functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the...

Molecular and Biochemical Parasitology, 45 ( 1991 ) 317-330 317

Elsevier

MOLBIO 01496

Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodiumfalciparum

in Escherichia coli

Stephen J. Hall, Paul F.G. Sims and John E. Hyde Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology,

Manchester, U.K.

(Received 4 September 1990; accepted 31 October 1990)

We have developed a recombinant system that directs the functional expression from Escherichia coli of both dihydrofolate reductase-thymidylate synthetase (DHFR-TS) and the isolated DFIFR domain from Plasmodiumfalciparum. Both products are inhibitory to a number of E. coli cell lines to the extent that cell growth ceases immediately upon induction. This dramatic inhibition is not seen in strain AB 1899, in which amounts of plasmodial protein of up to 100 times the basal E. coli TS level can be accumulated. However, as well as the full-length DHFR-TS molecule, smaller proteins carrying an intact TS substrate-binding site are produced. These represent ca. 60-75% of the total plasmodial protein expressed and are observed in every E. coli strain examined. We show that they are not derived by degradation of the parent DHFR-TS molecule, but can be correlated with the sizes of proteins expected to be produced if erroneous initiation of translation were occurring at 3 internal methionine residues.

Key words: Plasmodium falciparum; Dihydrofolate reductase-thymidylate synthetase (DHFR-TS); Heterologous expression; Escherichia coli; Aberrant translation initiation

Introduction

The dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.3) activity of the most pathogenic human malaria parasite, Plasmodium falciparum, is the target for the major antimalarial drugs pyrimethamine and cycloguanil (the toxic metabolic product of progu- anil). These substrate analogues bind to the parasite

Correspondence address: John E. Hyde, Department of Bio- chemistry and Applied Molecular Biology, UMIST, P.O. Box 88, Manchester, M60 1QD, U.K.

Abbreviations: BSA, bovine serum albumin; CH2FAH4, me- thylene tetrahydrofolate; DHFR, dihydrofolate reductase; EDTA, ethylenediaminetetraacetic acid, disodium salt; FdUMP, 5-fluorodeoxyuridine-5'-monophosphate; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IPTG, iso- propyl-13-D-thiogalactopyranoside; PCR, polymerase chain reaction; SDS, sodium dodecyl sulphate; TES, N-tris[hydroxy- methyl]methyl-2-aminoethanesulphonic acid; TMP, trimethoprim; TS, thymidylate synthetase.

enzyme several hundred times more tightly than to the mammalian homologue [1], blocking DNA synthesis and the transfer of 1-carbon units in other metabolic pathways. Resistance to these antifolate drugs is now widespread [2,3], severely compro- mising their use. A genetic basis for parasite resistance was recently elucidated [4-8] when it was shown that the degree of resistance to pyrimethamine correlated with a small number of amino acid changes in the DHFR sequence relative to that found in sensitive strains (reviewed in refs. 9 and 10). This work has recently been extended to cycloguanil resistance, where a slightly different subset of amino acid changes are involved [ 11,12].

The DHFR activity in P. falciparum is unusual in that, as in a number of other parasitic protozoa [13-15], it is found on a bifunctional protein in as- sociation with the thymidylate synthetase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyl- transferase, EC 2.1.1.45) activity that catalyses the preceding step in the folate metabolic pathway. The

0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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two activities are encoded by a single intronless gene [16]. It has been suggested that this organis- ation provides the opportunity for efficient chan- nelling of substrates between the two enzyme domains, as well as ensuring coordinate regulation of these sequential activities [15,17]. Analysis of the predicted protein sequence linking the DHFR and TS domains indicates that it is unstructured, and is poorly conserved both in length and sequence among P.falciparum [ 16], P. chabaudi [ 18], Leish- mania major [19,20] and Crithidia fasciculata [21].

Three-dimensional characterisation of the DHFR-TS molecule is mandatory for a complete understanding of the mechanisms of resistance to pyrimethamine and cycloguanil, and for the evolu- tion of any new antifolate inhibitors. Anti-TS com- pounds are also strong candidates for rational drug design [22]. For this reason, our experiments are directed towards the large-scale production of the bifunctional enzyme which will be necessary for proper biophysical analysis. In addition, the poorly conserved nature of the linker region of the bifunctional protein suggested to us that the DHFR and TS domains might be independently active. To test this possibility, we have used PCR amplification of specific segments of the DHFR-TS gene and cloned them into an Escherichia coli expression system. We demonstrate that a DHFR-encoding fragment of the bifunctional P.falciparum gene can function in E. coli, to the extent that the bacteria can be res- cued from the inhibitory effects of the antibacterial antifolate drug trimethoprim (TMP). Furthermore, we show that the complete DHFR-TS protein can be synthesised in E. coli with both functional DHFR and TS moieties.

