutathione. The sample was run in the presence of 100 pM sodium thioglycolate. After electrophoresis, the protein on the gel was electroblotted onto an Immobilon membrane using 10 mM CAPS, pH 11.0, 10% methanol as buffer. The membrane was stained with Coomassie Blue, and the protein band was cut out for amino-terminal sequencing. Amino acid sequence determination was performed by the Biomolecular Resource Center at UCSF. Samples containing -120-170 pmol of purified protein were subjected to Edman degradation using an Applied Biosystems 470A gas-phase sequencer. The PTH-derivatives were identified and quantitated by reverse-phase HPLC using an on-line Applied Biosystems 120A PTH analyzer. Construction of the Expression Vector-An 812-base pair BamHISspI fragment of the Smc-2 plasmid (4), containing the entire coding sequence for the schistosomal HGPRTase, was ligated to a 371-base pair EcoRI-BamHI fragment of the pTRAP plasmid (11) containing the alkaline phosphatase promoter and signal peptide of E. coli. The spliced gene was then inserted between the EcoRI site and a bluntended XbaI site in the polylinker of a Bluescript KS+ plasmid from Stratagene. This construct, referred to as “pBSprts,” permits induction of expression of the schistosomal HGPRTase by starving the transformed E. coli for phosphate in the culture medium. The signal peptide is expected to force secretion of the newly translated enzyme through the plasma membrane into the periplasmic space of the bacteria. Expression of the HGPRTase Gene in E. coli-The pBSprts-transformed GPlZOD cells (500 ml) were grown to lag phase at 37 “C in MOPS medium (12) plus 0.2% glucose, 1.5 pM thiamine, 20 pg/ml adenine, 0.2% “vitamin free” casamino acids, 0.1 mM phosphate, and
TABLE
of S. mansoni
13529
75 pg/ml ampicillin. The cells were pelleted and resuspended in 5 ml of TMK buffer (100 mM Tris-HCl, pH 7.0, 100 mM MgC12, 500 mM KCl). One-tenth volume of chloroform was added, and the tube was vortexed for 10 s and kept on ice for 10 min with occasional agitation. Supernatant from the periplasmic lysate was collected after centrifugation at 10,000 X g for 10 min leaving the protoplasts in the pellet fraction. As a control, 500 ml of Bluescript+-transformed GPl2OD (BS’) was grown and prepared under the same conditions. For determination of the distribution of the recombinant HGPRTase, equal numbers of cells were either treated with chloroform as described above to isolate periplasmic proteins, or lysed completely by sonication followed by assay of both periplasmic and cytosolic lysates for HGPRTase activity. For purification of the recombinant enzyme, the supernatant from the periplasmic preparation was heated to 80 “C for 2 min, chilled on ice, and centrifuged at 14,000 X g for 5 min. The supernatant was concentrated by Centricon (10,000 molecular weight cut-off, from Amicon) to approximately 0.5 ml, and diluted 10 times with MonoQ buffer A (50 mM Tris-HCl, pH 7.5,6 mM MgClp, 1 mMadithiothreitol, 10% glycerol). The sample was loaded onto the Mono& column and eluted with a linear gradient of KC1 (O-500 mM in 40 min) at a flow rate of 1 ml/min. One ~1 of 1 M PRPP was added to each l-ml fraction to stabilize the enzyme. Each fraction was assayed for phosphoribosyltransferase activities. Enzyme Kinetics-The kinetic experiments were performed using a Beckman DU-7 spectrophotometer equipped with a kinetics accessory according to the method of Hill (14). The synthesis of IMP and GMP from PRPP and the corresponding purine base were followed spectrophotometrically at 245 and 257.5 nm, respectively. All measurements were carried out in 300 mM Tris-HCl, pH 7.8,18 mM MgCl,, 1 mM PRPP at 37 “C. The extinction coefficients for the formation of IMP and GMP under these conditions were found to be 2930 f 50 and 5150 + 60 M-’ cm-‘, respectively. Ten substrate concentrations ranging from 0.5 to 10 pM were used. Double-reciprocal plots were used to analyze the data. RESULTS
