Note: Descriptions are shown in the official language in which they were submitted.
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Microtiter plate (MTP) based High Throughput Screening (HTS)
assays
Field of invention
s Technologies such as DNA shuffling, random DNA mutagenesis
and in vivo recombination have allowed the generation of enor-
mous populations of variant cells that produce variants of a
certain protein, RNA, or small molecule. In addition, it has
become possible in recombinant host strains to establish large
io libraries of natural enzymes cloned from other organisms. To-
gether these technologies have created a need for assays that
efficiently and accurately can screen large numbers of vari-
ants, high troughput screening (HTS) assays.
i5 Background of the invention
Historically, biological assays have been developed and op-
timized with regard to sensitivity, linearity and simplicity.
However, if these assays are to be transformed into high
throughput screens for substances produced by large populations
2o cell cultures, a number of difficulties must be overcome. Exam-
ples of difficulties encountered when using e.g. a microtiter
plate based screening assay include: a) differential growth
and/or gene expression of identical clones in the separate
wells of the microtiter plate, b) differential growth and/or
25 gene expression of different clones, c) differential stress ex-
posure (eg. heat treatment, humidity) across the plate, d) ef-
fects of cell-material or growth medium on the assay. In the
past, efficient and accurate high throughput screening has been
hampered by the inability to work around such difficulties.
so Below we describe a method of performing an assay that has
been specifically developed for the screening of large popula-
tions of clones producing variants of a given protein.
Summary of the invention
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The problem to be solved by the present invention is to pro-
vide a method to perform assays that efficiently and accurately
can screen large numbers of cell populations producing variants
of a molecule of interest.
In a first aspect the invention relates to a method for
high throughput screening (HTS) of a large population of host
cells for production of a molecule of interest, the method com-
prising the steps of:
a) arranging the host cells in a spatial array so each posi-
io tion in the spatial array is occupied by one cell,
b) cultivating the host cells under growth conditions suit-
able for HTS,
c) assaying each array position for production of the mole-
cule of interest, and
i5 d) selecting the cells from those positions where the mole-
cule was produced, as determined in step c).
In a second aspect the invention relates to a method for
screening a DNA library for DNA of interest, the method com
2o prising the steps of:
a) creating host cells comprising the DNA library,
b) using the method described in the first aspect to assay
for a host cell producing a molecule of interest; wherein
the host cell comprises the DNA of interest.
Further, in a third aspect the invention relates to a recom-
binant vector comprising DNA isolated by a method as defined in
the preceding aspect.
In a fourth aspect the invention also relates to a recombi
3o nant host cell comprising DNA isolated by a method as described
in the second aspect or the vector according to the third as
pect.
Also a fifth aspect relates to a transgenic animal contain
ing and expressing the DNA of interest according to any of the
second, third or fourth aspect.
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A sixth aspect relates to a transgenic plant containing and
expressing the DNA of interest according to any of the second,
third or fourth aspect.
A seventh aspect relates to a method of producing a molecule
s of interest, which method comprises cultivating a cell accord-
ing to any of the first or fourth aspects in suitable culture
medium under conditions permitting expression of the DNA of in-
terest and recovering the resulting molecule from the culture
medium.
io An eigth aspect relates to a method of producing a molecule
of interest, which method comprises recovering the molecule
from any part or secrete of/from the transgenic animal accord-
ing to the fifth aspect.
A final aspect relates to a method of producing a molecule
is of interest, which method comprises growing a cell of a trans-
genic plant according to the sixth aspect, and recovering the
molecule from the resulting plant.
Definitions
2o Prior to a discussion of the detailed embodiments of the in-
vention is provided a definition of specific terms related to
the main aspects of the invention.
In accordance with the present invention there may be em
ployed conventional molecular biology, microbiology, and recom
25 binant DNA techniques within the skill of the art. Such tech
niques are explained fully in the literature. See, e.g., Sam-
brook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Man-
ual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York (herein "Sambrook et al., 1989")
3o DNA Cloning: A Practical Approach, Volumes I and II /D. N.
Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed.
1984); Nucleic Acid Hybridization (B. D. Hames & S.J. Higgins
eds (1985)); Transcription And Translation (B. D. Hames & S.J.
Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed.
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(1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B.
Perbal, A Practical Guide To Molecular Cloning (1984).
