Vol. 56, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1990, p. 2847-28520099-2240/90/092847-06$02.00/0Copyright © 1990, American Society for Microbiology

Expression and Secretion in Aspergillus nidulans and Aspergillusniger of a Cell Surface Glycoprotein from the Cattle Tick, Boophilus

microplus, by Using the Fungal amdS Promoter SystemIAN F. TURNBULL,' DONALD R. J. SMITH,2 PHILLIP J. SHARP,2 GARY S. COBON,2

AND MICHAEL J. HYNES'*Department of Genetics, University of Melbourne, Parkville, Victoria 3052,1 and Biotechnology Australia Pty. Ltd.,

East Roseville, New South Wales 2069,2 Australia

Received 13 November 1989/Accepted 2 July 1990

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus,which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed andsecreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promotersystem. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide exceptfor the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformedcells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a

similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was

expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditionsused. No intracellular Bm86 was detected. A general relationship was observed between transformantscontaining a high number of copies of the expression plasmid and high expression levels.

In recent times, recombinant DNA technology has pro-vided new methodologies for the development of human andanimal vaccines. Once the potential antigen is identified, thegene can be cloned and expressed in any of a number ofexpression systems for heterologous proteins, such as Esch-erichia coli or Saccharomyces cerevisiae. Such a strategyhas major advantages when the native antigen is found at lowlevels or is particularly difficult to purify. The limitation ofthe recombinant DNA approach is that the selected host maynot express the foreign protein or, if it does, its posttransla-tion modifying machinery may produce a protein that has atertiary structure which is significantly different from that ofthe native protein due, for example, to incorrect folding ofthe recombinant protein or to nonnative glycosylation. Suchincorrectly processed recombinant polypeptides may not actas efficient immunogens to protect against the pathogen.

Immunization is usually carried out to provide protectionagainst microorganisms that enter the bloodstream, butrecently attention has been given to immunization againstectoparasites. A major agricultural pest in parts of NorthernAustralia is the cattle tick (Boophilus microplus). This par-asite is responsible for substantial losses in production.Willadsen et al. (31, 32) described the isolation from partiallyengorged adult female ticks of a glycoprotein (Bm86) whichwas shown to elicit a protective immunological response inthe bovine host. B. microplus fed on vaccinated cattlesuffered severe damage to its gut digestive cells (30). It wasshown that the antibodies against Bm86 in the blood boundto the glycoprotein on the surface of B. microplus gut cellswhen a blood meal was eaten. The result was that the tickssuffered severe damage to the gut, with resultant leakage ofbovine blood into their hemolymph, which in turn resulted inthe death of the majority of the ticks and decreased thefertility of the survivors. Bm86 is difficult to isolate from B.

* Corresponding author.

microplus because it is present in small quantities. Morethan 50,000 hand-picked ticks (1 kg) were required in orderto isolate 20 to 200 ,ug of the antigen (32). Native Bm86 is aglycoprotein with a molecular mass of 89 kilodaltons (kDa),containing glucosamine and mannose moieties in N- and0-glycosidic linkages.The Bm86 gene has been cloned and sequenced (18a). The

protein has a large number of cysteines (65 of 650 aminoacids), and the native protein is presumably highly folded.Expression of a modified Bm86 lacking the N-terminal leadersequence and the carboxy-terminal anchor (membrane-tra-versing) amino acids was achieved in E. coli. It had anapparent molecular mass of 69 kDa (18a). Immunization withE. coli-produced Bm86 inclusion bodies did not result in thesame degree of protection against B. microplus infestation aswas obtained with native Bm86 (18a). A eucaryote expres-sion system might produce Bm86 that is structurally (andtherefore antigenically) more closely related to native Bm86.One such eucaryote expression system that has created

interest and proved useful in expressing and secreting for-eign proteins is the filamentous fungus Aspergillus nidulans(5, 26, 28). It has been used to express a number ofheterologous proteins (4, 8, 25, 27). We have previouslydescribed the construction of an expression vector (pV3)derived from the A. nidulans amdS gene (25). Expressionusing the system was demonstrated with an E. coli protein,the heat-labile enterotoxin subunit B, which was not se-creted. In this article, we describe the construction of anexpression vector for Bm86 by using the amdS promotersystem (pS8), and the expression and secretion of a solubleform of Bm86 by transformed A. nidulans and Aspergillusniger.