Materials and Methods

Polymerase chain reaction amplification of P. falciparum genomic DNA. Pyrimethamine-resistant K1 (Thailand) parasites were maintained in culture [23] and DNA extracted [6] as previously described. The following PCR primers were designed from the complete K 1 DHFR-TS sequence [6]:

5' end of the DHFR coding sequence: 5 '-CTCCTTTTTACCATGGAACA AGTCTGC- GACG-3' (31 -mer)

(Residues underlined have been altered from the plasmodial sequence to introduce an NcoI site which incorporates the ATG initiation codon.)

3' end of the DHFR coding sequence: 5'-GTTCATITAACATIWAATT A T T C G T T T - TC-3' (29-mer) (The residue underlined has been altered to introduce an in-frame termination codon just beyond the border between the DHFR domain and the junction domain.)

3' end of the TS coding sequence: 5'-AATTTCAAGCTTAAGC A G C C A T A T C C - ATTG-3' (30-mer) (Residues underlined have been altered to incor- porate a HindIII site just downstream of the natural termination codon.)

The PCR [24] was set up in 100-~tl reactions containing 0.5 ~tg of genomic DNA, 200 ng of each primer, 200 ~tM of each dNTP, 50 mM Tris-HC1 (pH 9.0), 15 mM (NH4)2SO4, 7 mM MgC12, 50 mM KC1, 170 pg ml -~ nuclease-free BSA and 2 U Taq polymerase. The polymerase and 5× reaction buffer (containing everything except target DNA, primers, dNTPs and enzyme) were supplied by An- glian Biotech, Colchester, U.K. The reaction was made up in 0.5-ml microfuge tubes and covered with 50 ~tl of sterile liquid paraffin oil. In a Techne PHC-2 automatic temperature cycler, the reaction was heated at 95°C for 5 min to separate the DNA strands and then cooled to 45°C (2 min) to allow primer annealing. The reaction was then heated to 72°C (5 min) to allow extension of the primers and complete the first cycle. Subsequent denaturing cycles were at 90°C (1 min), for a total of 20-30 cycles.

DNA sequencing. As PCR products were being cloned into an expression vector, it was necessary to check for any sequence alterations due to misin- corporation by the Taq polymerase [25]. Sequen- cing was by the chain-termination method [26] using both universal and internal oligonucleotide primers. Cloned products of the DHFR domain corresponded exactly to the original K1 genomic sequence [6] except where changes had been delib- erately introduced via the PCR primers. In PCR-

3 1 9

derived clones of the entire DHFR-TS gene, a single nucleotide change was found upstream of a un- ique SpeI site close to the 5' end of the gene. As this would have altered the amino acid sequence of the product, the fragment from the 5' end of the gene to this SpeI site was replaced with the equivalent fragment from the DHFR construct, thus restoring the proper sequence (see Fig. 1 ).

Preparation of bacterial extracts for thymidylate synthetase assay. A 10-ml overnight culture of E. coli in L-broth containing the appropriate antibi- otics for selection was used to inoculate 200 ml of prewarmed medium. The cells were grown at 37°C to an A~x~ of ca. 0.5 and then the culture divided, one half being induced with 1 mM IPTG. After incubation for a further period, the cells were spun down and the pellet resuspended in 100 lal of 50 mM TES (pH 7.0), 1 mM EDTA, 75 mM 2- mercaptoethanol, together with the following protease inhibitors: 10 mM benzamidine, 20 ~g ml -~

• - I • - f .

leupeptm, 20 B~ ml pepstatm, 50 lag ml apron~ nin, 50 lag ml- trypsin inhibitor and 25 lag ml- phenylmethylsulphonyl fluoride [17,27] (all inhibitors from Sigma, Poole, U.K.). The cells were then flash frozen in liquid N2 and stored at -80°C if desired. Cell extracts were prepared by sonication on ice with 5 pulses of 5-s duration followed by 10- s intervals for cooling, using an MSE 150 W disin- tegrator. The cell debris was pelleted and the extract removed to a clean tube on ice. At this stage the protein concentration of the extracts was determined [28] by comparison with BSA standards. This allowed a standard amount of protein from each extract to be added to the TS assay or run on polyacrylamide ~els (see below), as well as nor- malisation of [6=H]FdUMP binding.

Thymidylate synthetase assay. TS substrate- binding activity was quantitated as a covalent complex of the substrate analogue [6-3H)FdUMP with the cofactor CH2FAH4 and the TS protein [29]. Assay mixtures of 0.4 ml contained 50 mM TES, pH 7.0, 25 mM MgC12, 1 mM EDTA, 75 mM 2- mercaptoethanol, 6.5 mM HCHO, 0.17 mM CH2FAH4 (Sigma, Poole, U.K.), 95 nM [6- 3H]FdUMP (24 Ci mmol -~, Moravek Biochemi- cals, Brea, CA) and 100 lai of bacterial cell extract. Negative controls included reaction mixtures that

were pretreated with unlabelled FdUMP (1 mM) for 10 min prior to addition of [6-3H]FdUMP or reaction mixtures that were made up without cell extract. The reactions were incubated at room temperature for 1 h. A 50-lal aliquot was removed for polyacrylamide gel analysis and 2 lal for the deter- mination of the total [6--H]FdUMP in a reaction. Samples of 100 lal from the remainder of each reaction were slowly filtered (0.5 ml min -~) through nitrocellulose membrane filters (24-mm Millipore HA 0.45 lain) previously soaked in 25 mM potas- sium phosphate buffer, pH 7.4. The filters were washed at the same filtration rate with 5 ml of this buffer. Filters were air dried and counted in 4 ml of liquid scintillant (Opti Phase Hi-safe 3, LKB).