Isolated: When applied to a protein, the term "isolated" indi-
Gates that the protein is found in a condition other than its
native environment, such as apart from blood and animal tissue.
In a preferred form, the isolated protein is substantially free
of other proteins, particularly other proteins of animal ori-
gin. It is preferred to provide the proteins in a highly puri-
io fied form, i.e., greater than 95o pure, more preferably greater
than 99% pure. When applied to a polynucleotide molecule, the
term "isolated" indicates that the molecule is removed from its
natural genetic milieu, and is thus free of other extraneous or
unwanted coding sequences, and is in a form suitable for use
i5 within genetically engineered protein production systems. Such
isolated molecules are those that are separated from their
natural environment and include cDNA and genomic clones. Iso-
lated DNA molecules of the present invention are free of other
genes with which they are ordinarily associated, and may in-
2o clude naturally occurring 5' and 3' un-translated regions such
as promoters and terminators. The identification of associated
regions will be evident to one of ordinary skill in the art
(see for example, Dynan and Tijan, Nature 316: 774-78, 1985).
25 A "polynucleotide" is a single- or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or pre-
pared from a combination of natural and synthetic molecules.
A "nucleic acid molecule" refers to the phosphate ester poly-
meric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxyribonucleosides (de-
oxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine;
"DNA molecules") in either single stranded form, or a double-
stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA
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helices are possible. The term nucleic acid molecule, and in
particular DNA or RNA molecule, refers only to the primary and
secondary structure of the molecule, and does not limit it to
any particular tertiary or quaternary forms. Thus, this term
s includes double-stranded DNA found, inter alia, in linear or
circular DNA molecules (e. g., restriction fragments), plasmids,
and chromosomes. In discussing the structure of particular dou-
ble-stranded DNA molecules, sequences may be described herein
according to the normal convention of giving only the sequence
io in the 5' to 3' direction along the non-transcribed strand of
DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
i5 A DNA "coding sequence" is a double-stranded DNA sequence which
is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
zo translation stop codon at the 3' (carboxyl) terminus. A coding
sequence can include, but is not limited to, prokaryotic se-
quences, cDNA from eukaryotic mRNA, genomic DNA sequences from
eukaryotic (e.g., mammalian) DNA, and even synthetic DNA se-
quences. If the coding sequence is intended for expression in a
25 eukaryotic cell, a polyadenylation signal and transcription
termination sequence will usually be located 3' to the coding
sequence.
Expression vector: A DNA molecule, linear or circular, that
3o comprises a segment encoding a polypeptide of interest operably
linked to additional segments that provide for its transcrip-
tion. Such additional segments may include promoter and termi-
nator sequences, and optionally one or more origins of replica-
tion, one or more selectable markers, an enhancer, a polyade-
3s nylation signal, and the like. Expression vectors are generally
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derived from plasmid or viral DNA, or may contain elements of
both.
Transcriptional and translational control sequences are DNA
s regulatory sequences, such as promoters, enhancers, termina-
tors, and the like, that provide for the expression of a coding
sequence in a host cell. In eukaryotic cells, polyadenylation
signals are control sequences.
io A "secretory signal sequence" is a DNA sequence that encodes a
polypeptide (a "secretory peptide" that, as a component of a
larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The
larger polypeptide is commonly cleaved to remove the secretory
i5 peptide during transit through the secretory pathway.
The term "promoter" is used herein for its art-recognized mean-
ing to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
zo transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
"Operably linked", when referring to DNA segments, indicates
that the segments are arranged so that they function in concert
2s for their intended purposes, e.g. transcription initiates in
the promoter and proceeds through the coding segment to the
terminator.
A coding sequence is "under the control" of transcriptional and
3o translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-
RNA spliced and translated into the protein encoded by the cod-
ing sequence.
35 "Isolated polypeptide": is a polypeptide which is essentially
free of other non-[enzyme] polypeptides, e.g., at least about
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20% pure, preferably at least about 40e pure, more preferably
about 60% pure, even more preferably about 80% pure, most pref-
erably about 90% pure, and even most preferably about 95°s pure,
as determined by SDS-PAGE.
