VIROLOGY

182, 513-521

Expression

(1991)

of Human Papillomavirus

Proteins

in Yeast Saccharomyces

Cerevisiae

JOSEPH J. CARTER,* NOBUO YAEGASHI,* STEVEN A. JENISON,* AND DENISE A. GALLOWAY*+’ *Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, and tDepartment of Pathology, University of Washington, Seattle, 98 195 Received November

28, 1990; accepted

February 5, 199 1

The Ll and L2 proteins of human papillomavirus (HPV) types 1,6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 Ll and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-a-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes. o ISSI Academic

Press.

Inc.

INTRODUCTION

of virions. Virus production in the natural infection is low and no suitable tissue culture system has been established to replicate virus. Studies on the human immune response have therefore relied on bacterial fusion proteins and synthetic peptides (Firzlaff et al., 1987, 1988; Li et al., 1987; Jenison et a/., 1988, 1990; Dillner et a/., 1989, Jochmus-Kudielka et al., 1989). These studies have demonstrated that the major and minor virion proteins of HPV 6 and 16 are the proteins most frequently recognized by immune human sera (Galloway and Jenison, 1990; Jenison et a/., 1990). One difficulty encountered when bacterial fusion proteins or peptides were used for serologic studies was that antibody reactivity to epitopes dependent upon the native conformation of the protein were not detected. It has been suggested that most human antibody responses to HPV type 1 may be restricted to the native form of the virion protein (Steele and Gallimore, 1990). To address the issue of the role of conformational epitopes and to alleviate other problems associated with bacterial fusion proteins, we have chosen to express HPV proteins in yeast. Here we describe the production of the proteins encoded by HPV 1, 6b, and 16 late ORFs and the HPV 16 E6 and E7 early proteins in Saccharomyces cerevisiae. Several HPV proteins were also produced in a soluble form by secretion from yeast by fusion with the yeast pre-pro-a-factor leader sequence.

There are currently more than 60 genotypes of human papillomaviruses (HPVs) recognized. The genotypes are quite divergent on the nucleotide level, however all have similar genetic organization (zur Hausen and Schneider, 1987). All are epitheliotropic with virus replication restricted to specific anatomical sites (Pfister and Fuchs, 1987). Most HPVs have been identified as causing benign epithelial tumors; however, several types have been associated with premalignant and malignant conditions (see, for review Koutsky et a/., 1988). Of the HPVs which infect the genital tract, types 6 and 11 are associated with benign condylomas whereas HPV types 16 and 18 have been associated with cancers and precancerous lesions. The HPV virion is composed of a circular double stranded DNA molecule encased in an icosahedral capsid. The principle protein components of the virion are two virus encoded proteins. The major capsid protein has a molecular weight of 55-60K and is encoded by open reading frame (ORF) Ll (Roseto et al., 1984; Doorbar and Gallimore, 1987; Tomita et a/., 1987). The minor capsid protein, of which there is substantially less, is encoded by the L2 ORF (Komly et a/., 1986; Doorbar and Gallimore, 1987). The predicted molecular weight of the L2 protein is 50-55K whereas the apparent molecular weight is 75-l OOK as determined by polyacrylamide gel electrophoresis. Immunological studies on the genital HPV types have been hindered by the lack of sufficient quantities ’ To whom correspondence dressed.

and reprint requests

METHODS Cloning strategy DNA fragments encoding the HPV ORFs were generated either by polymerase chain reaction (PCR) am-

should be ad-

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0042.6822/91

$3.00

CopyrIght 0 1991 by Academic Press. Inc. All rights of reproduction I” any form reserved.

