Gene, 122 (1992) 219-223 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

219

0378-I 119/92/$05.00

06773

An inducible

expression

Aspergillus (Recombinant

system for the production

of human lactoferrin

in

nidulans

DNA;

iron-binding

glycoprotein;

filamentous

fungi; al4

promoter;

secretion;

functional

assay)

Pauline P. Ward a, Gregory S. May a, Denis R. Headon b and Orla M. Conneely a ” Department qf Cell Biology, Baylor College of Medicine, Houston. TX 77030, USA; and ’ Cell and Molecular Biology Group, Department of‘ Biochemistry, lJniversit_vCollege, Galway, Ireland. Tel. (353~91)-27370 Received

by J.A. Gorman:

20 March

1992; Revised/Accepted:

2 July/3 July 1992; Received

at publishers:

3 August

1992

SUMMARY

The production and secretion of human lactoferrin (hLF) in Aspergillus niduluns is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (&A) promoter. Recombinant hLF (rehLF) is produced at levels up to 5 pg/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.

INTRODUCTION

Human lactoferrin (hLF) is an iron-binding 78-kDa glycoprotein (Anderson et al., 1989). It is present in milk and other exocrine secretions (Masson et al., 1966) and also in the secondary granules of polymorphonuclear granulocytes (Masson et al., 1969). A major role in iron regulation has been proposed for hLF based on its ability to reversibily

Correspondence to: Dr. O.M. Conneely, lor College of Medicine, Tel. (713) 798-6233;

Department

One Baylor Plaza,

of Cell Biology, Bay-

Houston,

TX 77030, USA.

Fax (713) 790-1275.

Abbreviations:

A., Aspergillus; ADHI,

alcohol

gene encoding

ADHI;

bp, base pair(s);

complementary munosorbent

Ap, ampicillin;

to RNA;

cpm, counts/min;

ELISA,

assay; hLF, human lactoferrin;

1; alcA,

cDNA,

DNA

enzyme-linked

im-

hZ_F,gene (DNA) encoding

or 1000 bp; N., Neurospora; MCS, multiple cloning

hLF; kb, kilobase site; nt, nucleotide(s); polyacrylamide-gel

dehydrogenase

ORF,

open reading

electrophoresis;

coli DNA polymerase

frame;

p, plasmid,

PAGE,

PolIk, Klenow (large) fragment

I; re, recombinant;

SSC, 0.15 M NaCI/O.OlS M Na,,citrate scription; UTR, untranslated region.

of E.

SDS, sodium dodecyl

sulfate;

pH 7.6; T, terminator

of tran-

bind iron with high affinity over a broad pH range (N&met and Simonovits., 1985). The functions proposed for lactoferrin include protection against microbial infection (Arnold et al., 1977) cellular growth promotion (Hashizume et al., 1983; Nichols et al., 1987) and regulation of intestinal iron homeostasis (Hu et al., 1990). To provide a source of biologically active hLF to facilitate investigations of possible therapeutic and nutritional uses of this protein, we have expressed the hLF cDNA in A. nidulans. Aspergillus strains naturally secrete a wide range of glycoproteins which makes these systems especially attractive for the production of extracellular eukaryotic glycoproteins. Several eukaryotic post-translational modifications, such as N-terminal processing and glycosylation, are performed correctly in Aspergillus (Van Brunt, 1988). For years these filamentous fungi have been used in the industrial production of glycoproteins (Bat-besgaard, 1977). Hence, largescale fermentation technology and downstream processing are already well established. The genetically well characterized A. niduluns has been used as a host for the heterologous production of a variety of proteins of both prokaryotic and eukaryotic origin

220 (Gwynne et al., 1987a; Cullen et al., 1987). Both constitutive and inducible used to direct recombinant protein et al., 1989). The controlled induction

1987; Upshall et al., promoters have been production (Sanders of recombinant pro-

EXPERIMENTAL

(a) Construction plasmid

AND DISCUSSION

of the Aspevgillus

niduluns

expression

teins at a particular stage in the growth curve in Aspergillus is advantageous for a number of reasons. Cultures can be grown to high cell density before induction of the recom-

