Expression of the pheromone 3-encoding gene of Euplotes octocarinatus using a novel bacterial secretion vector

Gene, 150(1994) 187-192 0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00

187

GENE 08125

Expression of the pheromone 3-encoding gene of Euplotes octocarinatus using a novel bacterial secretion vector (Kanamycin resistance; T7 promoter; hypotrichous isomerase)

ciliate; ZZ fusion protein; affinity chromatography;

protein disulfide

Claudia Briinen-Nieweler, Frank Meyer* and Klaus Heckmann Institutfiir

Allgemeine Zoologie

und Genetik, Universitdt Miinster, D-48149 Mtinster, Germany

Received by J.K.C. Knowles: 14 February 1994; Revised/Accepted: 11 April 1994; Received at publishers: 26 May 1994

SUMMARY

The pheromone 3-encoding gene (phr3) of Euplotes octocarinatus was expressed in Escherichia coli using a novel expression-secretion vector. The vector, pExSec1, contains a strong and tightly regulated T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The expression-secretion modules are embedded in the high-copy-number plasmid vector, pUK21, which carries a kanamycin-resistance marker (KmR). The produced ZZ-pheromone 3 (Phr3) fusion protein was secreted into the culture medium of the host cells. It was isolated by affinity chromatography and was further purified by gel filtration. After refolding with protein disulfide isomerase (PDI), the fusion protein exhibited the same high activity as the native pheromone.

INTRODUCTION

Phr3 is one of four known polypeptide pheromones secreted by mature cells of Euplotes octocarinatus, a hypotrichous ciliate (Heckmann and Kuhlmann, 1986). The protein is 99-aa long and contains ten Cys residues, three of which are encoded by UGA triplets (Meyer et al., 1991). It induces cells of other mating types of Eu. octocarinatus to prepare for conjugation and is biologically active in concentrations as low as 10-i’ M. The matingCorrespondence to: Dr. K. Heckmann, Institut fur Allgemeine Zoologie und Genetik, Universitlt Mnnster, Schlossplatz 5, D-48149 Monster, Germany. Tel. (49-251) 83-3841; Fax (49-251) 83-4723; e-mail: [email protected] *Present address: GENOMED GmbH, Wielandstr. 28a, D-32545 Bad Oeynhausen, Germany. Tel. (49-5731) 792-201.

Abbreviations: A, absorbance (1 cm); aa, amino acid(s); Ap, ampicillin; bp, base pair(s); E., Escherichia; EM., Euplotes; FPLC, fast protein liquid chromatography; IgG, immunoglobulin G; IPTG, isopropyl-S-o-thiogalactopyranoside; kb, kilobase or 1000 bp, Km,

SSDI 0378-1119(94)00336-Q

type-specific response is believed to be due to pheromonespecific receptors (Heckmann and Kuhlmann, 1986; Kuhlmann and Heckmann, 1989). The isolation of Phr3 from cultures of Eu. octocarinatus is very laborious. We therefore decided to express the corresponding gene in E. coli to produce large quantities of protein for structure-function analysis and the identification of the specific receptor. Because folding and correct disulfide bond formation is favoured in the oxidative environment outside the cytoplasm of E. coli, the use of kanamycin; MCS, multiple cloning site; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; Pl-P7, oligo primers l-7; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; PDI, protein disulfide isomerase; Phr3, pheromone 3; phr3, gene encoding Phr3, phr3 cm, mutated version of phr3 cDNA; phr3sm, mutated version of the cDNA sequence corresponding to secreted Phr3; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; R, resistance/resistant; RBS, ribosome-binding site(s); S. aureus, Staphylococcus aureus; SDS, sodium dodecyl sulfate; spa, gene encoding protein ASZZ, the leader sequence followed by the synthetic ZZ sequence of protein A; ZZ, an engineered IgG-binding region which is present twice; ::, novel junction (fusion or insertion); [I, denotes plasmid-carrier state.

188 an expression-secretion system appeared advantageous with respect to the many Cys residues found in Phr3. To improve the currently existing expression-secretion systems we developed the pExSec1 vector. After refolding with protein disulfide isomerase (PDI) a ZZ::Phr3 fusion protein produced with this novel vector was as active in inducing conjugation in Eu. octocarinutus as native Phr3.