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CARTER ET AL.

plification or by restriction enzyme digestion. The fragments were initially cloned into pUC19 or PATH 1 1. pATHl1 was a gift of T. J. Koerner (American Cancer Society, Atlanta, GA). PATH vectors contain 5’ transcription control elements and a portion of the first structural gene (trpE) of Escherichia co/i tryptophan synthetase operon. All constructs were engineered to contain a Sal1 site 3’ to the termination codon and a Ncol site or another restriction enzyme recognition sequence which yielded a compatible 5’ overhang. The fragments were inserted into NcollSall-digested pBS1OO.l or pa100 (described below). The plasmid pBSlOO.1 contains the alcohol dehydrogenase 2/ glyceraldehyde phosphate dehydrogenase (ADH2/ GAPDH) promoter upstream of a unique Ncol site and the glyceraldehyde phosphate dehydrogenase termination sequence downstream of a unique SalI site (Barr et al., 1987). The HPV ORFs together with the yeast promoter from pBSlOO.1 were then inserted as a BarnHI-Sal1 fragment into pBS24.1, a 2~ based plasmid which contains the Leu2 and Ura3 selectable genes (Barr et a/., 1987). Plasmids pBS100.1 and pBS24.1 were kindly provided by Dr. Philip J. Barr (Chiron Corp., Emeryville, CA). The S. cerevisiae strain used for these studies was BJ2168 which is Ura3-, Leu2- and protease deficient (Jones, 1990). Descriptions

of constructs

To express HPV 6 Ll, the pUCl9 polylinker was modified by the addition of a Ncol linker at the EcoRl site and removal of the BamHl site with SI nuclease (Boehringer Mannheim, Indianapolis, IN). A XballAccl fragment (5903-7815) from HPV 6b (de Villiers et al., 1981, Schwarz et a/., 1983) was initially inserted into the modified pUC19 plasmid. HPV 6 L2 was constructed by inserting a Ncol-BarnHI fragment (44214722) into pUCl9 which previously had a Ncol site added at the EcoRl site. The BarnHI-Xbal fragment (4722-5903) was inserted into the same pUCl9 plasmid. The HPV 16 Ll ORF was reconstructed in pUCl9 when the Avall-BamHI (5472-6151) and the BamHIEcoRl (partial digest) (6 15 l-7454) fragments of pHPV 16 (Durst et a/., 1983; Seedorf et al., 1985) were inserted into the same plasmid. lvcol linkers were added at the 5’ end but destroyed after cloning into pBS100.1 to allow translation initiation to occur at the authentic initiation codon. PCR directed

cloning

For amplification of the yeast pre-pro-a-factor leader sequence (Kurjan and Herskowitz, 1982) a 5’ primer was synthesized which converted the sequence surrounding the first ATG into a BspHl site. The 3’

primer was homologous with the a-factor sequence until nucleotide 256, followed by 16 nucleotides which included Ncol and Sal1 recognition sequences. The 16 nucleotides also included an alanine codon 5’ to the Ncol site. The amplimer was cut with BspHl and Sal1 and inserted into NcollSall-digested pBS100.1. The resulting plasmid was named pal 00. To clone the HPV ORFs the primers contained 20 nucleotides homologous with target sequences, and all 3’ primers contained SalI recognition sequences adjacent to the ORF termination sequence. The template for HPV la amplification was the plasmid KLuG28 (Danos et a/., 1982). The template for HPV 16 amplification was pHPV 16 (Durst et a/., 1983; Seedorf et al., 1985). The HPV 1 Ll oligonucleotide upstream primer included EcoRl and Ncol recognition sequences. The HPV 1 L2 5’ primer included EcoRl and Afllll recognition sequences. The HPV 16 L2 5’ primer contained an EcoRl site and modified the sequence encoding the first methionine (nt 4236) to create a &HI recognition site. HPV 16 E6 5’ primer had a Fokl recognition sequence 9 nucleotides 5’ of the presumed translation initiation site (nt 104) and nucleotide 103 was converted from an A to a C so that Fokl treatment would yield a Ncol compatible overhang. The HPV 16E7 synthetic primers provided an EcoRl in the upstream primer. The PCR was performed in buffer containing 50 mM KCI, 10 mM Tris (pH 8.3) 2.5 mM MgCI,, 10 ng target DNA for plasmid or 100 ng for yeast cellular DNA, 250 PLM primer, 150 pM dNTP’s, and 5 U Thermus aquaticus polymerase (Taq polymerase; Perkin-Elmer Cetus, Norwalk, CT) in 100 ~1 total volume. Cycling was controlled by a thermal cycler using a standard program (Perkin-Elmer Cetus). Following PCR the HPV 1 and HPV 16 E7 amplimers were digested with appropriate enzymes, inserted into pATH1 1 and screened for fusion protein expression. Subsequently the HPV sequences were cloned into pBSlOO.1 and/or pal 00. To clone HPV 16 E7 the pBS1OO.l and pal 00 plasmids were modified by digesting with Ncol, filling with Klenow polymerase (Promega Corp., Madison, WI) and religating (converting the Ncol sites into Nsil sites). The E7 sequence cloned into these vectors was the Nsil (nt 562 in HPV 16)lSall fragment (from the PATH 1 1 construct). The HPV 16 L2 amplimer was digested with appropriate restriction enzymes and cloned into pUC19 prior to cloning into pBS100.1 and palOO. The HPV 16 E6 amplimer was cloned directly into pBSlOO.1 and pal 00. Analysis