The plasmid used for expression of hLF cDNA is shown schematically in Fig.1. The complete cDNA was isolated from a human monocyte cDNA library in AgtlO (Clontech,

binant protein thereby minimizing the exposure time of the foreign protein to the cells. This shorter exposure time pro-

Palo Alto, CA) using a 32P-labelled DNA probe from the previously published partial sequence of hLF (Rado et al., 1987). The 2.3-kb clone contained the secretory signal se-

tects the recombinant protein against proteolysis by endogenous Aspergillus proteases. Furthermore, it is possible to

quence and complete translation frame. The sequence of the entire cDNA was confirmed by dideoxy sequence analysis (Sequenase version 2.0, U.S. Biochemical, Cleveland, OH). The cDNA was repaired using the PolIk and sub-

make recombinant proteins whose presence may be detrimental to host Aspergillus cells. Since lactoferrin is thought to have anti-fungal trolled expression

as well as antibacterial properties, conof this protein in A. niduluns may be

cloned into AccI-digested and blunt-ended pGEM4. The plasmid, pGEMhLF, was digested with Hind111 + Asp718 and repaired using PolIk. The resulting 2.3-kb hLF fragment was subcloned into a unique SmaI site located in the multiple cloning cassette of pAL3 downstream from the alcA promoter (Waring et al., 1989), generating pAL3hLF. The P-tubulin transcription terminator fragment was obtained by digesting the 3’-untranslated region of the benA gene (nt 2569-2665; May et al., 1987) with XbaI + NheI and subcloned into X&I-digested pAL3hLF generating pAL3hLFT. This plasmid was used to transform A. nidulans strain GR5 (pyrG89; wa3; pyroA4).

necessary for efficient fungal growth and protein production. The A. nidulans alcA gene, which encodes ADH 1, is expressed at high levels under inducing growth conditions (Pateman et al., 1983; Pickett et al., 1987). The alcA gene is induced by ethanol and is subject to strong catabolite repression by glucose, acetate or glycerol (Creaser et al., 1985; Waring et al., 1989). Extensive characterization of this gene has shown that all the regulatory promoter elements necessary for controlled gene expression are contained within the first 300 bp 5’ to the alcA start codon (Gwynne et al., 1987b). Here we describe the use of this 300-bp &A promoter fragment in an expression construct to direct the controlled production of biologically active hLF in A. nidulans.

(b) Transformation and Southern analysis Transformation was carried out as described (May, 1989). Protoplasts were transformed with 3 pg of the ex-

Pyr4 -2kb

(Xba Sma

Fig. 1. The A.niduluns expression

plasmid,

pAL3hLFT.

I/Nhe

I)

I

To express hLF in A. nidulans, we used pAL3 (Waring

et al., 1989). This plasmid

contains

300

bp of 5’4lanking sequence of the A. nidulans &A gene containing all the regulatory elements necessary pALhLFT, a 2.3.kb hLF cDNA fragment containing 17 nt of 5’-UI’R, the complete hLF ORF encoding

for controlled gene expression. To construct the secretory signal peptide and mature hLF,

followed by 209 nt of 3’ UTR was subcloned

promoter.

into a unique Smal

site in pAL3 downstream

the A. nidulans /I-tubulin-encoding (he&) gene was subcloned into a unique X&I site downstream an ApR marker and the N. cra.rsa pyr4 selectable marker (Waring et al., 1989).

from the &A

A 96-bp terminator

fragment

from

from the hLF cDNA sequence. The plasmid also contains

221 pression DNA.

plasmid

(c) Production of hLF in Aspergillus

with an efficiency of 40 transformants/pg

Transformants

obtained

were purified

Western

three times

through conidial spores. Southern blot analysis was performed to confirm that transformants contained integrated plasmid with hLF cDNA. The results are shown in Fig. 2. A hLF-specific radiolabelled band was detected at the expected size (2.3 kb) in lanes l-10 but not in DNA from control spores. These results demonstrate that hLF cDNA