HGPRTase-The chromatthe same as described previously (3). Our procedure involved: 1) SWAP used as the starting material; 2) a 75% ammonium sulfate precipitation step added before the heat treatment; 3) an additional MonoQ chromatography inserted following the heating step (see Table I). Further fractionation with a MonoP column resulted in a homogeneous schistosomal HGPRTase preparation, as revealed by SDS-PAGE (Fig. 1). The enzyme has a subunit molecular weight of 26,000 in SDS-PAGE, which is in agreement with the earlier predictions based on sequencing of both cDNA and genomic DNA (4, 5), but disagrees with the value of 64,000 estimated in the earlier study (see “Discussion”). The new procedure used to purify HGPRTase from S. mansoni, summarized in Table I, resulted in more than 12,700fold purification and a 23% recovery. Approximately 13 rg of the enzyme was obtained from 700 mg of protein extract. The specific activity for hypoxanthine was found to be 5,400 nmol/ Purification of the Schistosomal ographic conditions were essentially
I
Purification steps
SWAP (NH.&SO~ Heat Mono& MonoP
VOlUme
[Protein]”
of the schistosomal HGPRTase Total activity Specific activity
Total protein
HPRT
ml
&ml
86
8,200 34,202
705,200 684,044
4,104
41,043
20 10 8 2
HPRT
nmol/min
clt?
12 6.5
GPRT
97 13
292.4 291.0 232.8 133.9 69.8
’ Protein concentrations were measured fractions in which the protein concentration stained bands on SDS-PAGE with standard
nmol/minf 394.7 407.4 318.9 190.1 99.1
GPRT
GPRT
HPRT
-fold
mg
0.42 0.43 5.68 1.380 5;366
Yield
HPRT
0.56 0.60 7.78 1.960 7;623
1 1 13.5 3.286 12;776
by Bio-Rad protein assay using IgG as standard was determined by comparison of the densities protein using a Beckman laser densitometer.
GPRT
% 1 1 13.8 3.500 13;612
except of the
100.0 99.5
100.0 100.0
79.6 45.8 23.9
80.8 48.2 25.1
for MonoP Coomassie-
Hypoxanthine-Guanine
13530 A
B
C
Phosphoribosyltransferase
M, x10-3 -93 -66
-45
-31 aa -22
-14
FIG. 1. SDS-PAGE
of the purified HGPRTase of S. mansoni the recombinant HGPRTase isolated from periplasmic space (B); and the recombinant HGPRTase isolated from cytoplasm (C). The gel was stained with Coomassie Blue. Molecular
(A);
weight standards are indicated. min/mg
which
is roughly
one-third
of that
of the purified
human enzyme (15). Approximately 4.5 pg of the protein was run on a SDSPAGE and then blotted onto an Immobilon membrane. The n/i, 26,000 band was cut out and directly sequenced. A fivecycle analysis revealed a sequence of Met-Ser-Ser-Asn-Met. This sequence, shown in Table II, is identical to one of the four potential starting sequences that were identified by sequencing genomic DNA. The molecular weight of the protein translated from the cDNA with the correct initiation codon adds up to 26,062.24, thus further confirming that the cDNA and gene previously isolated and sequenced code for the HGPRTase of S. mansoni. Using Kozak’s rules (24), this amino terminus had been postulated by us in our previous paper (4) as the most likely amino terminus of the HGPRTase of S. mansoni. Expression of the Schistosomal HGPRTase in E. coli-Fig. 2 is a map of the pBSprts vector for the expression of the schistosomal HGPRTase in E. coli. Expression of this vector in E. coli is controlled by the alkaline phosphatase (Pho A) promoter which is de-repressed in the low phosphate culture medium (16). For this study, we have taken advantage of the E. coli mutant (GP120D), which has a deletion of the xanthine-guanine phosphoriboxyltransferase gene and has a relatively low level of guanine phosphoribosyltransferase activity in the bact.erial HPRTase (-10% of the G salvage activity of the xanthine-guanine phosphoriboxyltransferase).’ The bacteria thus provide a relatively low background in guanine salvage activity. As shown in Fig. 3, when the pBSprtstransformed cells are grown in low phosphate MOPS medium, there is a dramatic increase of guanine and hypoxanthine salvage activities in the periplasmic extract. This expression of the HGPRTase activity can be suppressed by increasing the phosphate concentration of the medium to 100 mM (data not shown). Also the whole cell lysates were shown to have approximately 6 times higher HGPRTase activity than do periplasmic extracts (see below). Isolation of the Recombinant HGPRTase-Chloroform shock provides a simple and rapid method to release periplasmic proteins from bacteria (17). We have successfully applied a slightly modified protocol to extract proteins from the periplasm of the GP120D cells, and have observed better recovery of enzymatic activity compared with other methods, ‘J. Gots, personal communication.