of proteins

and glycoproteins

Yeast cultures were grown in leucine-deficient dium containing 8% glucose. Protein expression

mewas

HPV PROTEINS EXPRESSED IN YEAST

induced by diluting the culture 1:25 in YEP (1% yeast extract, 29/o Peptone) with 1% glucose, and the cultures harvested 24 hr later as described by Barr et al. (1987). For intracellular constructs the yeast were pelleted, washed and disrupted by mechanical lysis using glass beads in a buffer containing 50 mMTris (pH 8.0) 1 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and 0.1 O/O Triton X-l 00. For secreted HPV 6 and 16 L2 proteins the yeast were removed by centrifugation and the proteins concentrated approximately lo-fold in an ultrafiltration cell (Amicon, Danvers, MA) using a Diaflo PM30 membrane (Amicon). Proteins were resolved on 10% SDS-polyacrylamide gels and molecular weight determinations were made by comparison with prestained molecular weight markers (Bethesda Research Laboratories Life Science Technologies, Gaithersburg, MD). For immunoblots, mouse monoclonal tissue culture media were diluted 1:200, rabbit antisera were diluted 1:5000, and human sera were diluted 1:200. The DAK0 anti-BPVl antiserum (DAK0 Corp., Santa Barbara, CA) was diluted 1 :lOOO prior to use. The alkaline phosphatase conjugated secondary antibodies (Boehringer Mannheim) were diluted 1: 1000 prior to use. The Mabs were generously provided by David A. Baker and Lorne Taichman (SUNY at Stony Brook). Western blot (immunoblot) assay was conducted as previously described (Jenison et a/., 1988). To test for glycosylation, the concentrated protein samples were treated with peptide-N4-(N-acetyl-figlucosaminyl)asparagine amidase F (PNGase F) (Genzyme, Boston, MA) as described by the manufacturer. Samples were incubated overnight at 37” and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immune blotting. Purification

of HPV 16 E7

Yeast containing the HPV 16 E7 secretion plasmid were grown in 400 ml media and induced as described above. Following removal of the cells by centrifugation an ammonium sulfate cut of 35-650/o was taken and dissolved in 10 mMTris (pH 8.0) 1 ml\/l EDTA, with 50 mh/l NaCI. The sample was dialyzed against 1 liter of the same buffer four times for a minimum of 4 hr each. The sample was applied to a DEAE sephadex (Sigma Chemical Co., St. Louis, MO) column (5 ml) and washed with 50 ml of the same buffer containing 300 mM NaCI. The 16E7 protein was eluted by washing with 700 mM NaCl and reconcentrated by ultrafiltration as described above using a PM 10 membrane (Amicon). Radiolabeling

and immune precipitation

of HPV 16E7 Yeast were cultured in 5 ml of leucine-free medium containing 8% glucose at 30” overnight. The yeast

515

cells were then transferred into 5 ml of YEP medium containing 2% ethanol and were incubated at 30” for 2 hr. The cultures were transferred to 5 ml of methionine-free minimal medium containing 2.5% ethanol and 0.25 mCi of [35S]methionine (Tran35S-label, 10 mCi/ml; ICN Radiochemicals, Irvine, CA) and were incubated at 30” for 30 min. Immunoprecipitation, SDSPAGE, and autoradiography were performed as described previously (Jenison et al., 1990) with the following modifications: the cells were lysed by mechanical disruption in lysis buffer and the lysates were precleared by incubation with 150 ~1 of Staphylococcus aureus cell suspension (Sigma) for 1 hr. Additionally, the blocking agent used to reduce nonspecific binding was made with a 50 ml culture of yeast containing pBS24.1.