to target the vector into a particular

nidulans

to determine

if the hLF

cDNA was expressed in the A.nidulans transformants under the control of the aZcA promoter. Conidia (1 x 106/ml) from transformant No. 5, which contained the highest number of copies of integrated hLF cDNAs, and from untransformed GR5 were inoculated into minimal medium utilizing glucose as the carbon source. After 18 h, the cultures were harvested, washed and reinoculated into minimal medium supplemented with 1.2% ethanol and grown for an

was integrated into the genome of all A. nidulans transformants tested and varied randomly from one copy (transformants Nos. 3, 6 and 10) to 20 copies (No. 5) per cell. The site of integration of the plasmid into the A. nidzdans genome is random due to the absence of homologous sequences

analysis was performed

additional 12 or 24 h before harvesting the cultures. Cell extracts and samples of the growth medium were resolved by SDS-PAGE, transferred to nitrocellulose and immunoblotted using a specfic polyclonal IgG directed against

site.

a i -

zoz z 0,

pAL3T Transformed

Control

E xc;

123456789100r’

5-l a, 0-u

-

al 07J

pAL3T Transform

Control 7--lal

u

ed

u a,

kb

Fig. 2. Fig. 2. Southern

blot analysis

formed spores as described to a nitrocellulose (hLF cDNA). tion solution

of transformed

(Rasmussen

filter and hybridized

Prehybridization contained

A.nidulans.

Extracts

~&3.

Cellular

Genomic

DNA was isolated

with a radiolabelled

hLF cDNA

probe (2.1-kb).

of the filter was performed

200 ng of 32P probe (2.1 kb; specfic activity 4 x 10” cpm/pg

on a 0.8% agarose

A sample (20 ng) of hLF cDNA

in 6 x SSC/O.l%

SDS/O.S%

of DNA).

to Kodak

and untrans-

gel and transferred

was used as a positive control

dried milk at 65 “C for 16 h. The hybridiza-

Filters were washed

SDS at 68°C for 30 min. The filter was dried and exposed

Medium

A.niduluns (GR5) transformants

et al., 1990). The DNA (1 pg) was digested with EcoRI, size fractionated

and hybridization

30 min followed by 0.5 x SSC/O.S%

Growth

from ten individual

in 2 x SSC/O.S% SDS at 68°C for

X-AR5 film at -70°C

for 30 min and devel-

oped by autoradiography. Fig. 3. Production

of re-hLF

in A.niduluns.

pg) from control

untransformed

GR5

Na.acetate

pH 6.5 as carbon

except for the addition CA). Mycelia

Methods: immunoblot

and transformant

source with or without

analysis

of recombinant

No. 5, respectively.

addition

Conidia

of 1.2% ethanol

hLF in cell extracts

to induce transcription

of 5 mM uridine and 10 mM uracil. Media and mycelia were harvested

(200 mg) were freeze-dried

nizer using 1 ml of phosphate-buffered

and lyophilized saline (PBS;

overnight.

Total cellular extracts

137 mM NaCl/2.7

mM KCl/4.3

(50 Ng) and growth medium

(1 x lO”/ml) were cultured

in minimal

of the hLF cDNA.

and separated

were prepared

(40

100 mM

GR5 was cultured

as above

using Miracloth

by homogenization

mM NazHP0,.7H,0/1.4

samples

media utilizing (Calbiochem,

San Diego,

in a glass teflon homoge-

mM K,HPO,

pH 7.4) in the presence

of phenylmethylsulfonylfluoride (PMSF, 10 pg). The homogenate was centrifuged at 12 000 x g for 30 min at 4°C and the supernatant containing the soluble fraction was recovered. The growth medium was concentrated by freeze drying and lyophilization and resuspended in l/30 vol. in PBS pH 7.4. Protein concentration containing

was determined 40 pg protein

using the Bradford

and soluble extracts

(Sigma, St. Louis, MO) was used as standard blot procedure

(Towbin

and then incubated

reagent according

(50 pg protein)

to manufacturer’s

were subjected

instructions

to 0.1 y0 SDS/7%

(hLF std). The resolved proteins were transferred

et al., 1979). Filters were blocked

for 2 h in the same with the addition

with Tris-buffered

(BioRad, PAGE

Richmond, (Laemmli,

to nitrocellulose

saline (TBS, 0.05 M Tris/O.lS

of a 1 pg/ml of a specific polyclonal

IgG directed

washes (5 x 10 min) were in TBS/0.05% Nonidet P-40 followed by incubation with 1 PCi of [ “‘Ilprotein (5 x 10 min) with TBS/O.OS% Nonidet P-40, dried and exposed overnight to Kodak XAR5 film at -70°C.

