Cloning of trichosanthin cDNA and its expression in Escherichia coli

Getle. 97 (1991) 267-272

267

Elsevier

GENE

03X27

Short Communications Cloning

of trichosanthin

(Recombinant

Pang-Chui

DNA;

cDNA

root tuber;

Shaw, Mei-Hing

and its expression

signal sequence;

Yung, Rong-Huan

nucleotide

in Escherichia sequence;

coli

expression)

Zhu, Walter K.-K. Ho, Tzi-Bun Ng and Hin-Wing

Department of Biochemistry and Biotechnology Laboratory,

Yeung

The Chinese University of Hong Kong, Shatin, N. T. (Hong Kong)

Received by R. Wu: 29 May 1990 Revised: 15 August 1990 Accepted: 16 August 1990

SUMMARY

Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A) -)-RNA of the root tuber of Trichosanthes kirilowii Maximowicz. The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product [Collins et al., J. Biol. Chem. 265 (1990) 8665-86691. Recombinant TCS (reTCS) was synthesized in Escherichiu coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.

INTRODUCTION

Trichosanthin (TCS) is an active component of THF, the root tuber of the Chinese medicinal herb Trichosanthes kirikowii Maximowicz of the Cucurbitaceae family. In the classical medical reference work in China, Compendium q/ Materiu Medica, written by Li Shi-Zhen in the late 14th century, THF was mentioned as a drug that resets

Correspondence Chinese

fe: Dr. P.-C. Shaw,

University

Tel. (852)695-2868; Abbreviations: double which

50:;

side; kb, kilobase

Hong

Department

Kong,

of Biochemistry,

Shatin,

N.T.

(Hong

The Kong)

Fax (852)603-5123.

aa, amino

strand(ed); causes

of

HIV,

acid(s); human

inhibition;

Ap, ampicillin;

bp, base pair(s);

immunodeliciency

IPTG,

virus;

ID,,,,

ds, dose

isopropyl-p-d-thiogalactopyrano-

or 1000 bp; LB, Luria-Bertani

(medium);

nt, nucle-

otide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide-gel electrophoresis;

re, recombinant;

chosanthin; moter;

SDS,

TCS, gene (DNA)

THF. Tian hua fen;

sodium

encoding

dodecyl TCS;

sulfate;

trc, hybrid

[ 1,denotes plasmid-carrier

TCS,

tri-

rrp/lac pro-

state.

menstruation and expels retained placentae. The prescription, which contains THF and seven to eight other Chinese herbs, has been used until recently for abortifacient purposes (Wang et al., 1986). In the early 1970s TCS was isolated and then it has been used to terminate early and mid-trimester pregnancies (Liu et al., 1985; Jin, 1985) to treat ectopic pregnancies, hydatidiform moles and trophoblastic tumors (Jin, 1985; Huang, 1987). Studies in Hong Kong on TCS and related proteins including c(- and b-momorcharin (Yeung et al., 1988) revealed many novel pharmacological activities of these proteins. Besides having abortifacient properties (Law et al., 1983; Chan et al., 1984) they also inhibit tumor growth (Tsao et al., 1986) suppress the immune responses (Leung et al., 1986; Yeung et al., 1987) and inactivate eukaryotic ribosomes (Maraganore et al., 1987; Yeung et al., 1988). Recently, TCS has been discovered to act selectively against HIV-infected cells (McGrath et al., 1989) and clinical studies are underway. To study the structure-function relationship of TCS and to improve its value in these applications, we have isolated

36X

and characterized cDNA clones encoding expressed the cDNA in Escherichiu coli.