Enzyme-linked immunosorbant assay The concentrated secreted 16L2 sample was diluted 1 :lOO in 0.1 lvI carbonate buffer (pH 9.6). Where indicated, the sample was made 0.1% SDS and the samples boiled for 5 min. Protein samples were incubated in the wells of an lmmulon 2 plate (Dynatech Laboratories, Chantilly, VA) at 100 PI/well. Control wells received an equal volume of concentrated culture media from cells containing the pBS24.1 vector. The plates were incubated overnight at 4” with rocking. The plates were washed, blocked, antibodies applied and the plates developed as described previously (Jenison et al., 1990).

RESULTS Cloning strategy and expression of HPV late proteins in yeast The HPV ORF sequences were cloned by restriction digests or PCR-directed cloning into pUCl9 or PATH 1 1. Figure 1 outlines the cloning strategy for HPV ORFs initially cloned into pUC19. The HPV sequences were inserted adjacent to the glucose repressible ADH2/GAPDH hybrid promoter in the pBS100.1 plasmid. The HPV late ORFs with the yeast promoters were cloned into the yeast expression plasmid pBS24.1. The HPV 1 Ll and L2, the HPV 6b L2, and the HPV 16 Ll and L2 ORFs expressed in yeast were predicted to encode the entire ORF encoded proteins assuming that initiation occurred at the first ATG or in the case of HPV 1 Ll at the second ATG (the presumed start of translation (Danos et al., 1983)). The HPV 6b Ll sequence was predicted to encode the 461 C-terminal amino acids of the 500 amino acid protein. The first 39 amino acids from the N-terminal were deleted and replaced with 9 amino acids of vector encoded se-

516

CARTER ET AL. NcoI

Cut with BornHI

+ Sal1

Cut with BamHI + Sal1 Isolate fragment

FIG. 1. Strategy for cloning HPV ORFs into yeast expression vectors HPV ORF sequences were cloned by PCR or restriction enzyme digests into pUC19 where DNA sequences could be modified as necessary. The ORFs were excised from pORF. 19 and inserted into pBS100.1 so that the ORF was in frame with the initiation methionine being encoded by the Ncol recognition sequence. This placed the ORF adjacent to the ADH2/GAPDH hybrid promoter. The ORF with the promoter was excised and inserted into the 2~ based yeast vector.

quence (MGIRARYPG). Table 1 summarizes the HPV sequences contained in the yeast expression vectors. To compare the proteins expressed in yeast with HPV 1 virion proteins, yeast lysates from cells containing expression vectors, bacterial lysates from bacteria expressing HPV 1 Ll and L2 trpE fusion proteins and purified HPV 1 virions were run on polyacrylamide gels and immune blots prepared. The monoclonal antibodies used in these experiments had been generated to purified HPV 1 virus and have been characterized previously (Yaegashi er al,, 1991). Figure 2A is a Coomassie-stained gel of purified virion proteins, total cell lysates from yeast containing the pY1 Ll, pY1 L2, and pBS24.1 expression vectors, and insoluble preparations from f. co/i expressing trpE fusion proteins 1 Ll and 1L2. The yeast 1Ll and 1 L2 proteins could not be detected by Coomassie blue staining of crude lysates. Identical gels were transferred to nitrocellulose and reacted with the DAK0 polyclonal anti-papillomavirus serum (Fig. 2B) or the Mabs, DW45 (Fig. 2C), or W275