Sodium dodecyl sulphate-polyacrylamide gel analysis of dihydrofolate reductase-thymidylate synthetase. Whole-cell samples were boiled for 10 rain in 10 mM Tris, pH 8.0, 1 mM EDTA, 1% SDS, 5% 2-mercaptoethanol, run into a 4% stack- . - F • - I mg gel at 9 V cm , then run for 90 mln, 20 V cm , on a 10% SDS-PAGE gel, and stained with Coomassie Blue to visualise total protein. To monitor radioactive labelling of the TS-active site, [6-3H]FdUMP-treated cell extracts (50 lal) prepared as described above were processed in the same way. After staining, the gel was rinsed in dis- tilled water for 30 min followed by 30 min in 1 M salicylic acid, 1 M NaOH. The gel was then dried down and autoradiographed at -80°C with intensi- fying screens for 1-2 weeks.

Results

Polymerase chain reaction amplification of the dihydrofolate reductase and dihydrofolate reductase-thymidylate synthetase regions and production of expression constructs. The con- siderable technical limitations of in vitro culture of P. falciparum and of enzyme purification there- from preclude the isolation of quantities of DHFR- TS from native material sufficient for biophysical analyses such as X-ray diffraction and nuclear magnetic resonance. We therefore aimed to obtain in the first instance molecules identical as far as possible to their in vivo equivalents, expressed as unfused proteins from a recombinant system. We chose to insert amplified gene fragments into the

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A

PCR PRODUCT8

D H F R ~ DHFR-T8

N N S N N S H _I_ I

eubolone Into Sine eubol • Into Sms cut pBIueeorlpt out pUBS

eequenoe N/H partial digest )

|

N/H partial digest eubclone Into pKK283.2

8ubclone Into pKK233.2 |

N 8 H C N 8 H

B N N S H N N S

e®quenoe remove 8-H t

fragment Isolate 8-H " ~ J fragment

Ilgste

D N N S H

H 1

Fig. 1. Strategy for construction of clones expressing the dihydrofolate reductase domain and full-length dihydrofolate reductase-thymidylate synthetase. Black areas indicate sequence derived from the PCR primers, the bold arrow represents the pKK233-2 promoter, and the asterisk in the DHFR-TS construct shows where a single nucleotide was wrongly incorporated by the Taq polymerase. This was corrected as shown. Restriction enzyme sites are N, Ncol; S, Spel;

H, HindllI. Maps are schematic and not to scale.

expression vector pKK233-2 (Pharmacia, Uppsala, Sweden), which possesses the strong trc promoter and a prokaryotic ribosomal binding site (Shine and Dalgarno sequence) upstream of the un- ique insertion sites. This vector has been used suc- cessfully in the high level expression of trypanothione reductase from Trypanosoma congolense [30], and a closely related vector, pKK223-3, in that of the DHFR-TS enzyme of Leishmania major [31].

Alignment of the primary sequence of the DHFR-TS molecule of P. falciparum with the DHFR and TS sequences of other organisms allows it to be subdivided as follows: a DHFR domain, ex- tending from the N-terminus to approximately

amino acid residue 225, a junction region ex- tending from residues 226 to 320, and a TS domain from residue 321 to the C-terminus at 607 [10,16]. To obtain just the DHFR domain, PCR primers were designed that incorporated not only the requi- site restriction enzyme sites for facile cloning, but also a termination codon corresponding to residue 231. The 718-bp PCR fragment corresponding to the DHFR domain was first ligated via its blunt ends into pBluescript (Stratagene, La Jolla, CA). It was then excised using the NcoI site incorporated within the 5' PCR primer, together with HindIII (Fig. 1). The aforementioned NcoI site is not un- ique to the construct: a further site is found 136 bp into the DHFR coding region. This allowed us to create not only the full-length DHFR construct after partial digestion with NcoI, but also, after complete digestion, a truncated construct carrying all but these 136 bp. In this case, the ATG sequence within the NcoI site was now out of register with respect to the DHFR reading frame, thus providing a useful negative control. These two fragments were ligated into pKK233-2 cut with NcoI and HindIII to give clones which we term DHFR(+) and DHFR(-) respectively (Fig. 1B and A). The NcoI site en- compasses the ATG initiation codon, which is thereby placed at the correct distance from the promoter sequence, enabling synthesis of an unfused protein product. The open reading frame of P. falciparum DHFR-TS begins with 2 adjacent methionine residues, the second of which we believe to be the true initiation codon, both from the nucleotide sequence context [6] and by comparison with the nucleotide sequence of the DHFR-TS gene from the rodent parasite P. chabaudi [18]. It is this second Met that we have retained in our PCR construct.