EXPERIMENTAL

AND DISCUSSION

(a) Construction of the expression-secretion vector The construction of the pExSec1 vector is shown in Fig. 1. The novel expression-secretion vector is derived from the T7 expression system developed by Studier et al. (1990) and from the protein A gene-fusion system developed by Nilsson and Abrahmsen (1990). The expression module consists of the inducible T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region of the vector pET3b. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The two Z domains are derivatives of the B domain of protein A in which the Asn-Gly motifs were replaced by Asn-Ala yielding a protein A fragment being resistant to hydroxylamine cleavage (Moks et al., 1987). They do not contain Cys residues and are therefore ideal fusion partners of recombinant proteins containing disulfide bonds. The pExSec1 vector allows expression of a foreign gene in the bacterial cytoplasm and secretion of the respective polypeptide as a ZZ fusion protein. For production of a heterologous protein in the cytoplasm the encoding gene should be cloned into the vector linearized with NdeI and a restriction enzyme with specificity in the MCS. For secretion as a ZZ fusion protein the gene has to be inserted into the MCS in the correct reading frame. To allow effective selection of only those host cells which carry the expression plasmid we have chosen the gene encoding KmR of the pUK21 plasmid (Vieira and Messing, 1991) as a selective marker for our pExSec1 expression vector. In addition the pExSec1 vector contains the Ml3 origin of replication and the M 13 intergenic region from pUK21 allowing the production of phagemid single-stranded DNA. (b) Engineeering of phr3 cDNA for expression The phr3 cDNA of Eu. octocarinatus contains three TGA triplets which do not function as stop codons but are translated as Cys (Meyer et al., 1991). To allow heterologous expression in E. co/i these triplets had to be changed to TGCs coding for Cys according to the universal genetic code. The three point mutations were introduced into the phr3 cDNA by the use of the overlap

extension method (Ho et al., 1989; Fig. 2). Six specific oligo primers (Pl-P6) were designed based on the published sequence of the phr3 cDNA (Meyer et al., 1991). Pl and P6, carrying restriction sites for EcoRI and BamHI at their 5’ ends, flank the cDNA, while P2-P5 are internal primers each of them containing one point mutation. In the primary PCR with Pl/P2, P3/P4 and P5/P6, respectively, three phr3 fragments were amplified, showing one (Pl/P2, P5/P6) or two (P3/P4) mutations and overlapping ends. The PCR products were gelpurified and used for a second PCR with the primers Pl and P6. The resulting fusion product was cleaved with EcoRI + BumHI and cloned into the pT7T3-19U vector. By sequencing both strands of the double-stranded insert (Sanger et al., 1977) we showed that the point mutations were set successfully and that the mutated version of phr3 cDNA (phr3 cm) contained no PCR-based replication errors. To omit the phr3 leader sequence part of phr3 cm was amplified with primers P6 and P7 (Fig. 2). P7 contains the first 16 nt of the sequence corresponding to secreted Phr3 preceded by the codons for Asn-Gly and an EcoRI site. The Asn-Gly motif allows cleavage of the corresponding fusion protein with hydroxylamine. The PCR product (phr3sm) was gel-purified, digested with EcoRI $BamHI and ligated into the EcoRI + BamHIcleaved pExSec1 vector. The resulting construct was transformed into the T7 expression hosts E. coli BL21(DE3), BL21 (DE3)[ pLysS] and BL21(DE3)[pLysE] (Studier, 1991). (c) Production of the ZZ::Phr3 fusion protein For the production of the ZZ::Phr3 fusion protein E. coli strain BL21(DE3)[pLysE] transformed with pExSec1 was grown to an Aeoonm of 0.6-l and induced with IPTG for 1 to 4 h. To determine the fate of the ZZ::Phr3 fusion, proteins from culture medium, periplasmic fraction and spheroplasts of uninduced (TO) and induced cultures (Tl-T4) were analysed by SDS-PAGE (Fig. 3). After 1 h induction the fusion protein appeared in the spheroplasts and was secreted into the periplasm from where it was probably leaking into the culture medium. After 3 h induction most of ZZ::Phr3 was found in the culture medium, while the periplasmic fraction and the spheroplasts contained only small amounts of it. The fusion protein located in the spheroplasts migrates with an apparent molecular mass (about 36 kDa) which is slightly higher than that of the fusion protein found in the periplasmic fraction or the culture medium (about 32 kDa). This indicates that the spheroplast-associated form of ZZ::Phr3 containing the 36-aa-long signal sequence of protein A is probably correctly processed and that the secretion of the fusion protein into the medium

189

F’CRwith SZZ-primers: 5’

Purification of

271-bp fragment

primer:

(

S"CAGGGGGGATCCATAn;AAAAAGAAAAACATT 3' primer: I S-GG'KGAC?CTAGAGGAT

1

BarnHI /Ndel

BamHl

modified SZZsequence

q q q q

l7 regulatory

EJ

lx/spa promoter

q

Ml3 intergenic region Ml3 origin of replication Ndei L?amHl

K~R sequences

signal sequence and ZZ sequence of protein A

ligate

I

Mcs multiple cloning site

Fig. 1. Construction of the expression-secretion vector, pExSec1. Enzymes used for DNA manipulation were obtained from Boehringer-Mannheim (Mannheim, Germany), NE Biolabs (Schwalbach, Germany) or Gibco BRL (Eggenstein, Germany). The pUK21 plasmid was a gift from .I. Messing (Rutgers University, Piscatatway, NJ, USA). The pET3b vector and the E. coli hosts BL21(DE3), BLZl(DE3)[pLysS] and BL21(DE3)[pLysE] were from Novagen (Madison, WI, USA) and the pEZZl8 vector was from Pharmacia (Freiburg, Germany). The E. coli strain DHSa (Gibco BRL) was chosen for propagation of the intermediate products during construction. PCR (Saiki et al., 1988) was carried out using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) according to the instructions of the manufacturer. Recognition sites in the PCR primers for the restriction enzymes BamHI/NdeI (5’ primer) and BamHl (3’ primer) are underlined. Methods: Manipulations of DNA followed standard procedures (Sambrook et al., 1989). The 271-bp BglII-EcoRV fragment of pET3b (Studier et al., 1990) carrying the T7 promoter, the RBS, NdeI and BamHI sites and the T7 terminator region was treated with Pollk, gel-purified and cloned between the two PuuII sites of pUK21 (Vieira and Messing, 1991). The resulting plasmid pUKT7 was cleaved with NdeI + BamHI. The DNA encoding the signal sequence and the ZZ fragment of protein A of Staphylococcus aureus, including part of MCS. was amplified from pEZZ18 (Pharmacia). Two PCR primers were synthesized to obtain the SZZ sequence. The 5’ primer contained a BumHI and Ndel site preceding the start triplet of the protein A signal sequence, which was changed from a TTG, often used by Gram+ bacteria such as S. aureus, to an ATG, the typical start codon of E. coli. The 3’ primer hybridized to a part of MCS of pEZZl8, including the sites for BarnHI, XbaI and SalI. The resulting PCR product was gel-purified, digested with BamHI and cloned into pT7T3-19U (Pharmacia). By sequencing both strands of the double-stranded SZZ sequence according to the method of Sanger et al. (1977) we proved that the amplified DNA contained no PCR artefacts. The insert was excised with NdeI+ EamHI and cloned into NdeI+BamHI-cleaved pUKT7 resulting in the expression-secretion vector pExSec1.

190 TGA TGA I I I leader sequence of phr3 cDNA

TGA I

A

y///1 downstream region of phr3 cDNA

cDNA sequence corresponding to secreted Phr3

M

TO

Tl

T2

T3

T4

M

TO

Tl

T2

T3

T4

M TO m. .LW

Tl

T2

T3

T4

kDa 66 45 -

primary PCR

36 1 -%

29 r;: + P4

Bf * P3

2

20 102 bp

269 bp 169 bp overlap

extension

4 TGC TGC

PCR

B

TGC

kDa mutated

version

496 bp of phr3 cDNA (phr3cm)

66 45 36 -

engineering of the S-end of phr3cm with PCR

P7

i

P6

29 r 347 bp mutated version of the cDNA sequence corresponding

P 1: P2 : P3: P4: P5 : P6 : P7 :

to secreted

20 -

Phr3 (phr3sm)

5’-CGGAATKATGAAAGCCA’17TTCATTA’MTTAGCCATCCT 5’-GTClTGA-fCAl-TTCCAGTTACGCTgCAATCG 5’-AACTGGAAATGATCAAGACAAATGcAATAAT 5’-TTCTGGATTTGAgCATCCACAAAAT 5’-ATTfTGTGGATGcACAAATCCAGAA S~ATW’l-TACTTGTCTGTCTGAAG 5’-GCGAATTCGAAlXXXl-ATTATTGTTGGGAAG