(Fig. 2D), which have previously been shown to be reactive with HPV 1 Ll and L2 polypeptides, respectively (Yaegashi et a/., 1991). Figures 2B and 2C showed similar patterns of reactivity in that both reagents recognized proteins of the appropriate size to be the HPV majorvirion protein. In Fig. 20 only proteins in the virion yeast containing pY1 L2 and bacterial trpE1 L2 lanes reacted. The immunoreactive proteins produced in yeast were identical in size with virion proteins. There were, however, several smaller bands recognized by anti-l Ll antibodies in the virion lanes which may be proteolytic fragments. The bacterially derived proteins, although abundant, were largely degraded. The HPV 1 Ll proteins migrated at the size predicted from the coding sequence (57K). The HPV 1 L2 protein migrated more slowly than predicted by sequence analysis, 92K apparent molecular weight compared to the predicted 55K. The HPV 6 and 16 Ll and L2 ORFs were cloned into the yeast expression plasmid. Figure 3 is a series of immunoblots of lysates from yeast transformed with plasmids containing the HPV ORFs. The primary antisera were generated in rabbits by immunization with HPV ORF-trpE bacterial fusion proteins (Firzlaff et al., 1987). There was a large variation in the quantities of the proteins detected products. This variation was principly due to differences in the levels of proteins expressed by the yeast and not due to differences in antibody titer which had been previously determined (not shown). There was also a wide variation in the integrity of the products with some proteins migrating as single bands (HPV 6 Ll and HPV 16 Ll) and others having multiple bands (HPV 6 and 16 L2). The high molecular weight band seen in yeast expressing HPV TABLE 1 HPV

Viral Type

ORF

HPV la

Ll L2 Ll L2 L2 E6 E6 E7 E7 Ll L2 L2

HPV 6b

HPV 16

YEAST

EXPRESSION

PLASMIDS

HPV coordinants

Restriction sites’

5431-6939 3897-5402 5903-7815 4421-5903

Ncol-Sal1 Afilll-Sal1 Ncol-Sal1

144-556

Fokl-Sal1

562-856

Nsil-Sal1

5472-7454 4240-5744

Ncol-Sal1 BspHI-Sal1

Expression constructs pYlL1 pY1 L2 pY6Ll pY6L2 paY6L2 pY16E6 porY16E6 pY16E7 pcuY16E7 pYl6Ll pY16L2 paY16L2

a All restriction sites were added by PCR cloning or with synthetic linkers, with the exception of the HPV 6b L2 Ncol and the HPV 16 E7 Nsil sites.

517

PROTEINS EXPRESSED IN YEAST

26-

_\e

FIG. 2. Analysis of HPV 1 virion proteins and HPV 1 ORF sequences expressed in yeast. (A) Coomassie blue-stained gel of purified virion, prestained molecular weight markers, total cell lysates from yeast containrng the pY1 Ll , pY1 L2, and pBS24.1 vectors, and insoluble fractions from bacteria containing the plLlBB1 or pl L2SBl fusion protein expression plasmids. The arrows point out the major and minor virion proterns and the 1Ll and lL2 fusion proteins. Replicate blots were transfered and immunoblotted using anti-BPVl (B), anti-HPV 1 Ll Mab (C), or anti-HPV 1 L2 Mab (D).

16 Ll (Fig. 3c) not consistently weights of the HPV 6 Ll , 55K characteristic HPV 16 L2.

was likely due to dimerization and was observed. The apparent molecular yeast expressed proteins were 50K for for HPV 16 Ll , 82K for HPV 6 L2 with a smaller product of 70K, and 1OOK for

Cloning and expression secreted proteins

pressed in yeast, the proteins would be secreted into the medium and the leader would be cleaved. When HPV 6 Ll was expressed in this manner all of the protein remained intracellular (not shown). When the HPV 16 Ll ORF was cloned into the secretion vector by PCR-directed cloning it was secreted; however, the amount of protein expressed was barely detectable by immune blotting (not shown). When HPV 6 and HPV 16 L2 ORFs were expressed via the secretion vector the levels of protein were readily detected in the supernatant after 5- to 1O-fold concentration by ultrafiltration (Fig. 4). lmmunoblot analysis of the culture medium from yeast containing the paY6L2 identified a protein with an apparent molecular weight of about 1OOK (Fig. 4a, lane 1) which was larger than that of the intercellular product expressed by yeast containing the pY6L2 plasmid (Fig. 4a, lane 2). The larger size was not due to N-linked carbohydrate because PNGase F, which deglycosylates asparagine-linked glycans (Tarentino et a/., 1985), had no effect on migration (not shown). Treatment of HPV 16 L2 with PNGase F reduced the apparent molecular weight from 130-l 50K (Fig. 4b, lane 1) to approximately 1OOK(lane 2), the same size as the largest intracellular product (lane 4, faintly visible).