MATERIALS AND METHODSBiological materials, chemicals, and antibodies. All E. coli

transformations were into strain MC1061. A. nidulansstrains used were J9 (paba Al prn-309) and VH1 (yAl

2847

on Septem

ber 1, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2848 TURNBULL ET AL.

(wsI0

BpiU..

1l

FIG. 1. Construction of the expression vector pS8. (a) Diagram of the region of BTA 1751 containing the Bm86 gene and showing therelevant features including the leader sequence, relevant restriction enzyme recognition sites, and the regions encoding the amino acidsequences from which synthetic peptides SP1 and SP2 were derived. The coding region is boxed, and the region encoding the deleted carboxytail is indicated (*). (b) amdS expression vector pV3. (c) Construct PS8 containing the BamHI-HindIII (1.94-kb) fragment of BTA 1751. E,EcoRI; B, BamHI; P, PstI; H, HindIII; S, Sacd; Sl, Sall.

amdRJ04c amdIJ8 areA102 amdA7facB88). pPL3 containedthe riboB+ gene (17) and was provided by B. R. Oakleigh,Department of Microbiology, Ohio State University.pAN222 was a clone of the prn+ gene cluster in pBR322 (6).pAmph was a gift from B. Tyler, Research School ofBiological Sciences, Australian National University.pAmph, a plasmid which carries a gene for bleomycinresistance fused to the Neurospora crassa am gene pro-moter, confers resistance to blenoxane (B. Tyler and B.Austin, personal communication). BTA 1751 was a clonecontaining a 1.92-kilobase (kb) EcoRI-PstI fragment (18a) ofDNA encoding all except the 21 hydrophobic carboxy-terminal amino acids of the B. microplus Bm86 gene inpUC13. The clone contained an additional 31 bases 5' to theATG (Fig. la). The vector pV3 (Fig. lb) contained the amdSpromoter and terminator sequences with a multiple cloningsite, replacing most of the coding region, cloned intopBR322, as previously described (25).DNA-modifying enzymes were from Bethesda Research

Laboratories, Bresatec Ltd., Adelaide, South Australia, andBoehringer Mannheim. [a-32P]dATP was from Bresatec.Nitrocellulose BA85 was from Schleicher & Schuell. [35S]cysteine was from Amersham Corp. Blenoxane was agift from Ruth Hall, CSIRO Division of Biotechnology.X-ray film was from Fuji. All other fine chemicals wereof molecular biological or analytical grades. The Protoblotimmunoblot detection system, containing alkaline phos-phatase-conjugated anti-rabbit immunoglobulin G, was fromPromega Biotec. Antibodies were obtained as postinocula-tion sera from cattle inoculated with full-length bacterialBm86 or from rabbits inoculated with synthetic peptides ofBm86: SP1, the N-terminal 18 amino acids of the matureBm86 conjugated to ovalbumin, and SP2, the carboxy-terminal 18 amino acids (before the anchor) conjugated tobovine serum albumin (Fig. la).A. nidulans cultures and transformation. A. nidulans was

cultured, as described previously, in Aspergillus minimal

(ANM) medium (9, 10) or in Carter-Bull medium (2) modifiedby using (NH4)2SO4 as the nitrogen source and sodiumcitrate as the chelating agent. Protoplast isolation and trans-formation procedures were as previously described (23). Thesame procedures were used for A. niger transformations. J9was cotransformed with pAN222 (6), and transformantswere selected on medium containing L-proline (10 mM) asthe sole nitrogen source. VH1 was cotransformed with pPL3(17), and transformants were selected on medium lackingriboflavin. A. niger transformants were cotransformed withpAmph and selected by growth on blenoxane (2 [Lg/ml).Controls throughout were transformed with the cotransform-ing plasmid only.

Molecular methods. Routine molecular biology procedureswere used (16). For Southern blot analysis (21), A. nidulansor A. niger DNA was prepared (10), digested with restrictionendonucleases, size fractionated by agarose gel electropho-resis, and transferred to nitrocellulose. Labeled probes wereprepared by nick translation (19) and hybridized at 42°C insolutions containing 50% formamide and 10% dextran sul-fate. The final wash was in 0.1x SSC (lx SSC is 0.15 MNaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecylsulfate (SDS) at 65°C. Total RNA was prepared as describedpreviously (7, 25). RNA (6 ,ug) was size fractionated byelectrophoresis in submarine gels (1.5% agarose-2.2 M form-aldehyde) at 50 mA for 2 h in MOPS (morpholinepropane-sulfonic acid) buffer (15). After ethidium bromide staining,the RNA was transferred to nitrocellulose by the Northernblot (RNA blot) procedure (22), and the filters were hybrid-ized by the same procedure used above for Southern blots.Final washes were in 0.1x SSC and 0.1% SDS at 65°C.Autoradiograph exposure times were normally overnight.