EXPERIMENTAL

TCS and have

AND DISCUSSION

(a) Strains, plasmids and antisera References for E. coli strains and vectors are as follows. (1) For the generation of a cDNA library: BNN93. BNN102, and /2gtlO (Huynh et al., 1985); (2)for nt sequencing: pUC18 (Yanisch-Perron et al., 1985) and DHSx (Sambrook et al., 1989); (3) for the expression of TCS cDNA: RB791 and pTRC99A (Amann et al., 1988). Oligos used were made with an in-house DNA synthesizer (Applied Biosystems EP391) or from Dr. S.A. Chow of the University of Hong Kong. Polyclonal antiserum against TCS was generated as follows: 400 pgiml of crystallized TCS in saline with OS”;, SDS was diluted to 200 ,ng/ml after heating at 80°C for 10 min. An equal volume of Freund’s complete adjuvant was added and the emulsion was injected intradermally into a 2.5-kg albino rabbit. Five booster injections emulsified with incomplete adjuvant were subsequently given and the antiserum was prepared according to Harlow and Lane (1988). Kits, enzymes and biochemicals were obtained from various commercial sources and used according to the suppliers’ recommendation s. (b) Isolation of poly(A)+RNA, cDNA library construction and selection of TCS cDNA clones Fresh root tubers of T. kirilowii Maximowicz were obtained from Guangdong (China). They were cut into small pieces and stored at -70°C. 100 g of frozen root tubers of T. kirilowii added to 20 g of alumina were ground to a fine powder in a grinding buffer [6”,, (w/v) Na .4-aminosalicylate/l ”;, WV) Na . triisopropylnaphthalene sulfonic phenol/l.25”, Na’ . dteth$l?h?car~~m~ (w/v) ’ ‘acid/50 mM Tris . HCl pH 8.41. After the homogenate had been thawed, SDS and EDTA (pH 8.0) were added to final concentrations of 1 and 2 mM, respectively. The slurry was then centrifuged at 15 000 x g at 4 aC for 10 min. The supernatant was extracted twice with an equal volume of phenol/chloroform. The RNA in the aqueous phase was precipitated by ethanol. The precipitated RNA was washed twice with 3 M Na. acetate (pH 5.2) and then with 70”, ethanol, and the RNA pellet was dissolved in TE buffer (10 mM Tris . HCI pH 8.011 mM EDTA). Then the RNA was further purified by CsCl gradient ultracentrifugation (GliSin et al., 1974). Poly(A) + RNA was subsequently isolated from the total cellular RNA by oligo(dT)-cellulose chromatography according to the method of Jacobson (1987). The ds cDNA was made by using a cDNA synthesis kit

Fig. I. Restriction (a) Restriction pTCS5021

map

map

and

sequencing

of TCS cDNA

does not include the dotted

of K’S cDNA in(i) pTCS5021 clones were obtained by oligo primers

for

T(‘S

region. (b) Sequencing ofvarious

to appropriate

subclones

regions

cDN.4.

TCS cDNA

and (ii) pTCS482 IO. Sequences

by ds sequencing

hybridized

strategw

in pTCS48210:

in

strategies of the two

in pUClX or

of the TC’S cDNA.

(Amersham). The cDNA was methylated. ligated with EcoRI linkers and size-fractionated according to Huynh et al. (1985). The cDNA with size above 0.6 kb was cloned into i,gt 10 vector. A cDNA library with 4.9 x 10’ recombinants was obtained. It was screened by plaque hybridization (Mason and Williams, 1985) with a 120-bp cDNA 32P probe of r-momorcharin. Its deduced primary sequence is 72.5”,, homologous to TCS (W.K.-K. H., S.-C. Liu, P.-C. S.. H.-W. Y., T.-B. N. and W.-Y. Chan. manuscript in prcparation). A total of 5 1 positive clones were found among 6 x 10’ recombinants. (c) Nucleotide sequences and deduced aa sequence Two oppositely oriented clones pTCS48210 and pTCS5021 were constructed by cloning two independent TCS cDNA into pUC18 (Fig. 1). The complete nt sequences were determined by the dideoxynucleotide chaintermination method (Sanger et al., 1977) and ds sequencing technique (Chen and Seeburg, 1985) using the T7 DNA polymerase sequencing kit (Pharmacia). The sequences of TCS cDNA from both clones contain an ORF of 289 aa (Fig. 2). Except for three nt changes, both sequences are identical. Nevertheless, these changes do not alter the code aa. Comparison of the deduced aa sequence with that obtained by Collins et al. (1990) shows that our TCS cDNA codes for a precursor molecule with a signal peptide of 23 aa and an extra 19 aa at the C terminus. The sequences for the mature protein obtained in both studies are identical. The sequence of TCS in pTCS48210 also agrees with that of the genomic DNA (Chow et al., 1990) except for six nt changes (Fig. 2). Four do not affect the coded aa. Two of them, G + C at nt 710 and C + T at nt 749, lead to the change Ser + Thr and Thr --f Met, respectively. Root tubers of our work and that of Chow et al. (1990) were obtained from Southern China, while leaves for generating the genomic library in Collins et al. (1990) were