A similar PCR procedure was followed to construct clones carrying a 1846-bp fragment incor- porating the full-length DHFR-TS-coding region (DHFR-TS(+)), together with a corresponding truncated version (1710 bp, DHFR-TS(-)) to be used as a negative control (Fig. 1D and C). The latter was derived by deletion of the 5' 136-bp Ncol fragment as for the DHFR construct above. Se- quencing of the cloned products confirmed the fidelity of the coding sequences, except for a single nucleotide change, which was corrected as indicated in Materials and Methods and Fig. 1.

Cloning of the independent DHFR domain sequence proved to be straightforward in pBlue- script, and the required fragment was easily trans- ferred to the expression vector. The complete DHFR-TS-coding sequence was however unstable in pBluescript. We therefore resorted to pUBS 1, a pUC-type vector from which certain M I3 sequences have been deleted to increase stability (G. Murphy, pers. comm.). However the insert was still unstable both in this vector and the expression vector unless TMP was maintained in the medium (see below).

Rescue of E. coli in trimethoprim by expression of P. falciparum dihydrofolate reductase activity. TMP is an antifolate compound which bears a structural resemblance to the antimalarial pyrimethamine, but which binds about 70-fold less tightly to antifolate-sensitive malaria parasites

321

than to that of bacterial DHFR [1]. It binds even less strongly to the enzyme from antifolate-resiso tant parasites, from which our construct is derived. To test whether the cloned DHFR domain from P. falciparum could rescue E. coli cells from inhibitory doses of TMP, the growth of cells carrying the plasmid-encoded malarial gene was monitored for evidence of complementation both on solid agar and in liquid cultures. In initial experiments, clones containing either the complete DHFR region (DHFR(+) clone), the truncated, out of frame DHFR sequence (DHFR(-) clone) or just the expression vector without insert were grown in E. coli strain TG2 on minimal medium plates. All clones grew in the absence of TMP, but only DHFR(+) clones were able to grow in the presence of 5 ~tg ml ~ TMP or higher (Fig. 2A); indeed growth could still be observed at a level of 100 ~tg ml t TMP. The pKK233-2 vector system used provides a means of

A - T M P B - -TMP

a b C1 C2 Cl C2

I i I ;

-I- TM P -I-TMP Fig. 2. (A) Rescue ofE. coli from trimethoprim inhibition by the dihydrofolate reductase activity ofP.falciparum. TG2 cells transformed with either pKK233-2 alone (a), the DHFR(-) clone (b), or 2 independent DHFR(+) clones (c~ and c2) were patched onto minimal plates containing no TMP (-TMP) or 50 lag ml ~ TMP (+TMP) and incubated at 37°C for 16 h. TMP was initially dissolved

in 0.01 M HCI at 10 mg ml ~. (B) As for A, but with 1 mM IPTG added to the plates as inducer.

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controlling expression of the foreign gene by IPTG induction. Interestingly, the above results were obtained in the absence of IPTG, suggesting that the low, uninduced level of expression from the trc promoter leads to production of sufficient parasite enzyme to overcome the TMP inhibition. Para- doxically however, if IPTG was added to the plates, cell growth of the DHFR(+) transformants was blocked whether TMP was present or not (Fig. 2B). Under these conditions, the control clones were unaffected and grew normally in the absence of TMP. This alerted us to the possibility that the plasmodial DHFR product, while able to rescue E. coli from the inhibitory effect of TMP at low levels, itself became toxic to the cells at higher levels.

To eliminate any possible artefacts resulting from local changes in inhibitor concentration within solid media, and to show conclusively that the plasmodial enzyme was not just titering out the drug, the above experiments were duplicated in liquid culture (Fig. 3). DHFR(+) and D H F R ( - ) clones were grown to early exponential phase in minimal medium, then the cultures were divided and TMP added to one half. After a further 3-h incubation, samples were again divided and IPTG added to one half. As in the plate assays, TMP at a low level (10 gg ml ~ in this case) completely inhibited DHFR(- ) -conta in ing cells, whereas DHFR(+) cells continued to grow in the presence of the drug (tolerating at least 50 gg ml ), albeit at a reduced rate. Once again, IPTG induction immediately terminated the growth of TG2 cells containing DHFR(+), even in the absence of TMP. The results of the plate and liquid culture assays show that the functional activity of DHFR in P.falciparurn is independent of the covalent linkage of the DHFR domain to the TS domain and can be expressed in a heterologous system.