Protein preparations. Postculture medium was obtained byfiltering the cultures through Whatman no. 4 filter paper in aBuchner funnel. The mycelia were weighed, and the filtratewas concentrated either by using Centricon-10 microconcen-trators (Amicon Corp.) or by freeze-drying until the volume

APPL. ENVIRON. MICROBIOL.

on Septem

ber 1, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

EXPRESSION OF TICK GLYCOPROTEIN BY ASPERGILLUS SPP. 2849

was reduced to one-tenth. Phenylmethylsulfonyl fluoridewas added to the concentrated medium (final concentration,10 ,ug/ml). Cellular soluble protein extracts were prepared byhomogenizing mycelium (wet weight, 0.5 g) in a minimumvolume of extraction buffer containing Tris hydrochloride (5mM), pH 8.0, and phenylmethylsulfonyl fluoride (10 ,ug/ml).Cells were disrupted by grinding with a mortar and pestleunder liquid nitrogen. The homogenate was centrifuged at27,000 x g in a Beckman J2-21 high-speed centrifuge for 10min. Protein concentrations were determined by using theBio-Rad reagents.SDS-PAGE, Western blots (immunoblots), and immunode-

tection. Concentrated media or cellular protein extracts werereduced, SDS-denatured, and fractionated by SDS-poly-acrylamide gel electrophoresis (PAGE) (14). Routinely, gelswere 14 by 12 cm by 1.5 mm and consisted of 8 or 10% totalacrylamide and 2.7% cross-linker with a 1-cm stacking layer(4% acrylamide). Gels were electrophoresed at 20 mA for 16h at 4°C with the Bio-Rad Protean apparatus. Molecularweights were determined against the midrange Rainbowprotein molecular weight markers from Amersham. Proteinwas transferred to nitrocellulose electrophoretically by usingthe Western blot procedure (24) and an LKB Instruments2117-250 Novablot electrophoretic transfer apparatus intheir continuous buffer system. Bm86 was detected by usinga Protoblot immunoblotting system (Promega Biotec) andthe method of the supplier. The primary antibodies, anti-SP1and anti-SP2, were used at 1:200 dilutions after preadsorp-tion with A. nidulans control extract. For preabsorption,media from control cultures was concentrated 10 times byfreeze-drying, and a nitrocellulose filter was soaked in theconcentrate for 1 h, blotted dry, and then soaked in thediluted (1:200) sera for 1 h. Gels were stained in 0.75%Coomassie brilliant blue R-250 (Sigma Chemical Co.) in 50%methanol and 10% acetic acid or by using the Bio-Rad silverstain procedure. Carbohydrate was detected with the peri-odic acid-Schiff reagent, in either gels or nitrocellulosemembranes, by the method of Segrest and Jackson (20).Enzyme-linked immunosorbent assay determinations.

Bm86 determinations were made by using an antibodycapture enzyme-linked immunosorbent assay and detectedwith antibody conjugated to horseradish peroxidase. Theantibodies used were raised in the cow against full-lengthBm86 produced in bacteria and affinity purified. Purifiedfull-length bacterial Bm86 (standardized against bovinegamma globulin) was used as a standard, and estimates ofBm86 concentration were made from the standard curve.

Affinity chromatography. An antibody affinity column wasprepared by using cow antibodies to full-length bacterialBm86 coupled to CNBr-activated Sepharose 4B (Pharma-cia).Medium (40 ml) from a 3-day culture of the best Bm86-

producing strain was pumped onto the affinity column at aflow rate of 1.2 ml/min after the addition of protease inhibi-tors (10'- M leupeptin, 10' M pepstatin, 2 x 10-' Mantipain, 5 x 10-3 M EDTA, 10-3 M phenylmethylsulfonylfluoride). The column was washed with Tris-saline and theneluted with 3 M MgCI2.