of 5’. mansoni
such as osmotic shock or lysozyme treatment (data not shown). Since the periplasmic lysate contains both recombinant HGPRTase and bacterial HPRTase activities, the sample was subjected to ion exchange HPLC (MonoQ). Fig. 4 shows that the recombinant HGPRTase (fractions 9 and 10, Fig. 4B) was well separated from the bacterial HPRTase (fractions 17 and 18, Fig. 4, A and B). Also note that only the recombinant enzyme possesses comparable levels of hypoxanthine and guanine salvage activities. When fractions 9 and 10 were combined, concentrated, and run on a SDS-PAGE, a major protein band of M, 26,000 was revealed (Fig. 1). The 26,000dalton band (-120 pmol) was cut out from Immobilon after electroblotting and sequenced as described above. The first cycle revealed a Met with high background. The second to fourth cycles gave a clear sequence of Ser-Ser-Asn. No amino acid could be positively identified at the fifth cycle, but the sixth cycle revealed an Ile (Table II). This sequence is identical to the amino-terminal sequence of the authentic schistosomal HGPRTase with methionine missing from the fifth cycle. The loss of methionine at the fifth cycle could be due to the oxidation of methionine (13). Whole cell lysates possess a significantly higher level of the active recombinant enzyme. Therefore, we also purified the enzyme from the cytosol of the bacteria. When the isolated cytosolic enzyme was examined by SDS-PAGE, an unexpected M, 28,000 band was revealed (Fig. 1). Direct aminoterminal sequencing of this protein showed that it is indeed the recombinant enzyme but that part of the Pho A signal peptide, FTPVTKARIRD, is still attached to the amino terminus (Table II). Table III summarizes the isolation scheme for the recombinant enzyme. Because the total HPRTase activity in the crude periplasmic lysate represents both schistosomal and bacterial enzymes, the calculated yield for the recombinant enzyme was relatively low (11.5%). The recombinant enzyme has a specific activity of 5900 nmol/min/mg for hypoxanthine, which is in good agreement with the native enzyme, indicating that the recombinant enzyme is probably correctly folded after being synthesized and secreted by the bacterial host. Kinetic Properties of the Nutiue and Recombinant HGPRTase-Kinetic studies of the enzyme-catalyzed reaction were carried out in 300 mM Tris-HCl, pH 7.8, 18 mM MgClz at 37 “C in the presence of 1 mM PRPP. According to the study by Salerno et al. (18), magnesium ion plays a critical role for the catalysis of phosphoribosyltransferases. The concentration of free Mg2+ in the presence of PRPP was calculated without taking into account the formation of MpPRPP since the concentration of PRPP was kept at about 20 times lower than that for magnesium in all assays. The K,,, and k,,, values for both native and recombinant enzymes were calculated by least squares analysis of these data and are given in Table IV. No discernible difference was found between the two enzyme samples. DISCUSSION
A four-step procedure has been developed which results in the purification of the schistosomal HGPRTase to apparent homogeneity starting with a crude extract of adult worms (SWAP). The ammonium sulfate fractionation, the heat treatment, and the two chromatographic procedures were very effective in increasing the specific activity of the enzyme (Table I). However, we have found that the HGPRTase could not be purified to homogeneity from 85 “C heated adult worm protein extracts by one-step chromatography (MonoP or MonoQ) as described before (3). As reported previously (3),
Hypoxanthine-Guanine
Phosphoribosyltransferase
of S. mansoni
TABLE II Amino-terminal analysis of the native and recombinant HGPRTase The possible amino terminus is listed according to the open reading frame in the genomic sequence Craig et al. (4, 5). Italic is used for residue that lacks of absolute positive identification; X, unidentified -56 -M-L-T-S-L
Possible native enzyme sequence Schistosome adult worm HGPRTase Periplasmic recombinant HGPRTase Cytosolic recombinant HGPRTase
-18
. . . -M-N-S-S-V
reported residue.