a

1234

C

1234

b

1234

d 1234

of HPV late ORFs as

In order to allow for simplified purification and to avoid proteolysis, the HPV ORFs were subcloned into a yeast secretion plasmid. The secretion constructs were made by modifying the pBS100.1 plasmid so that sequences cloned into the unique NcollSall site would result in a protein coding sequence fused to the yeast pre-pro-a-factor leader sequence. Based on previous studies (Brake et a/., 1984) it was predicted that when protein sequences were fused in this manner and ex-

FIG. 3. lmmunoblots of HPV 6 and HPV 16 late ORF proteins expressed in yeast. Polyclonal rabbit antibodies raised against bacterially expressed fusion proteins containing HPV 6 Ll (a), HPV 6 L2 (b), HPV 16 Ll (c), and HPV 16 L2 (d) were used for immune detection. The proteins were from yeast transformed with pY16L2 (1) pY 16Ll (2) pY6L2 (3) or pY6Ll (4).

518

CARTER ET AL.

b

Cl

b

0 1

2

3

1

2

3

-196-

-106-

FIG. 4. lmmunoblot analysis of secreted HPV proteins. (a) The HPV 6 L2 proteins from yeast containing the pYa6L2 (lane 1) or pY6L2 plasmids (lane 2) were analyzed by immunoblot with rabbit anti-HPV 6 L2 sera. (b) HPV 16 L2 proteins were analyzed by immunoblot using rabbit antisera. The lanes are: supernatant from yeast containing pYa16L2 (1) the same preparation treated with PNGase F (2) the supernatant from yeast transformed with pYal6Ll (3) cellular lysate from yeast containing pY16L2 (4) and cellular lysate from yeast containing pY16Ll (5).

Expression of HPV E6 and E7 in yeast The entire HPV 16 E6 and E7 protein coding sequences were cloned into the yeast expression vector to be expressed either as intracellular or secreted products. Figure 5 shows the protein products from these constructions. Figure 5a is a Western blot of 16E6 us-

a

b 1

2

3

C

4

FIG. 6. Immune recognition of yeast expressed HPV proteins by human sera. Protein from yeast transformed with vectors containing no insert (1) pY6Ll (2) or pY6L2 (3) were reacted with a human serum. In (b) the serum was preabsorbed with bacterially derived HPV GLl-trpE fusion protein. (a) is the same serum nonabsorbed.

ing immune rabbit sera. The 16E6 protein was readily detected from the intracellular expression construct (Fig. 5a, lane 2) and was not detected in concentrated medium from the yeast containing the secretion vector (Fig. 5a, lane 3). The 16E6 protein was not detected by Coomassie blue staining in whole cell lysates (not shown). Figure 5b compares the 16E7 product produced intracellularly and the partially purified protein from yeast secreting 16E7. The intracellular product (Fig. 5b, lane 1) is indistinguishable in size from the secreted 16E7 (Fig. 5b, lane 2). The 16E7 secreted protein was partially purified by ammonium sulfate precipitation and DEAE gel chromatography. As can be seen in Figure 5c, this procedure resulted in a sample in which 16E7 was the only protein detected by Coomassie blue staining (lane 2). Reactivity of yeast expressed HPV proteins with human sera

21 18

14

FIG. 5. Analysis of HPV 16 E6 and E7 proteins made by yeast. (a) Whole cells lysates (lanes 2 and 4) or concentrated medium (lane 3) from yeast containing pY16E6 (2) pYc~l6E6 (3), and pY16E7 (4) were separated by SDS-PAGE and immunoblotted using anti-l 6 E6 rabbit serum. Molecular weight standards were run in lane 1. (b) A whole cell lysate (lane 1) and partially purified HPV 16 E7 from yeast containing the pY0ll6E7 construct (lane 2) were immunoblotted using anti-l 6E7 rabbit serum. (c)A Coomassie blue-stained gel of partially purified HPV 16 E7 from yeast containing pYc~16E7 (lane 2) run with prestained molecular weight markers (lane 1).