RESULTS

Expression vector constructions. A number of vectors wereconstructed in the amdS vector pV3 to obtain expression ofBm86 in A. nidulans, but the construct used in this study wasPS8* Construct pS8 (Fig. lc) contained a 1.94-kb partialBamHI-HindIII Bm86-containing fragment in the BamHI-

HindIll sites of pV3. pS8 encoded an additional five non-Bm86 amino acids (Gly-Met-Glu-Ala-Trp) at the carboxyend, before the first stop codon. The construct had 68 basesfrom the putative amdS transcription start (3) to the Bm86ATG. This compares with 41 for native amdS and 27 for pL2,a previously described expression vector (25). There wereno ATG false starts in the construct. The construct encodedthe 19-amino-acid amino N-terminal signal sequence ofBm86. If transcription went through to the putative amdStermination sequence (3) of pV3, then the mRNA size for pS8should be approximately 2.20 kb.A. nidulans transformations and Bm86 DNA detection.

Transformants for p8S were obtained in strain J9 and strainVH1, a strain developed for its multiple cis- and trans-actingmutations leading to increased amdS expression. After ini-tial selection for the cotransforming plasmid on selectionmedium, transformants with multiple copies of pS8 (30 to70% of those with copies of the cotransforming plasmid [13])were detected by their reduced ability to grow on 2-pyrroli-dinone (10 mM) as a result of the extra amdS 5' regionssequestering the amdR gene product which is required for2-pyrrolidinone utilization (13, 25). Poor growth on pyrroli-dinone (high copy number) was observed in 7/70 J9 transfor-mants and 43/137 VH1 transformants.Genomic DNA was extracted from mycelia of high-copy-

number pS8 strains. Figure 2a shows the autoradiographfrom Southern blot analysis of some PS8 high-copy-numberstrains and shows the expected 3.84-kb EcoRI fragment in allof them. The band was not seen in DNA from controls (datanot presented).

Detection of Bm86 mRNA in transformants. Total RNAwas prepared from mycelia from 1-day cultures of sometransformant strains. Figure 2b shows a single transcript of2.20 kb in RNA from all PS8 high-copy-number transfor-mants (VH1) examined but not in RNA from the control.There was some variation between transformant strains, andsome strains failed to show detectable Bm86 mRNA, but thelevels were generally far in excess of that which would havebeen expected for amdS. The transcription levels in J9strains grown under acetate-induced conditions (9) and theamdS-deregulated VH1 strains were similar in their levels ofBm86 mRNA. Levels of Bm86 mRNA for J9 strains grownunder acetate-induced conditions were 5- to 10-fold greateron a molar basis than for the heat-labile enterotoxin subunitB mRNA that was previously described for this expressionsystem (25).

Detection of Bm86 secreted into medium. Cultures (100 ml)were prepared from five high-copy-number pS8 transfor-mants (VH1 strains) and two control strains (transformedwith pPL3 only). Samples of media were obtained after 3days when the protein secretion rate was at a maximum(protein concentration, 32.5 + 19.5 ,ug/ml [n = 7]). Theywere concentrated 10-fold with the Centricon-10. DuplicateSDS-PAGE (10% acrylamide) gels were prepared, 50-,ugsamples of total protein were loaded, and the proteins wereseparated by electrophoresis. A standard of Bm86, solubi-lized inclusion bodies from E. coli (2 ,ug), was run in additionto standard size markers. The proteins were transferred ontonitrocellulose and visualized with rabbit anti-SP1 and anti-SP2. A strong band was detected in the concentrated mediaof two of the five transformants (Fig. 3, transformants 3 and4), and a weaker band was detected in another (transformant1), while both controls (Cl and C2) and two other transfor-mants (2 and 5) did not react. The band was very broad, witha retarded electrophoretic mobility compared with the E.coli Bm86 (lane S) and an apparent molecular mass of 83 to

VOL. 56, 1990

on Septem

ber 1, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2850 TURNBULL ET AL.

FIG. 2. DNA and mRNA from A. nidulans transformants, en-

coding Bm86 sequences, were detected in extracts from transfor-mants. (a) Southern blot analysis revealed that a 3.84-kb EcoRIfragment was detected by the Bm86-specific probe (1.92-kb BamHIfragment) in DNA from four PS8 transformants (lanes 2 through 5).Untransformed controls did not show the band (not shown). Lane 2is overloaded for DNA, resulting in a smear. (b) Northern blotanalysis revealed a transcript of 2.20 kb detected by the Bm86-specific probe in total RNA from five transformant strains (lanes 1through 5) but not in the control (lane C). Samples 2 through 5 (lanes2 through 5) correspond to those in panel a. In each panel, theethidium bromide-stained gel is shown on the left and the equivalentautoradiograph is on the right. The marker lanes (M) contain 32p-labeled HindIll-digested lambda DNA. For the Northern blot, themarker DNA was denatured prior to being run on the gel.