CA). Concentrated

media samples

1970). 250 ng of purified lactoferrin

filters electrophoretically

using the Western

M NaCl pH 7.5) containing against

hLF (Sigma,

2% dried milk

St. Louis, MO). Filter

A in TBS/Z% dried milk. The filter was washed The film was then developed by autoradiography.

222

hLF

Control

Fig. 4. Iron binding analysis (40 pg protein)

of re-hLF.

from transformant

shows the analysis

of purified standard iron-free

remove any unbound

lactoferrin.

standard

media-

The left panel shows the “‘Fe filter-binding

No. 5, containing

pH 2.0 to prepare for 30 min. Samples

hLF

Transformed-

1 Ng of re-hLF,

assay of duplicate

and of induced

untransformed

samples from growth GR5 (40 pg protein),

medium of induced respectively.

hLF at the concentrations indicated. All samples including hLF standard were dialysed Excess 5’Fe (0.2 PCi) was added to the samples in an equal volume of 1 M NaHCO,,

were then applied to a nitrocellulose 59Fe. The filter was developed

filter by manifold

by autoradiography

hLF. The results of the Western analysis are shown in Fig. 3. An immunoreactive band indistinguishable from native hLF was evident in the cells and growth medium from transformant No. 5 after 12 and 24 h growth only after ethanol induction. Cell extracts or growth medium obtained from untransformed GR5 did not contain an immunoreactive band even after addition of ethanol. These results demonstrate that hLF is expressed in transformed A. niduluns under the control of the &A promoter. Western analysis revealed hLF in the cells in all of the remaining transformants (data not shown). In general there was a correlation between the plasmid copy number and the expression levels obtained. In the medium re-hLF was detected only with transformants containing multiple copies of integrated expression plasmid (Nos. 1, 5, 7 and 10). In order to monitor the levels of hLF produced in the system, a pilot fermentation of transformant No. 5 was carried out using the growth parameters described above. ELISA analysis (Vilja et al., 1985) using a specific biotinylated IgG directed against hLF demonstrated that the total level of recombinant hLF produced was 5 pg/ml with approx. 30% (1.5-2.0 pg/ml) of this material secreted into the medium. (d) Iron binding analysis of re-hLF To test if recombinant lactoferrin synthesized and secreted in A. nidulans has an iron binding capacity similar to authentic human lactoferrin, samples of the growth medium of transformant No. 5 and untransformed GR5 spores were examined using an 59Fe microfilter-binding assay to detect 59Fe-bound lactoferrin. The results are shown in Fig. 4. Iron-binding (59Fe) is detected in the medium from transformant No. 5 but not in the medium from control untransformed GR5 spores. The distortion in recombinant LF dot pattern seen in this figure is due to the high salt concentration in concentrated media samples. These re-

dot-blot

procedure.

and iron-binding

The filter was washed

capacity

was determined

cultures

The right panel

against 0. I M citric acid and incubated at 37°C

several times in

I M NaHCO,

using solid scintillation

to

counting.

sults indicate that re-hLF produced in A.nidulans logically active in its capacity to bind 59Fe.

is bio-

(e) Conclusions The data presented in this study demonstrate the successful production of biologically active hLF in A. niduluns. The levels of hLF produced in A. nidulans were approx. 5 pg/ml with 30% of the re-hFL secreted into the growth medium. The secreted re-hLF was identical to native breast milk hLF with regard to size and immunoreactivity. Furthermore, the re-hLF was capable of binding iron. Although hLF has been reported to contain anti-fungal properties, neither the re-hLF nor native hLF when added to the growth medium, retarded the growth of this strain of A. niduluns. The production of biologically active re-hLF in A. nidulans will facilitate testing of possible nutritional and therapeutic uses of this protein.

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