1 5 . . . M-S-S-N-M-I-K-A-D ii-S-S-N-ii
by
..
. . .
M-S-S-N-X-I ...
F-T-P-V-T-K-A-R-I-R-D-M-S
4.1O/OKb I
13531
...
50 -g P
40
'5 2g
30
p
20
8
2 g
10
0 0
20
10
Fraction
..a ACAWA Gee CGGATCCGCGACAT0 TCT AGTA&c AT0 A.. TKARIRDMSSNM
The regulatory sequences and phosphatase (Pho A) were fused The amino acid and nucleotide the two proteins are shown in
pBSprts
FIG. 3. Hypoxanthine (stippled) and guanine activities in the periplasmic lysates. Fifty-ml script+-transformed (BS+) and pBSprts-transformed cells were prepared as describedunder “Experimental
(solid) salvage cultures of BlueE. coli GPlPOD Procedures.”
we also observed a major band of M, 64,000 in SDS-PAGE after one-step MonoP chromatography. However, this protein band could be totally separated from the HGPRTase activity by further fractionation using MonoQ. Evidence presented here indicates that the schistosomal HGPRTase has a subunit molecular weight of 26,000 with a specific activity of 5,400 nmol/min/mg for hypoxanthine (Fig. 1 and Table I). Thus, the subunit M, of 64,000 for the schistosomal HGPRTase reported previously must be due to a contaminant protein. Furthermore, the difference in specific enzymatic activity may be partly due to incorrect estimation of the amount of the enzyme protein in the previous study, in which protein concentration was determined by scanning the M, 64,000 protein band in a silver stained SDS-PAGE gel. Since substrate gel
5 .-
60
$ a E e 2
40
30
40
Number
z
250 y
20 0
0 0
10
20
30
110
Fraction Number FIG. 4. MonoQ HPLC of the periplasmic preparations. A, control (BS); B, pBSprts-transformed GPlZOD. Hypoxanthine (l3) and guanine (0) salvage activities were assayed as described under “Experimental Procedures.” The broken lines (- - - -) indicate the linear gradient of KC1 (from 0 to 500 mM).
electrophoresis was used to demonstrate that the schistosomal HGPRTase has a native molecular weight of 105,000 (3), we now suggest that the native enzyme is probably a tetramer of the M, 26,000 subunit as are the enzymes of mammals (19). According to earlier studies (4, 5), the complete gene sequence of the schistosomal HGPRTase revealed four possible translation initiation positions (Table II). The best way to determine, with certainty, the amino terminus for the schistosomal HGPRTase was to sequence the purified enzyme. Here we report the sequence for the amino terminus of the enzyme from adult worms. The sequence, Met-Ser-Ser-AsnMet, indicates that the methionine formerly predicted to initiate translation is indeed the amino acid on the amino terminus of the native enzyme. This result also confirms that the M, 26,000 protein is the schistosomal HGPRTase since the sequence was obtained from the M, 26,000 band which was electroblotted to an Immobilon membrane. As shown in Table I, HGPRTase represents less than 0.008% of total soluble proteins of the adult worm. Considering the difficulties of obtaining large quantities of adult worms, expression of the schistosomal cDNA for HGPRTase in bacteria provides a suitable alternative for generating the enzyme en masse. Several early attempts at over-expression of the schistosomal HGPRTase within the bacterial cytoplasm were unsuccessfu1.3 To overcome this problem, we 3 L. Yuan, lished data.