87 kDa. The band was detected with both anti-SP2 (Fig. 3a)and anti-SP1 (Fig. 3b).The broadness of the band and the fact that it was retarded

behind the E. coli Bm86 (69 kDa) and had an apparentmolecular mass of 83 to 87 kDa, whereas the DNA sequencepredicts 69 kDa for the protein alone, indicated that it wasprobably glycosylated and possibly to a similar extent as thenative Bm86, which has an apparent molecular mass of 89kDa (32) (the native Bm86 had additional carboxy-terminalamino acids not present in the A. nidulans protein). Evi-dence from gels and Western blots stained with a carbohy-drate stain (periodic acid-Schiff reagent) did result in a faintbroad band of the same mobility as the putative Bm86, thussupporting the presence of a glycoprotein of that mobility(data not presented).

It was not possible to reliably detect the recombinantBm86 in stained gels of culture media as there was materialin the media of control cultures in the 83- to 87-kDa regionwhich also stained substantially with Coomassie blue. Underappropriate conditions of electrophoresis, this componentcould sometimes be resolved from Bm86 which could bedetected as an additional band in the media of transformantcultures. However, the band was very broad, and there wasalways the possibility that some of the Bm86 was stillmasked by the other medium components. It was therefore

U

FIG. 3. Bm86 polypeptide was detected in culture media from A.nidulans transformants. Western blot analysis of duplicate gelsresolved with anti-SP2 (a) or anti-SP, (b) antibodies. Each gelcontained concentrated media (50 p.g of protein) from 3-day culturesof five transformant strains (lanes 1 to 5) and two controls (lanes Cland C2). A strong 83- to 87-kDa band was seen in transformants 1,3, and 4 (arrows) but not in transformants 2 or 5 or in the controls Clor C2. It was a broad band and was retarded in relation to E. ccliBm86 inclusion bodies (lane S). Solubilization of the inclusionbodies was not complete, so that some E. ccli material failed to enterthe gel.

very difficult to quantitate the levels of expression of Bm86by the transformants by using gel analysis. Attempts to do soby using Western blots and comparing the intensity ofstaining with that of known amounts of the E. coli-producedprotein led to estimates of up to 1.3 mg/liter, or approxi-mately 4% of the secreted protein in the culture media fromthe best producers. However, these estimates were notaccurate, because they were not quantitative.

Determination of the expression levels on the basis ofenzyme-linked immunosorbent assays, however, supportsthis estimate. The three Bm86-producing strains shown inFig. 3 were cultured in two different media, and the amountof Bm86 was estimated by using bacterially produced mate-rial as a standard. An expression level of 1.34 ±+ 0.08 mg/liter(n = 3) was obtained for transformant 3 in ANM medium,while 2-day cultures in Carter-Bull medium gave 0.85 ±+ 0.04mg/liter (n = 3) for transformant 1, 1.78 ±+ 0.03 mg/liter (n =

3) for transformant 3, and 1.68 ±+ 0.03 mg/liter (n = 3) fortransformant 4. These estimates are accurate, however, onlyif all of the Bm86 is intact in the culture media, and there wasevidence from Western blots that some degradation mayhave occurred in the Carter-Bull medium. The relative levelswere supported by the Western blot data (Fig. 3). The bestexpression achieved was 1.8 mg/liter (6% of secreted pro-tein).To substantiate the estimates, antibody affinity chroma-

tography was used to purify the Bm86 from a transformant-3culture medium which contained a relatively large amount ofthe product. Antibodies from a cow vaccinated with the E.coli-produced Bm86 were affinity purified on a column ofrecombinant protein and then coupled to cyanogen bromide-

APPL. ENVIRON. MICROBIOL.

on Septem

ber 1, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

EXPRESSION OF TICK GLYCOPROTEIN BY ASPERGILLUS SPP. 2851

activated Sepharose. Culture medium (100 ml) was thenpassed over the column, and after the unbound proteinswere washed off, specifically bound Bm86 was eluted withMgCl2. SDS-PAGE analysis of the affinity-purified productshowed that there were a number of minor bands presentwith a major high-molecular-weight band, Western blotanalysis showed that it reacted with antibodies againstBm86, and very little reactivity could be detected in thenonbound fraction (data not shown).