ATG ATCJ AGA TTC TTA GTC CTC TCT TTC CTA ATT CTC RCG CTC ‘FTC CTA ACA ACT &et

Ife

Arg

Phe teu

YaI teu

SW

Lm

LOU ffe

Lf?if Thr Lea Phe Leu Tftr

Thr

-20 t CCT GCT CTG GAG CCC CAT CTT AGC TTC COT TTA TC?%T GCA XCA ACC XGT TCC pro Ala Val Gfu Gty ASP Val SOP Pho Awg Lm Sex Gly AlrrTBr Ser.sersor -r f TAT CGA CTT TTC ATT TCA AAT CTC Tyr Gly Val Phs Xl% Ser ~L;T&L&t

ACA AAA CCT CTT CCA AAP GAA AGO XAA CTO Arg tys Ala LettPm Am Clu Irg Lys Leu

TTA CCT TCC TCT2%” CCA GFT TCT CA4 CCC TAG! ‘33 TTG Letr Leu Arg far S’s: Leu Pro GIy Sor G&i Arg Tyr ALlaLeu

TAC CAT ATC CCT WC Tyt

Asp

Ite

Pro

Al’C

CAT CR ACA AAT TAG GCC CAT CAA

Xl@ His Leu Thy Asn Tyr

Ala Asp

ICC ATT TCA GTG CCC AT& GAC CTA ACG Glu Thr Ifs Ser Yal Al% fle Aq Yal Tkr CAT ACA TCC TAT TTT TTC AAC CA@ Ala Gly Asp Thr ,%a&Tyr P&e Phe AS?&GLit

AAC GTC TAT ATT ATG CGA 9% CCC GCT CGC Am

YaX Tyr

Ile

H6t Giy

Tyr

Arg

270

EcoRI

1. digest with NCOI + BarnHI

/ 1. remove 0.88-kb PstI fragment 2. PCR with oligos A and B 3. digest PCR product NcoI + PstI NcoI

2. isolate large fragment

\ 1. digest with PstI

+ BumHI

7

2. isolate 0.88-kb fragment

with PstI

BCZJTIHI

PstI

ligation )

Fig. 3. Construction

of expression

plasmid

pTRC48210.

Step1. pTCS48210

kit (BiolOl) and religated to form pST48210. A 35.nt oligo A with the sequence and a 17-nt -40 universal primer (oligo B) was obtained from NW England in a reaction

mixture

of 50 pl (Sambrook

was digested

with MI,

the large fragment

was isolated

by the Geneclean

5’-TGTGGCCATGGATGTTAGCTTCCGTTTATCAGGTG was made Biolabs. 500 ng of pST48210 and 50 pmol of oligos A and B uere mixed

et al., 1989) and 21 cycles of PCR were performed.

The PCR product

was then extracted

three times with

water-saturated chloroform and digested by JVCOI+ PP/I and purified by the Geneclean kit. Step 2.The O.&Y-kb Pstl-BurnHI fragment was isolated from pTCS48210 by the Gene&an kit. This fragment was first ligated to the large AJcoI-BnmHI fragment of pTRC99A and then to the ,VMI + MI-digested PCR product. The ligation mixture v,as tran3formcd to RI3791 made competent by standard techniques. Step 3. To assess that no mutation had occur-red

271 collected from South Korea. Therefore, differences between the former two aa sequences with the latter one may be due to a minor variation of the plant species used. Differences of our two cDNA sequences and to that of the genomic DNA may also be attributed to that TCS in T. kirilowii exists as a multigenic family. Southern-blot analysis of EcoRI-digested T. kirilowii DNA shows at least four bands (Chow et al., 1990). From the seeds of T. kirilowii, another ribosome-inactivating protein, trichokirin, with N-terminal sequence similar to that of TCS has been isolated (Casellas ct al., 1988). A TCS sequence with certain regions dramatically different from ours (and hence from Collins and Chow’s group) has also been obtained (Wang ct al., 19X6) and discussed