The same types of experiment were at tempted with the equivalent DHFR-TS(+) construct. How- ever, we observed in this case that this sequence was unstable in the absence of TMP selection, both in the pKK233-2 /TG2 expression system, or when transformed into the highly recombinat ion-deficient E. coli SURE host (Stratagene, La Jolla, CA). Moreover, unlike the DHFR(+) moiety, this construct could only rescue TMP-inhibited E. coli cells when grown in a rich medium (L-broth); transformants grown in minimal medium apparently

A O00

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0 ,3

0 .2

0.1~ I

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0

10

RO (1 I

TMP IPTG TIME (h)

Fig. 3. Assay ofE. coli rescue from trimethoprim inhibition in liquid culture. The growth of TG2 cells transformed with either the DHFR(-) clone (control), or the DHFR(+) clone in minimal medium was monitored from absorbance measurements at 600 nm as follows: DHFR(+) clone in the absence of TMP (boxes) was induced at the indicated time with 1 mM IPTG (asterisks). A parallel culture grown in the presence of 10 lag ml -~ TMP (crosses) was similarly induced (diamonds). Growth of the control culture of DHFR(-) cells was completely inhibited in the presence of the same concentration of TMP (tri-

angles).

produced insufficient enzyme to overcome inhibition. As expected, the D H F R - T S ( - ) transformants were inviable in the presence of TMP, irres- pective of the culture medium.

Detection of protein products. The above experiments suggested that only low levels of expression of the DHFR(+) or DHFR-TS(+) products were necessary to complement E. coli cells whose endogenous DHFR enzyme had been completely inhibited by TMP, and that high levels of these proteins were toxic. To place these observations on a more quantitative basis, whole-cell samples of the transformants both before and at various intervals after induction were examined on denaturing protein gels. In no case was a new band or an elevated

level of an existing band evident upon Coomassie Blue staining (data not shown). Thus, there was no evidence that large quantities of protein were being produced, either in soluble form or deposited as inclusion bodies. Moreover, the level of DHFR activity that could be assayed by a standard procedure in the spectrophotometer [32] was not significantly above background NADPH-oxidase levels. In all subsequent experiments, we therefore made use of a highly sensitive quantitative assay in which the substrate analogue FdUMP, labelled with tritium, reacts stoichiometrically with the TS active site to give a covalent complex [29] which resembles a steady-state intermediate of the normal enzymic reaction [33]. After growth with or without IPTG induction, cells carrying the DHFR-TS(+) construct were sonicated, the cell debris pelleted, and the supernatant assayed with the radioactive label. Fig. 4 shows the autoradiograph from an SDS gel of cell extracts processed in this way. In the negative control (containing just the DHFR domain), only the endogenous E. coli TS band at 31 kDa was seen. In the uninduced culture, the DHFR-TS(+) clone gave rise to a faint new band at 72 kDa, corresponding to the full-length enzyme (not visible in Fig. 4, but see Fig. 7a). This band was much more intense in the sample taken from the IPTG-induced culture (Fig. 4, lane 3, labelled I,DT+). As well as the full- length product, at least 3 other smaller products carrying an intact TS active site were observed: a faint band at 59 kDa (not clearly visible in Fig. 4, but see Fig. 7a), a band at 40 kDa, and a strong band at 34 kDa. This suggested that the full-length DHFR-TS product may be processed in a highly specific degradative manner, which could possibly explain the low level of foreign product that we observe. Quantitation of these bands by densitometry indicated that the proportion of the total foreign product present in the cell at the time of assay, relative to the endogenous E. coli TS enzyme, was only about 1.8:1. Nevertheless, the TS substrate-binding activity expressed from the PCR construct is clearly functional in E. coli, as was found for the DHFR activity.

Control experiments were performed to exclude the possibility that these smaller products arose under the assay conditions themselves. Sonication of the E. coli cells was performed in buffers containing (i) no protease inhibitors, (ii) the standard

323

I HIIIP - 72 D T ( P f )

-59

-40 -34

-31 T(Ec)

I U I D + DT+ DT+

Fig. 4. Analysis of expression products by polyacrylamide gel electrophoresis and autoradiography. Cell extracts (E. coli SURE) were labelled at the TS active site and run on polyacrylamide gels as described in Materials and Methods. I, culture induced by IPTG; U, uninduced. D+, cells transformed with the DHFR(+) construct, as negative control; DT+, cells transformed with the DHFR-TS(+) construct. DT(Pf) is the full-length malarial DHFR-TS molecule, T(Ec) is the endogenous E. coli TS molecule. Smaller plasmodial products carrying an intact TS binding site are indicated at the sizes shown (in kDa, calcu-

lated by comparison with standard protein markers).

level of inhibitors for the TS assay [17,27] as described in Materials and Methods, and (iii) 5 times this level. For each of these samples, incubation with [3H]FdUMP was systematically varied up to 3 h. The autoradiograph pattern was identical in all cases (data not shown). Moreover, the pattern obtained was not dependent upon the time elapsed before or after sonication of individual samples. We therefore conclude that the products observed are not an artefact arising during the processing of the E. coli cells after culture.