Previous work with this system had suggested that thebest producers of heterologous proteins were also, in generalterms, those transformants with the highest copy number forthe heterologous gene (25). Data supporting that view wasobtained from dot blot analysis using anti-SP2 to detectBm86 as color above that in controls. The extent of growthon 2-pyrrolidinone was used as a measure of copy number.High-copy-number transformants grew very poorly on2-pyrrolidinone, while moderate-copy-number transfor-mants grew relatively well. For the nonexpressors, 7/8 (88%)had a moderate copy number, compared with only 18/41(45%) for those that produced detectable levels of Bm86.The result was just significant (X21 = 5.09; P < 0.025). Thesedata support the view that gene copy number may influenceexpression level in this system.No intracellular soluble Bm86 was detected. No Bm86 was

detected in cellular extracts (soluble or particulate fractions)from A. nidulans strains transformed with Bm86 expressionvectors by Western blot analysis using anti-SP1 or anti-SP2.Gels were loaded with between 50 and 400 ,ug of totalprotein, but no soluble intracellular Bm86 was detected inany transformed strain tested, including high-copy-numbergood and poor Bm86 secretors. The limits of detection wereestimated to be approximately 0.01% of intracellular solubleprotein. Bm86 has a very high cysteine content and could bereadily labeled by culturing E. coli cells in medium contain-ing [35S]cysteine (18a); therefore, A. nidulans transformantswere cultured in medium containing [35S]cysteine (10), butno significant product with the predicted molecular weightcould be seen in autoradiographs from SDS-PAGE gels ofcellular protein extracts (data not shown). These resultssuggest that the Bm86 does not build up in the cells but, rather,passes quickly into the medium where it does accumulate.Bm86 secreted by pS8-transformed A. niger. A. niger is

considered to be a better secreter than A. nidulans, althoughits genetics are less well understood. It does not possess anamdS gene (12) and cannot grow on acetamide as the solenitrogen or carbon source, but it has previously been trans-formed with the A. nidulans amdS gene, and regulatedexpression has been observed (12). Wild-type A. niger wastransformed with pS8 by using cotransformation withpAmph and selection on blenoxane. Thirty-two transfor-mants were obtained, and DNA was prepared and dottedonto nitrocellulose. DNA from 10 transformants hybridizedstrongly with the Bm86-specific probe, and 8 of these wereselected for further testing. EcoRI-digested DNA, run on a1% agarose gel and subjected to Southern blot analysis,revealed the expected 3.84-kb Bm86 band in all the selectedtransformants. A band with the mobility of A. nidulansBm86 was detected by Western blot analysis in media fromall the selected transformants (data not presented). The levelof Bm86 expression was estimated to be approximately 0.5% ofsecreted protein, less than in the best A. nidulans expressors.

DISCUSSIONA cell surface glycoprotein (Bm86) found on cells of the

gut of the cattle tick, B. microplus, was expressed in the

filamentous fungi A. nidulans and A. niger by using theamdS promoter system. The construct pS8 was transcribedreadily to give high Bm86 mRNA levels. This was so for A.nidulans J9 under the acetate-induced growth conditionsexpected to enhance amdS expression via the 19 mutation (9)in the construct and for VH1 (deregulated for amdS) understandard growth conditions. The Bm86 mRNA levels ap-peared substantially higher than those previously described(25) for the E. coli heat-labile enterotoxin subunit B mRNAin J9 (acetate induced) for similarly high-copy-number trans-formants.Bm86 was readily secreted in reasonable quantities by A.