S. P. Craig,
J. H. McKerrow,
and C. C. Wang,
unpub-
13532
Hypoxanthine-Guanine
Phosphoribosyltransferase TABLE
Isolation steps
Periplasmic Heat MonoQ
Volume
lysate
ml
Pdml
I#
5 5 2
321 101 5.3
1,605 505 10.6
TABLE
Kinetic Assays assuming
Total protein
[Protein]
of the natiue HGPRTo..se recombinant HGPRTase were performed in duplicate. Values an M, of 26,000 for the enzyme.
Native Recombinant
NM 3.7 4.2
kc., s-1 2.4 2.6
HPRT 540.3 548.8 62.1
for k,., were
LlKm S-’ mM-’ 649 619
calculated
Gamine Km pM 2.1 2.7
kc., s-1 4.3 5.0
LlKm se’ mM-’ 2048 1852
modified an expression vector previously designed to express and secrete mammalian trypsin into the periplasmic space of E. coli (11). Also, the construct reported here uses the Bluescript+ plasmid which permits the rapid preparation of large amounts of either double- or single-stranded DNA for sequencing and/or site-directed mutagenesis. When introduced into a bacterial strain deficient in xanthine-guanine phosphoriboxyltransferase, this expression system provides a simple and rapid way to obtain active schistosomal HGPRTase from bacteria. The bacterial Pho A promoter and signal peptide has been used to secrete a variety of enzymes into the periplasmic space of E. coli (21,22,11). After being secreted into the periplasmic space, the signal peptide is expected to be cleaved at an arginine residue (see Fig. 2) (11). If the Pho A signal peptide cleavage occurs at this arginine or at a second arginine residue two positions downstream in the Smc-2 insert, there should be one to four additional amino acids remaining on the amino terminus of the recombinant HGPRTase (i.e. Asp-, ArgAsp-, Ile-Arg-Asp-, or Arg-Ile-Arg-Asp-). Direct sequencing of the amino terminus of the recombinant enzyme revealed an amino acid sequence identical to that for the native enzyme, a somewhat unexpected result. This result could be explained if the signal peptide was cleaved at the last Asp residue by a secondary cleavage activity of the bacterial signal peptidase (ll), thereby leaving a correct amino terminus for the recombinant enzyme. Variability in Pho A signal peptide cleavage has been reported previously (20, 22). Approximately 80% of the recombinant enzyme activity remains in the cytoplasm. However, this recombinant enzyme has part of the Pho A signal peptide attached to the amino terminus. Studies on signal peptide-directed protein secretion indicate that a basic amino acid such as arginine near the mature amino terminus of a secreted protein can block secretion (23). Thus, the arginine residue in the junction peptide between the Pho A signal peptide and the start methionine for the HGPRTase may significantly reduce the efficiency of secretion. Therefore, we have recently modified the expression vector by removal of the junction peptide. This modified vector has been induced in E. coli and has been shown to have an approximate 6-fold increase in efficiency of secretion. This
HGPRTase Specific
GPRT
nmol/min
and the secreted
Hypoxanthine Km
activity
IV
constants
III
of the recombinant Total
of S. mansoni
260.3 273.2 80.7