Comparison of different E. eoli cell lines. The results above indicated that although easily detect- able, the absolute level of DHFR-TS that could be obtained from induced cultures of the standard reeA strain TG2 or the highly recombination-de-

324

ficient SURE strain was small (less than 1% total cell protein). In addition, such preparations contained smaller TS substrate-binding products, as well as the full-length species. Moreover, induction of the DHFR-TS gene by IPTG led to almost immediate cessation of cell growth. We therefore transformed our DHFR-TS(+) construct into a number of different strains of E. coli which have proved useful in the high level expression of other foreign proteins. These included the protease-deficient strains E. coli B, AR68 and AB 1899, since the small bands seen in the electrophoresis experiments above could be the products of specific pro- teolytic activity. Of the strains tested, one (AR68) proved completely refractory to transformation with the DHFR-TS(+) construct, although transformation with vector alone was normal. SURE, E. coli B and EC1321 all transformed with a low efficiency (ca. 20%) relative to the control plasmid, while one strain alone, AB 1899, transformed with near normal (ca. 80%) efficiency.

When the growth characteristics of the transformed strains were examined (Fig. 5), their responses fell into 2 classes. Thus, while all uninduced cultures grew normally, induction of the plasmodial DHFR-TS gene in E. coli B and EC1321 led to an immediate cessation in the increase of cell density, as observed for TG2 and SURE above, while AB 1899 alone continued to grow at a near-normal rate. The classification of the E. coli strains in these terms thus mirrors their relative transformation efficiencies. These different growth characteristics prompted us to examine the cell morphology of the transformed strains in the light microscope (Fig. 6). In all cases except AB 1899 (panel A), induction led to the formation of elongated cells which associate into filaments (panel B). In these strains at least, the plasmodial protein product clearly affects com- pletion of the normal life-cycle of E. coli. Similar cell morphology has been reported in cases involving the expression of other heterologous proteins in E. coli and is often associated with the formation of inclusion bodies [34]. However, since no new or enhanced bands could be identified on Coomassie Blue-stained gels of our induced whole-cell preparations, only a minimal amount of product, if any, could be accumulating in inclusion bodies in these strains. It thus seems more likely that expression of the heterologous proteins may be interfering di-

1.4

1.2 A eoo

D

0.8 l

0.6

I 0.4

0.2 j (EC18211

0 1 2 3 4 8 6 7 t TIME (h)

IPTG

Fig. 5. Effect of induction on different E. coli cell lines in liquid culture containing the dihydrofolate reductase-thymidylate synthetase (+) construct. Crosses, uninduced E. coli SURE; boxes, uninduced AB1899; triangles, induced SURE; asterisks, induced AB 1899. E. coli B and EC 1321 gave curves

essentially identical to those of SURE.

rectly with the E. coli cell cycle. We further examined the status of the DHFR-TS

product in the 2 classes of cells by gel electrophoresis and autoradiography as described above. Ali- quots were examined in a series of time points up to 20 h after induction. In all cases, the same set of smaller TS binding site-containing products was seen, as well as the full length DHFR-TS molecule (Fig. 7a). Those strains that ceased growth upon induction, and subsequently produced elongated cells, displayed a peak level of the foreign species after about 1 h, as measured by the ratio of their sum to the endogenous TS band. In contrast, AB 1899 cells, which showed no evidence of modi- fied morphology, produced increasing amounts of the foreign products up to about 12 h after induction. Stationary phase was reached in these cells at ca. 5 h after induction. These results were substan- tiated by quantitative filter assays of the TS corn-

325

Fig. 6. Morphological eflbcts of induction of the plasmodial dihydrofolate r ~ f t tion, cell line AB 1899 (A) appeared identical to uninduced cells, while SURE (B) er induc-

and filamentous. Magnification 5(~onxd. all other E. co//strains tested became elongated A

I* U 1 U I U l U 1

Fig. 7. (a) Pattern o f t h ' m - • - - ~-" f~o 180 240 • Y ~ayJate synthetase binding site-containing products b mducOon A AB 1899 E EC 1321 B, E. ~ oh B, S, SURE. The track marked l ~ expressed m various cell lines befor re°re clearly the exlstenc;ofthe 59. kDa band (b)T,.me course of mduc!mn o;STf;bmnad~:[te~cl~oStdiau~grpdidgu;~i!~ ~

as momtored by autoradioeraphy F~gures represent t~me after mduct~on m minutes. Annotation and sizes of the bands

normalised to total cell protein (data not the level of the endogenous E. coli TS. However, shown). Thus, while expression in E. coli is limited from the gel analyses, only ca. 25--40% of the p/as- by the toxicity of the DHFR-TS product to the host cell, in AB 1899, the most favourable strain examined, the amount ofplasmodial protein determined either by scanning of autoradiographs or quantitative assays increased to between 30 and I00 times

modial protein that accumulated in these cells corresponded to full-length DHFR-TS.