Fig. 2. Mutagenesis of phr3 cDNA by PCR. PCR (Saiki et al., 1988) was carried out using Taq DNA polymerase (Gibco BRL) according to the instructions of the manufacturer. Oligo primers are represented by the arrows adjacent to their annealing sites in the target DNA. Recognition sites for the restriction enzymes EcoRI (Pl, P7) and BarnHI (P6) are underlined. The mismatches in the primers are shown as lower case letters. The sequence of P7 coding for Asn-Gly is written in bold letters. For the primary PCR 10 ng of plasmid DNA isolated from a phr3 cDNA clone were used as template. The phi-3 cDNA was obtained from a cDNA library constructed from poly(A)+RNA of Euplotes cells that were homozygous for the phr3 gene and was cloned into pUC12 (Meyer et al., 1991).

is not due to the cell lysis. Localization studies presented by Abrahmstn et al. (1985) revealed that the expression of protein A fragments induces changes in the morphology of the E. coli host cells, resulting in filamentous growth and a more ‘leaky’ outer membrane. We assume that a similar phenomenon is responsible for the secretion of the ZZ::Phr3 fusion protein from the periplasmic space to the culture medium.

C kDa 66 45 36 29 -

20 -

Fig. 3. Distribution of the ZZ:Phr3 fusion protein in the medium (A), periplasmic space (B) and spheroplasts (C) of transformed host cells before (TO) and l-4 h after induction (Tl-T4). Lane M contains protein markers. Proteins were resolved on 0.1% SDS-12% PAGE under reducing

conditions

(Laemmli,

1970)

and

stained

with

Coomassie

Methods Transformed host cells of E. coli strain BL21(DE3)[pLysE] were grown in terrific broth (Tartof and Hobbs,

brilliant

blue.

1987) at 37°C with 80 ug Km/ml to an A600 nm of 0.6-l and induced with 0.4 mM IPTG (final concentration). The cells were separated from the medium by centrifugation, the periplasmic fraction was obtained by cold osmotic spheroplasts were

shock (Bosse et al., 1993) and the remaining suspended in SDS-lysis buffer (50 mM TrivHCI

191 1

M

kDa 97-

3

2

4

5

w

-

(e) Biological activity and refolding of ZZzPhr3

14 -

.-

Fig. 4. Purification Proteins

gene in E. coli strain BL21(DE3)[pLysS] and especially in strain BL21(DE3)[pLysE] by T7 lysozyme (Studier, 1991) leads to an increase in the yield of the fusion protein. This effect could be due to an overload of the translation machinery at high transcription rates, but it is also possible that the translocation machinery is working at full capacity in strain BL21(DE3)[pLysE] and that the secretory pathway is overloaded at higher expression levels.

M

and

were resolved

refolding

(lanes 1 and 2) or non-reducing and stained

of the

on a 0.1%

ZZ::Phr3

SDS-12%

PAGE

(lanes 3-5) conditions

with Coomassie

brilliant

1, affinity purification of the culture Flow (Pharmacia); 2, fusion protein

fusion

protein.

under

reducing

(Laemmli,

blue. Lanes: M, protein

1970)

markers;

medium on IgG SepharoseR 6 Fast gel purified on Superdex 75 FPLC

(Pharmacia); 3, fusion protein gel purified on Superdex 75 FPLC (in contrast to 2 SDS-PAGE under non-reducing conditions); 4, fusion protein

treated

Biomedicals, Superdex

with

PDI

(homodimer

of

Japan);

5, refolded

ZZ::Phr3

Methods:

Transformed

host cells of E. cali strain

Kyoto,

75 FPLC.

BLZl(DE3)[pLysE]

were grown

in terrific broth

57 kDa)

(TaKaRa

gel-purified

(Tartof

on

and Hobbs,

1987) at 37°C with 80 pg Km/ml to an AGO,,nm of 0.6-l and induced with 0.4 mM IPTG (final concentration) for 3 h. The cells were separated

from the medium

by centrifugation.

For affinity

purification

of

the fusion protein 8 ml of IgG Sepharose (Pharmacia) were added to 1 litre of cell-free culture medium. After mixing for l-2 h the IgG Sepharose

was sedimented,

washed

extensively

with the equilibration

buffer (50 mM Tris.HCI pH 7.6/150 mM NaCI) and filled into a column. The column was washed with 5 mM ammonium acetate pH 5.0 and bound proteins were eluted with 0.5 M acetic acid pH 3.4.