One reason the HPV proteins were expressed in yeast was for use in serologic studies. It was therefore important to show that sera from HPV infected humans would react with the recombinant proteins expressed in yeast. In Figure 6 HPV 6 Ll was identified by western blot using human sera (Fig. 6a, lane 2). The reactivity was shown to be specific for HPV 6 Ll by preabsorption of the sera with bacterially expressed HPV 6 Ll fusion protein (Fig. 6b). Reactivity was not blocked by the HPV 6 L2 bacterial fusion protein (not shown). The bands at 7 1K were assumed to be cross reactivity with yeast protein as they appear in all lanes. One of the rationales for expressing HPV proteins in yeast was to examine the presence of epitopes on native proteins. The 16L2 secreted protein was used in

HPV PROTEINS

519

EXPRESSED IN YEAST

8

Titer

it reacted against untreated (open squares) and denatured (filled circles) antigen. Although denaturation reduced the reactivity it was significantly above background (open triangles). Another assay which can be used to detect native epitopes is immune precipitation. Figure 8 shows an immune precipitation of 16E7 from yeast containing the intracellular pY16E7 expression plasmid. The human serum used in this experiment had been previously identified as seropositive by immunoblot. The E7 protein was precipitated from yeast containing pY 16E7 (Fig. 8, lane 4) and not from vector containing control cells (lane 3). Nonimmune sera did not precipitate any detectable 16E7 (lanes 1 and 2). Although this experiment did not examine the presence of conformational epitopes on 16E7, the ability to immune precipitate 16E7 from yeast will enable us to perform these experiments.

DISCUSSION

8

Titer FIG. 7. Human antibody responses to native and denatured 16L2 as determined by ELISA. Yeast secreted 16L2 protein was added to the wells of a microtiter plate untreated (open squares), SDS-denatured (filled circles), or buffer only (filled triangles). Human sera which were negative (A) or positive (B) by Western blot were serially diluted and used as the primary antibody in ELISA.

ELISA to test its usefulness in serologic studies. Human sera which had been screened previously for reactivity to 16L2-trpE fusion proteins were tested by ELISA for reactivity to secreted 16L2. One serum was identified (of eight sera tested) which reacted strongly in ELISA but had been found to be negative by immunoblot analysis. To determine if this discrepancy was due to the presence of epitopes which were destroyed by denaturation, an ELISA was conducted using native and denatured 16L2. The test serum (Fig. 7A) only recognized native proteins (open squares). Reactivity to denatured antigen (filled circles) was lower than when no antigen was present (open triangles). A serum which was positive by immunoblot was also tested and

Due to the lack of availability of HPV proteins from natural sources this laboratory and others have employed molecular cloning strategies to study the HPV ORFs. The majority of these studies have employed bacterial expression systems to produce the ORFs as fusion proteins (Banks et al., 1987; Firzlaff et a/., 1987, 1988; Li et a/., 1987; Thompson and Roman, 1987; Tomita era/., 1987; Jenison eta/., 1988, 1990b; Cason, et a/., 1989; Jochmus-Kudielka eta/., 1989; Pate1 et al., 1989). Some HPV ORFs have also been expressed in mammalian cell culture (Browne ef a/., 1988) and the E2 ORF of HPV 16 was expressed in S. cerevisiae (Lambert eta/., 1989). Although bacteria often produce high levels of foreign protein there are several prob-

FIG. 8. lmmunoprecipitation of 16E7 by human sera. Human sera which were seronegative (1 and 2) or seropositive (3 and 4) to 16E7 by Western blot were used to precipitate protein from total cell lysates of yeast containing the pBS24.1 (1 and 3) or pY16E7 expression plasmids (2 and 4). The arrow indicates the 16E7 band.