(Collins

A

B

C

D

F

E

kDa

-43

-30

-20.5

et al.. 1990). Fig. 4. Expression

(d) Expression of TCS in Escherichia coli By mutagenic PCR, a NcoI site was created to add a Met codon at the -1 position of the mature protein. At the same time, the N-terminal signal sequence was removed. The TCS gene was then placed under the control of the trc promoter of pTRC99A (Fig. 3). RB79l[pTRC48210] after induction was lysed and proteins were analysed by SDS-PAGE (Laemmli, 1970) and Western blotting (Bjerrum, 1986). A distinct band immunoreactive to a rabbit anti-TCS antiserum was detected in the soluble fraction of the cell lysate (Fig. 4). This band corresponds to a 29-kDa protein, and matches the size of TCS as deduced from the cDNA sequence. Since the C-terminal 19 aa have not been removed by E. coli, the recombinant TCS is larger than the 27-kDa natural protein. Its biological activity is being studied. The amount of TCS synthesized in E. coli was about 0.01 y0 of the total cellular protein. This was much less than the 5 y0 expression of the unfused placental protein 4 by the same system (Amann et al., 1988). TCS is related to ricin A and is a eukaryotic ribosome-inactivdting protein (Zhang and Wang, 1986; Maraganore et al., 1987; Yeung et al., 1988). It is not known if TCS also has a detrimental effect on the translation of E. coli. We are in the process of removing the sequence coding for the extra 19 aa and the non-essential 0.4-kb region between the stop codon of TCS and the T 1 and T2 transcriptional termination signals of the vector. Whether or not the latter clone has an increased efficiency for the synthesis of TCS will be investigated.

of reTCS in E. cofi. 5 ml of E. coli RB791 [pTRC99A]

or RB791[pTRC48210]

was cultured

at 37°C

in LB (Sambrook

1989) with 50 pg Apjml until the cell density reached IPTG was added collected

to 1 mM and the cells were cultured

and resuspended

in 0.3 ml loading

a 35.~1 aliquot

was analysed

localize the synthesized and resuspended Tris

protein,

containing

of 5 pg of DNase

incubated

at 2O’C

loading

buffer was added.

evaporate

the

SDS-PAGE. loading

three times.

11600

at

liquid

and

the

content

was

analysed

buffer; 30 ~1 was extracted

anti-TCS

antiserum Madison.

and boiled at IOO’C for 3 min before the protein \vas transferred

paper by electroblotting using

a Protoblot

WI). Lanes:

B, pTRC48210,

induced,

by

in 0.5 ml

At the same time, the pellet was resuspended

(Promega,

x g for

to a new tube and 30 ~1 of

The tube was boiled at IOO’C for 15 min to

excessive

a piece of nitrocellulose

mM

and the lysate was

centrifugation

loading onto the gel. After electrophoresis,

fraction;

in liquid nitrogen

was transferred

To

them on an ice bath for

and RNase was added

for 30 min. After

15 min. 30 111of supernatant

PAGE.

cells was collected

0.5 mg lysozgme/30

After placing

15 min. the cells were frozen and thawed An aliquot

2 h,

1970). It

for 30 s at 100 W output.

by O.loo SDS-12.5”,,

5 ml of the induced

in 0.5 ml buffer

HCI pH 8.0:5 mM EDTA.

for another

buffer (Laemmli,

was boiled at 100°C for 3 min and sonicated Then.

et al.,

A.,,,, ,1,,1= 0.8. Then

Ih’

A, pTRC48210. supernatant

to

and TCS detected by an immunoscreening system induced.

fraction;

sedimented

C, pTRC48210,

uninduced, total protein; D, crystallized TCS, 500 ng: E, pTRC48210, induced, total protein: F, pTRC99A, induced. total protein.

nt changes which do not alter the coded aa. These together with evidences from related work of others suggest that TCS exists as a multigenic family. (3) ReTCS has been produced at low level in E. coli by putting the TCS cDNA under the control of trc promoter.

ACKNOWLEDGEMENTS

(e) Conclusions (1) We have isolated several TCS cDNA clones and have sequenced two of them. (2) The two cDNA contain three

4 in the PCR reaction, examined

pTRC48210

by nt sequencing

was drgcsted

by P.rrI, the large fragment

using two primers with sequences

to the 5’ and 3’ ends. respectively,

of the polylinker

T2 the transcriptional

of the rrrrB operon.

terminators

This work was supported University and Polytechnic

was religated

5’-GGCTCGTATAATGTGTGG

region of pTRC99A.

and transformed

to DH5r.

by a research grant from the Grants Committee of Hong

The PCR-generated

and 5’-TTAATCTGTATCAGGCTG.

In the maps of pTRC99A

and pTRC48210,

‘5s’ denotes

region was then each hybridizing

the 5S rRNA;

Tl and

272 Kong. The strains RB791 and pTRC99A are gifts from Dr. E. Amann. We are grateful for the help offered by Dr. H.-S. Kwan in the PCR experiment, Dr. C.-C. Wong for raising the antiserum against TCS, Dr. V. Lam for analysis of the TCS sequence and Mr. S.-H. Wong for the artwork. We also thank the Croucher Foundation for a donation to establish the Biotechnology Laboratory.

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