Despite the ability of AB 1899 cells to continue with near-normal growth after induction, the relatively high levels of foreign product attained, and

326

6 R A T

I 0 4

2

0

(a)

0 1 2 3 4 5 6 7 8 9 10 20 TIME (h)

R A 6 T I

O

4

(b)

0 i 2 ' 3 ~ 4

TIME (h) Fig. 8. (a) Ratio of the total amount of smaller thymidylate synthetase binding site-containing products to full-length dihydrofolate reductase-thymidylate synthetase expressed from dihydrofolate reductase-thymidylate synthetase (+)-transformed AB 1899 cells, over a 20-h period post induction. Autora- diographs of Fig. 7b and tracks of later time points were scan- ned by densitometry and peak areas corresponding to the relevant bands calculated. (b) The same ratio expressed from identical cells following translational block by chloramphen-

icol (time point 0), 3 h after induction.

the protease-deficient status of this strain, the same class of small products was observed as seen in all other strains, in roughly similar proportions (Fig. 7b). Thus, no strain significantly influenced the ratio of smaller products to the full-length product. Surprisingly, this ratio stayed quite constant, within experimental error, across the whole time course for AB 1899 right up to the 20-h time point (Fig. 8a). This did not suggest a simple precursor/ product relationship between the full-length product and the smaller bands, since the maintenance of

a steady state from the moment the cells are induced in early exponential phase, through to 15 h after stationary phase is reached seems highly unlikely.

OHgin of the smaller products carrying intact thymidylate synthetase-binding sites. To shed further light on the origin of the smaller products, we added chloramphenicol to one half of a DHFR- TS(+)-transformed AB1899 culture, 3 h after induction, when the production of foreign protein was at a high level. This antibiotic binds to the large ribosomal subunit and immediately blocks any further translation. We also wished to compare in this way the stability of our recombinant DHFR-TS product with that of the native molecule in the parasite itself, which has been measured to be stable for over 6 h [35]. We examined the DHFR-TS products at intervals up to 4 h after the block to deter- mine at what rate the full length and smaller products decayed, and the relationship between them (Fig. 8b). Under these conditions of arrested protein synthesis, species related by degradation would be revealed as their distribution shifts from high to lower molecular weight. No evidence of such a shift was seen in the data obtained, with the ratio of smaller products to the full-length product staying relatively constant over the entire post- blockage period. Indeed, all the products appeared to be quite stable to decay over the 4 h following the block. We therefore conclude that there is no degradation-induced, product-precursor relationship amongst any of the proteins expressed from the plasmodial DHFR-TS(+) construct.

A family of related proteins could alternatively be the product of different, in-frame, translational initiation events. Such proteins, having similar and significant half-lives, would be expected to be maintained at ratios that reflect the efficiencies of individual initiation sites. Provided that the relative efficiencies of such sites did not change during growth, exactly the observed, constant, ratios of the levels of the individual protein species would be expected and these should, again as observed, be unaffected by translational inhibitors such as chloramphenicol.

Important factors in defining and determining the efficiency of an E. coli translational start site are the degree of complementarity between the ribosome-binding site and the 3' end of the 16S

327

TABLE I Analysis of potential translational initiation sites within the P.falciparum dihydrofolate reductase-thymidylate synthetase gene

Position of Putative SD Separation Intervening sequence ( ISf %A+U Predict- Mr of Intensity of initiation sequence ~ (bp) c of IS ed Mr of observed product codon a product product

(kDa) (kDa)

55 (AUG) uGGAaa 15 UGUAAUUCCCUAGAU 67 66 - not seen 92 (AUG) uGGAua 11 AUGUAAAUGAU 82 61 59 faint

249 (AUG) AGGAGa 16 AGAAAAAAAUAAUGAU 88 43 40 medium 358 (AUG) cGGAua 4 UAUU 100 29 34 strong

aAmino acid residue numbered according to ref. 6. bUpper case denotes homology to the consensus Shine and Dalgarno (SD) sequence AGGAGG [36]. CDistance between SD motif and initiation codon. dSequence between SD motif and initiation codon, taken from ref. 6.

rRNA and the spatial relationship of this sequence to the initiation codon [36]. For a sequence to function as a potential start signal, all that is required is that it bears some homology to the Shine and Dal- garno sequence and is a short distance upstream of an initiation codon. We therefore analysed the plasmodial DHFR-TS nucleotide sequence, ident- ifying in-frame initiation codons that might function in E. coli (ATG and GTG), and examining the immediate upstream region for possible ribosome- binding sites. Of the 18 possible in-frame initiation codons, only 5 have at least 3 contiguous base matches to the consensus Shine and Dalgarno motif AGGAGG located from 4 to 16 nucleotides upstream of them. Of these 5, only 4 would be expected to give rise to polypeptides containing an intact FdUMP-binding domain, and these are listed in Table I. From the DHFR-TS sequence, the molecular weights of products that would arise from initiation at these codons were calculated. We note that there is close agreement between the protein products whose synthesis would be initiated from 3 of the 4 start codons preceded by sequences that meet the parameters defining a minimal ribosome- binding site [36], and the size of the additional bands measured in Figs. 4 and 7.