520

CARTER ET AL.

lems inherent in the use of bacterial fusion proteins. First, it has been generally observed that the bacterially derived proteins are insoluble and require denaturing conditions to solubilize. Second, although the proteins are made in abundance they often undergo extensive proteolysis. A third problem with bacterial expression systems is that the post-translational modifications such as disulfide bond formation, phosphorylation, and glycosylation do not occur in bacteria. The bacterial products may, therefore, be substantially different from the naturally occurring protein. In order to overcome these problems we chose to express the HPV ORFs in a yeast expression system. We chose yeast for several reasons: because high levels of foreign proteins have been produced using S. cerevisiae and the vector system (Barr et al., 1989) employed here; because post-translation modifications occur in a manner similar to mammalian cells; and because the proteins may be secreted into the medium in a soluble form when fused to the pre-pro-afactor leader sequence. In this paper we describe the expression of HPV ORFs sequences which are similar or identical to the major and minor virion proteins of HPVs 1, 6b, and 16 in S. cerevisiae based on two criteria. First, the Ll and L2 ORFs of HPV 1 when expressed in yeast were identical to the purified virion proteins in size and were recognized by monoclonal antibodies generated against purified HPV 1 virions. Second, although it was not possible to do a similar comparison with HPV 6 and 16 virion proteins due to the small number of virions produced in natural infections (Grussendorf-Conen et a/., 1983), we have shown here that the HPV proteins expressed in yeast were of the molecular weight expected for the major and minor virion proteins. Additionally, several of these proteins were specifically recognized by immune human sera. We have demonstrated here that levels of HPV proteins produced in yeast were readily detected by immunoblots. The products were largely intact (with the exception of 16L2) when expressed intracellularly. Although not shown here, the majority of protein was found to be insoluble when the cells were lysed in the absence of denaturing reagents. While this attribute may allow for a simplified purification scheme, it will make examination of native epitopes of these proteins difficult. When the HPV proteins were expressed behind the a-factor leader sequence HPV 6 and 16 L2 and HPV 16 E7 secreted detectable levels of protein into the medium. This may expedite purification of the recombinant proteins as shown by the partial purification of 16E7. The HPV 16 L2 protein, when secreted from yeast, was found to have N-linked oligosaccharides

which was not unexpected based on sequence analysis. The presence of carbohydrate was demonstrated by reducing the apparent molecular weight of the proteins by treatment with PNGase F (Tarentino et a/., 1985). The secreted HPV 6 L2 was larger than the intracellular protein; however, its migration on polyacrylamide gels was not affected by PNGase F treatment. This result suggested that other modifications occurred or that the a-factor leader sequence was not cleaved. The presence of oligosaccharide is a potential problem if it masks native epitopes. Glycosylation and other potential modifications should not, however, create a problem when linear epitopes are being examined. In ELISA experiments the secreted 16L2 was identified by sera known to be positive for reactivity to the bacterial fusion protein. In addition one serum was identified that apparently recognized an epitope which was only present on the native protein. This result suggests that the presence of oligosaccharide will not inhibit binding to native epitopes although further investigation is required in this regard, and more importantly that yeast expressed proteins will provide a source of proteins for the detection and characterization of conformational epitopes. It appears that the HPV proteins expressed by yeast will be valuable tools for examining humoral immunity to these viruses. In the future these proteins may also serve as tools for the study of cellular immunity and for structure/function studies of the proteins themselves. ACKNOWLEDGMENTS We thank Philip J. Barr for the yeast expression plasmids; Jocelyn Wright, Mike Conrad, and Kurt Runge of the Zakian lab for advice on expressing recombinant protein in yeast; King Holmes and Janet Daling for providing human sera; Lorne Taichman and David Baker for HPV 1 virions and monoclonal antibodies; Elizabeth Etscheid forcritical reading of the manuscript; Marci Wright for typing; Paul Su for assistance with the figures. This work was conducted with funds provided by the National Cancer Institute CA 42792 and CA 35568 to D.A.G. S.A.J. is supported by Physician Scientist Training Grant CA01391.

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