nidulans under the direction of the B. microplus secretorysignal. It appears that the B. microplus 19-amino-acid Bm86secretory signal functions to some extent in A. nidulans. Thesequence conforms to the -3, -1 rule (29), with Ala at -1and Gly at -3, and has a putative hydrophobic core se-quence (18) of Leu-Phe-Val-Ala-Ala beginning at the sixthamino acid. However, the high Bm86 mRNA levels andapparent lack of buildup of internal Bm86 together suggestthat higher levels of secreted product could be expected andthat the signal sequence may have limited the amount ofBm86 in the media. Therefore, it is possible that levels ofsecretion could be increased, for example, by using the A.niger glucoamylase signal sequence (1).Under the standard growth conditions used, the best

expression achieved resulted in Bm86 being secreted atgreater than 4% of total secreted protein and 1.3 mg/liter.This was increased to 1.8 mg/liter (6% secreted protein)when a more complex medium was used. The total secretionof protein could probably be increased under optimizedculture conditions, and the level of Bm86 would probablyincrease proportionally. A. nidulans systems have producedsecreted chymosin at up to 2.4 mg/liter (4), interferon at 1mg/liter (8), and tissue plasminogen activator at 0.1 mg/liter(27). At 6% of total secreted protein, Bm86 was secreted at1.8 mg/liter, which is comparable to levels reported for theseother systems. The A. nidulans-produced Bm86 is glycosy-lated (unlike the E. coli inclusion body material). Theconstructs in this study lacked the carboxy-terminal anchor,and the product was found in the media along with only a fewmajor, distinct protein components, which aids its purifica-tion.The A. nidulans Bm86 resolved by SDS-PAGE as a broad

band with a lower mobility than predicted from the proteinsequence and the E. coli-produced Bm86. The Bm86 stainedwith the periodic acid-Schiff reagent carbohydrate stain,which suggests that it was glycosylated. The A. niger-secreted protein glucoamylase is glycosylated at serine andthreonine residues via 0-glycosidic and N-glycosidic link-ages to asparagine residues (11). Thus Bm86 from PS8 A.nidulans transformants had the potential for both 0- andN-glycosidic linkages, both of which are predicted for thenative glycoprotein. The types of carbohydrate moietiesadded were not determined. The important question ofwhether the A. nidulans glycosylation of Bm86 makes itmore or less immunogenic than the E. coli Bm86 and morelike the B. microplus Bm86 can be addressed by testingpurified A. nidulans Bm86 in cattle vaccination trials. Af-finity chromotography followed by polymer-reversed-phasehigh-pressure liquid chromatography was used to obtain A.nidulans Bm86 as a 95% pure product that might be suitablefor such trials in the near future.

ACKNOWLEDGMENTThis project was funded by Biotechnology Australia Pty. Ltd.

VOL. 56, 1990

on Septem

ber 1, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2852 TURNBULL ET AL.

LITERATURE CITED1. Boel, E., I. Hjort, B. Svensson, F. Norris, K. E. Norris, and N. P.

Fill. 1984. Glucoamylases Gl and G2 from Aspergillus niger aresynthesized from two different but closely related mRNAs.EMBO J. 3:1092-1102.

2. Carter, B. L. A., and A. T. Bull. 1969. Studies on fungal growthand intermediary carbon metabolism under steady and non-steady state conditions. Biotechnol. Bioeng. 11:785-804.

3. Corrick, C. M., A. P. Twomey, and M. J. Hynes. 1987. Thenucleotide sequence of the amdS gene of Aspergillus nidulansand the molecular characterization of 5' mutations. Gene 53:63-71.

4. Cullen, D., G. L. Gray, L. J. Wilson, K. J. Hayenga, M. H.Lamsa, M. W. Rey, S. Norton, and R. M. Berka. 1987. Con-trolled expression and secretion of bovine chymosin in Aspergil-lus nidulans. Bio/Technology 5:369-376.

5. Cullen, D., and S. Leong. 1986. Recent advances in the molec-ular genetics of industrial filamentous fungi. Trends Biotechnol.4:285-288.

6. Durrens, P., P. M. Green, H. N. Arst, and C. Scazzocchio. 1986.Heterologous insertion of transforming DNA and generation ofnew deletions associated with transformation in Aspergillusnidulans. Mol. Gen. Genet. 203:544-549.

7. Glisin, V., R. Crkvenjakov, and C. Byus. 1974. Ribonucleic acidisolated by cesium chloride centrifugation. Biochemistry 13:2633-2637.

8. Gwynne, D. I., F. P. Buxton, S. A. Williams, S. Garven, andR. W. Davies. 1987. Genetically engineered secretion of activehuman interferon and a bacterial endoglucanase from Aspergil-lus nidulans. Bio/Technology 5:713-719.

9. Hynes, M. J. 1977. Induction of the acetamidase of Aspergillusnidulans by acetate metabolism. J. Bacteriol. 131:770-775.