The efficiency of translation has also been correlated with the incidence of A and U residues in the RNA sequence between the Shine and Dalgarno motif and the initiation codon [37]. In this respect, the trend in the intensities of the protein products that we observe broadly mirrors the percentage A+U content of these 4 regions (Table I), although the absolute intensities will be a complex function

of this A+U content, the extent and nature of the base pairing at the Shine and Dalgarno region, and the distance from the latter to the AUG codon. On the basis of our data, we would predict that site-directed mutagenesis of the regions in question to re- duce as far as possible the similarity to the Shine and Dalgarno consensus sequence, as well as the A+U content of the adjacent region, whilst still pre- serving the encoded amino acid sequence, should decrease the incidence of false initiation whilst increasing the level of the full-length product. Er- roneous initiation of translation at internal methionine codons of heterologous genes has been previously observed in E. coli [38,39].

Discussion

We have reported the production of significant levels of functional protein expressed from a recombinant plasmodial DHFR-TS gene within E. coll. However, over half of this material appears to consist of N-terminal truncated species. It is likely that these TS substrate-binding molecules retain the normal C-terminus, since even very limited proteolysis at this end of the corresponding Leish- mania enzyme results in loss of TS activity [40]. Although the production of the truncated molecules compromises the yield of the full-length product, it might be possible to achieve selective purification of the latter using methotrexate affin- ity chromatography, which requires an intact DHFR domain.

It is interesting to contrast our results with those obtained using a recombinant Leishmania DHFR-

328

TS gene [31]. In the latter case, a high level of expression in E. coli was achieved (7-40% of total cell protein, depending upon the promoter). In our case, both the full-length plasmodial DHFR-TS as well as the DHFR domain alone were toxic to the host and could only be expressed at much lower levels. We note that the DHFR domain of P.falciparum contains 2 long insertions that are absent from the Leishmania and E. coli sequences (as well as from those of other organisms), namely amino acid residues 21-37 and 63-85. Both these regions are quite highly charged (35% and 43% charged residues respectively). It is possible that interactions of these charged regions with E. coli proteins result in the observed inhibition of cell growth.

Even in strain AB 1899, the growth of which is comparatively unaffected by DHFR-TS expression, only moderate levels are accumulated. Thus, additional factors affecting either transcrip- tion or translation may be limiting expression in this case. The nature of the promoter that is present in the expression vector should ensure that tran- scription is not the limiting factor. However, mask- ing of the trc promoter's ribosome-binding site by secondary structural motifs could limit the translation of even abundant transcripts [41]. Such structural features could arise from the artificial nature of this region, which consists of sequence from the coding region of the eukaryotic gene joined to the prokaryotic promoter. In the absence of normal levels of translation, it is likely that the recognition of fortuitous ribosome-binding sites may be more fre- quent. Normally, such recognition might be sup- pressed by the presence of polysomes, which would not be formed under circumstances of reduced initiation.

It seems unlikely that the comparatively low level of expression that we have obtained results from any fundamental problem associated with the expression of functional plasmodial enzymes in E. coli, since the expression in this host of 2 other non- antigen genes from P.falciparum has been recently reported. The HPRT gene was obtained in quite large amounts from a recombinant plasmid [42], demonstrating that there is no intrinsic difficulty arising from the fact that codon usage in E. coli is quite different from that in the parasite [43]. The recombinant HPRT was active both in E. coli and in Salmonella typhimurium, and could complement

an hpt mutation in the latter. However, that the levels of expression of plasmodial proteins in bacteria are highly variable is reinforced by the study of expression of the aldolase gene in E. coli, where much lower levels were attained [44].

It is likely that more efficient expression of full- length plasmodial DHFR-TS will be necessary to produce the amounts required for biophysical stud- ies. As well as the site-directed mutagenesis experiments outlined in the previous section, we therefore propose to investigate the use of alternative strategies involving different E. coli promoters, the expression of DHFR-TS fused at its N-terminus with suitable (cleavable) prokaryotic polypeptides, and alternative hosts recognising different sequence motifs for translational initiation. We note however that production of sufficient full-length material does not guarantee successful crystallis- ation. In particular, proteins consisting of independent domains linked by a hinge region are often dif- ficult to crystallise because of the flexibility of the latter. The usual procedure then is to attempt crys- tallisation of individual domains. In this respect, our successful expression of the isolated DHFR domain may offer a solution if such difficulty is en- countered.

Acknowledgements

We are most grateful to Dr. Colin Dykes of Glaxo Group Research, for gifts of E. coli strains AB1899, E. coli B and EC1321, to Dr. Allan Schatzman of Smith Kline Beecham for strain AR68, to Martin Read for maintaining parasite stocks and providing general assistance, and to the Medical Research Council, U.K., for financial sup- port.

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