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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