(d) Purification of the ZZxPhr3 fusion protein

The synthetic ZZ domain of protein A is tightly bound by immunoglobulin G (IgG), allowing affinity purification of the secreted fusion protein on IgG Sepharose. SDS-PAGE of the resulting proteins showed a major band (32 kDa), representing the full-length ZZ::Phr3 fusion protein, faint bands at about 60 kDa and faster migrating bands probably representing degradation products (Fig. 4, lane 1). Two-step gel filtration of this fraction yielded fusion protein almost electrophoretically pure (Fig. 4, lane 2). We obtained 1 mg, 2 mg and 2.5 mg of purified ZZ::Phr3 per liter of the culture using the E. coli strains BL21 (DE3), BL21 (DE3)[ pLysS] and BL21(DE3)[pLysE], respectively. This demonstrates that the reduction of the transcription level of the target

pH 6.8/100 mM dithiothreitol/2% SDS/IO% glycerol). Fusion proteins in the periplasmic and medium samples were enriched by affinity purification with 0.5 ml IgG Sepharose (Pharmacia) per 100 ml culture according to the instructions of the manufacturer (batch procedure).

The biological activity of the ZZ::Phr3 fusion protein was determined by comparing its ability to induce cells of Eu. octocarinatus to form conjugation pairs with that of Phr3 isolated from Eu. octocarinatus cultures (Schulze Dieckhoff et al., 1987). We tested cells of the four mating types homozygous at their mating-type locus: VII (mt’mt’), VIII (mt’mt’), IX (mt3mt3) and X (mt4mt4). The reaction patterns were the same as those for native Phr3, i.e., ZZ::Phr3 induced conjugation in cells of all mating types but type IX. Phr3 isolated from Eu. octocarinutus cultures was active in concentrations as low as lo-” M, while the activity of ZZ::Phr3 was tenfold lower. As Phr3 is a secretory protein containing disulfide bonds, it was tested whether the reduced biological activity of ZZ::Phr3 resulted from lacking or incorrect disulfide bond formation. SDS-PAGE of the fusion protein under non-reducing conditions showed that the 32-kDa band of the fusion protein observed under reducing conditions separated into several faster migrating bands (Fig. 4, lane 3). These bands probably represent different conformations of the fusion protein with varying numbers of disulfide bonds indicating that the efficiency of proper disulfide bond formation in the eukaryotic protein synthesized in E. coli is reduced. Although protein folding and correct disulfide bond formation are favoured in the oxidative environment outside the cytoplasm of E. coli, secretion of eukaryotic proteins with correct disulfide bonds is more difficult than the export of native bacterial exoproteins (Stader and Silhavy, 1990). Until now only a few eukaryotic proteins containing disulfide bonds were shown to be exported from E. coli as correctly folded, biologically active molecules, as demonstrated for human insulin-like growth factor (Moks et al., 1987), bovine pancreatic trypsin inhibitor (Nilsson and AbrahmsCn, 1990), human epidermal growth factor (Oka et al., 1985), human growth hormone (Hsiung et al., 1986) and Fab fragments (Better et al., 1988). Differences in the structure and oxidizing properties of the prokaryotic and eukaryotic catalysts for disulfide-linked protein folding, DsbA and PDI (Bardwell and Beckwith, 1993), may account for this phenomenon.

192 PDI is directly involved in disulfide bond formation and isomerization of disulfides in eukaryotes (LaMantia and Lennarz, 1993) and is especially useful when working with recombinant proteins that lack the tertiary structure of the native protein. To improve the biological activity the ZZ::Phr3 fusion protein was refolded with PDI (Hillson et al., 1984) and analysed by non-reducing SDSPAGE (Fig. 4, lane 4). Two bands could be detected corresponding to PDI and ZZ::Phr3 which now shows the same migration pattern as the fastest migrating band of the untreated probe. After separating the PDT from the fusion protein by FPLC gel filtration the biological activity of the purified, refolded fusion protein (Fig. 4, lane 5) was tested. It exhibited the same specificity and activity as native Phr3, i.e., it was active in concentrations as low as 10-r’ M.

cob secretion

(I) We have constructed a novel vector, pExSec1, to combine the advantages of the T7 expression system developed by Studier et al. (1990) and the protein A gene fusion system developed by Nilsson and Abrahmsen (1990). The expression of the ZZ fusion protein is controlled by the very strong and inducible T7 promoter. The KmR marker allows effective selection of only those host cells which carry the expression plasmid. (2) The pExSec1 vector was used for the expression of phr3 of Eu. octocarinatus. The produced ZZ::Phr3 fusion protein was secreted to the periplasm from where it was leaking into the medium. It was effectively purified from the culture medium by affinity chromatography and gel filtration. (3) The ZZ::Phr3 fusion protein was refolded with PDI because part of it lacked the correct tertiary structure. After the refolding reaction the fusion protein exhibited the same biological activity as the native pheromone.

Heckmann,

K. and Kuhlmann,

ing substances

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