10. Hynes, M. J., C. M. Corrick, and J. A. King. 1983. Isolation ofgenomic clones containing the amdS gene of Aspergillus nidu-lans and their use in the analysis of structural and regulatorymutations. Mol. Cell. Biol. 3:1430-1439.

11. Innis, M. A., M. J. Holland, P. C. McCabe, G. E. Cole, V. P.Wittman, R. Tal, K. W. K. Watt, D. H. Gelfand, J. P. Holland,and J. H. Meade. 1985. Expression, glycosylation, and secretionof an Aspergillus glucoamylase by Saccharomyces cerevisiae.Science 228:21-26.

12. Kelly, J. M., and M. J. Hynes. 1985. Transformation ofAspergil-lus niger by the amdS gene of Aspergillus nidulans. EMBO J.4:475-479.

13. Kelly, J. M., and M. J. Hynes. 1987. Multiple copies of the amdSgene of Aspergillus nidulans cause titration of trans-actingregulatory proteins. Curr. Genet. 11:21-31.

14. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

15. Lehrach, H., D. Diamond, J. M. Wozney, and H. Boedtker. 1977.RNA molecular weight determinations by gel electrophoresisunder denaturing conditions, a critical re-examination. Bio-chem. J. 16:4743-4751.

16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

17. Oakleigh, C. E., C. F. Neil, P. L. Kretz, and B. R. Oakley. 1987.Cloning of the riboB locus ofAspergillus nidulans. Gene 53:293-298.

18. Perlman, D., and H. 0. Halvorson. 1983. A putative signalpeptidase recognition site and sequence in eukaryotic andprokaryotic signal peptides. J. Mol. Biol. 167:391-409.

18a.Rand, K. N., T. Moore, A. Sriskantha, K. Spring, R. Tellam, P.Willadsen, and G. S. Cobon. 1989. Cloning and expression of aprotective antigen from the cattle tick Boophilus microplus.Proc. Natl. Acad. Sci. USA 86:9657-9661.

19. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977.Labelling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

20. Segrest, J. P., and R. L. Jackson. 1972. Molecular weightdetermination of glycoproteins by polyacrylamide gel electro-phoresis in sodium dodecyl sulfate. Methods Enzymol. 28:54-63.

21. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

22. Thomas, P. S. 1980. Hybridization of denatured RNA and smallDNA fragments transferred to nitrocellulose. Proc. Natl. Acad.Sci. USA 77:5201-5205.

23. Tilburn, J., C. Scazzocchio, G. G. Taylor, J. H. Zabicky-Zissman, R. A. Lockington, and R. W. Davies. 1983. Transfor-mation by integration in Aspergillus nidulans. Gene 26:205-221.

24. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

25. Turnbull, I. F., K. Rand, N. S. Willetts, and M. J. Hynes. 1989.Expression of the Escherichia coli enterotoxin subunit B gene inAspergillus nidulans directed by the amdS promoter. Bio/Technology 7:169-173.

26. Upshall, A. 1986. Filamentous fungi in biotechnology. Biotech-niques 4:158-164.

27. Upshall, A., A. A. Kumar, M. C. Bailey, M. D. Parker, M. A.Favreau, K. P. Lewison, M. L. Joseph, J. M. Maraganore, andG. L. McKnight. 1987. Secretion of active human tissue plas-minogen activator from the filamentous fungus Aspergillusnidulans. Bio/Technology 5:1301-1304.

28. Van Brunt, J. 1986. Fungi: the perfect hosts? Bio/Technology4:1057-1062.

29. Von Heijne, G. 1984. How signal sequences maintain cleavagespecificity. J. Mol. Biol. 173:243-251.

30. Willadsen, P., and D. H. Kemp. 1988. Vaccination with 'con-cealed' antigens for tick control. Parasitol. Today 4:196-198.

31. Willadsen, P., R. V. McKenna, and G. A. Riding. 1988. Isolationfrom the cattle tick, Boophilus microplus, of antigenic materialcapable of eliciting a protective immunological response in thebovine host. Int. J. Parasitol. 18:183-187.

32. Willadsen, P., G. A. Riding, R. V. McKenna, D. H. Kemp, R. L.Tellam, J. N. Nielsen, J. Lahnstein, G. S. Cobon, and J. M.Gough. 1989. Immunological control of a parasitic arthropod:identification of a protective antigen from Boophilus microplus.J. Immunol. 143:1346-1351.

APPL. ENVIRON. MICROBIOL.

on Septem

ber 1, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from