activity
HPRT
GPRT nmol/min/mg 337.6 162.7 1.086.7 541.0 5;875.2 8,070.O
HPRT
Yield
GPRT HPRT -fold
1 3.2 17.4
GPRT %
1 3.3 49.6
100 100 11.4
100 100 31.0
modified expression system will be reported in a future communication. Nevertheless, our present construct does secrete recombinant enzyme (in excess of 0.5 mg/liter bacteria) with the correct amino-terminal sequence and essentially identical kinetic properties to those of the native HGPRTase of S. mansoni. Therefore the secreted recombinant schistosomal HGPRTase should be useful for further investigations, including more extensive enzymological studies, x-ray crystallographic analysis, and antibody production. Acknowledgments-We would like to thank Dr. Douglas Kuntz for his helpful suggestions on isolation of the periplasmic proteins, Dr. Charles Craik and John Vasquez for their advice and the generous gift of the pTRAP plasmid. We also thank Sara Heiny and Dr. Alan Sher for kindly providing the protein extracts from adult S. mansoni, and Dr. Joseph Gots for his generous gift of E. coli GP12OD. REFERENCES 1. Senft, A. W., Miech, R. P., Brown, P. R., and Senft, D. G. (1972) Znt. J. Parasitol. 2, 249-260 2. Dovey, H. F., McKerrow, J. H., and Wang, C. C. (1984) Mol. Biochem. Parasitol. 11, 157-167 3. Dovey, H. F., McKerrow, J. H., Aldritt, S. M., and Wang, C. C. (1986) J. Biol. Chem. 261,944-948 4. Craig, S. P., McKerrow, J. H., Newport, G. R., and Wang, C. C. (1988) Nucleic Acids Res. 16, 7087-7101 5. Craig, S. P., Muralidhar, M. G., McKerrow, J. H., and Wang, C. C. (1989) Nucleic Acids Res. 17, 1635-1647 6. Konecki, D. S., Brennand, J. C., Caskey, C. T., and Chinault, A. C. (1982) Nucleic Acids Res. 10, 6763-6775 7. King, A., and Melton, D. W. (1987) Nucleic Acids Res. 15,1046910481 8. Sher, A. Pearce,S., Heiny, S., and James,S. (1986) J. Zmmunol.
136.3878-3884 9. Schmidt, R., Foret, M., and Reichert, V. (1986) Clin. Chem. 227, 67-69 10. Laemmli, U. K. (1970) Nature 227,680-685 11. Graf, L., Craik, C. S., Patthy, A., Roczniak, S., Fletterick, R. J., and Rutter, W. J. (1987) Biochemistry 26,2616-2623 12. Neidardt, F. C., Bloch, P. L., and Smith, D. F. (1974) J. Bacterial.
119,736-747 13. Brot, N., and Weissbach, H. (1983) Arch. Biochem. Biophys. 223, 271-281 14. Hill, D. L. (1970) &o&em. Pharmacol. 19.545-557 15. Olsen, A. S., and Milman, G. (1977) Biochemistry 16,2501-2505 16. Torriani, A. (1960) Biochim. Biophys. Actu 38,460-469 17. Ames, G. F., Prody, C., and Kustu, S. (1984) J. Bacterial. 160, 1181-1183 18. Salerno, C., and Giacomello, A. (1981) J. Biol. Chem. 256, 36713673 19. Johnson, G. G., Eisenberg, L. R., and Migeon, B. R. (1979) Science 203,174-176 20. Vasquez, J. R., Evnin, L. B., Higaki, H. N., and Craik, C. S. (1989) J. Cell Biochem. 39, 265-267 21. Oka, T., Sakamoto, S., Miyoshi, K., Fuwa, T., Yoda, K., Yamasaki. M.. Tamura. G.. and Mivake. T. (1985) Proc. Natl. Acad. Sci. ‘U. s. A. 82,7212-7216 22. Shortle. D. (1986) J. Cell Biochem. 30.281-289 23. Summers, R. G., Harris, C. R., and Knowles, J. R. (1989) J. Biol. Chem. 264,20082-20088 24. Kozak, M. (1986) Cell 47,481-483