"Heterologous" DNA refers to DNA not naturally located in the
cell, or in a chromosomal site of the cell. Preferably, the he-
terologous DNA includes a gene foreign to the cell.
io A cell has been "transfected" by exogenous or heterologous DNA
when such DNA has been introduced inside the cell. A cell has
been "transformed" by exogenous or heterologous DNA when the
transfected DNA effects a phenotypic change. Preferably, the
transforming DNA should be integrated (covalently linked) into
i5 chromosomal DNA making up the genome of the cell.
A "clone" is a population of cells derived from a single cell
or common ancestor by mitosis.
20 "Homologous recombination" refers to the insertion of a foreign
DNA sequence of a vector in a chromosome. Preferably, the vec-
tor targets a specific chromosomal site for homologous
recombination. For specific homologous recombination, the
vector will contain sufficiently long regions of homology to
2s sequences of the chromosome to allow complementary binding and
incorporation of the vector into the chromosome. Longer regions
of homology, and greater degrees of sequence similarity, may
increase the efficiency of homologous recombination.
3o Nucleic Acid Sequence
The techniques used to isolate or clone a nucleic acid
sequence encoding a polypeptide are known in the art and in-
clude isolation from genomic DNA, preparation from cDNA, or a
combination thereof. The cloning of the nucleic acid sequences
35 of the present invention from such genomic DNA can be effected,
e.g., by using the well-known polymerase chain reaction (PCR)
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or antibody screening of expression libraries to detect cloned
DNA fragments with shared structural features. See, e.g., In-
nis et al., 1990, A Guide to Methods and Application, Academic
Press, New York. Other nucleic acid amplification procedures
s such as ligase chain reaction (LCR), ligated activated tran-
scription (LAT) and nuceic acid sequence-based amplification
(NASBA) may be used. The nucleic acid sequence may be cloned
from a strain. producing the polypeptide, or from another re-
lated organism and thus, for example, may be an allelic or spe-
io cies variant of the polypeptide encoding region of the nucleic
acid sequence.
The term "isolated" nucleic acid sequence as used herein
refers to a nucleic acid sequence which is essentially free of
other nucleic acid sequences, e.g., at least about 20o pure,
i5 preferably at least about 40% pure, more preferably about 60%
pure, even more preferably about 80o pure, most preferably
about 90% pure, and even most preferably about 95o pure, as de-
termined by agarose gel electorphoresis. For example, an iso-
lated nucleic acid sequence can be obtained by standard cloning
zo procedures used in genetic engineering to relocate the nucleic
acid sequence from its natural location to a different site
where it will be reproduced. The cloning procedures may involve
excision and isolation of a desired nucleic acid fragment com-
prising the nucleic acid sequence encoding the polypeptide, in-
25 sertion of the fragment into a vector molecule, and incorpora-
tion of the recombinant vector into a host cell where multiple
copies or clones of the nucleic acid sequence will be repli-
cated. The nucleic acid sequence may be of genomic, cDNA, RNA,
semi-synthetic, synthetic origin, or any combinations thereof.
Nucleic Acid Construct
As used herein the term "nucleic acid construct" is in
tended to indicate any nucleic acid molecule of cDNA, genomic
DNA, synthetic DNA or RNA origin. The term "construct" is in
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tended to indicate a nucleic acid segment which may be single
or double-stranded, and which may be based on a complete or
partial naturally occurring nucleotide sequence encoding a po
lypeptide of interest. The construct may optionally contain
s other nucleic acid segments.
The DNA of interest may suitably be of genomic or cDNA origin,
for instance obtained by preparing a genomic or cDNA library
and screening for DNA sequences coding for all or part of the
polypeptide by hybridization using synthetic oligonucleotide
io probes in accordance with standard techniques (cf. Sambrook et
al., supra).
The nucleic acid construct may also be prepared syntheti-
cally by established standard methods, e.g. the phosphoamidite
method described by Beaucage and Caruthers, Tetrahedron Letters
i5 22 (1981), 1859 - 1869, or the method described by Matthes et
al., EMBO Journal 3 (1984), 801 - 805. According to the phos-
phoamidite method, oligonucleotides are synthesized, e.g. in an
automatic DNA synthesizer, purified, annealed, ligated and clo-
ned in suitable vectors.
2o Furthermore, the nucleic acid construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or mixed ge
nomic and cDNA origin prepared by ligating fragments of syn
thetic, genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid con
25 struct, in accordance with standard techniques.
The nucleic acid construct may also be prepared by poly-
merase chain reaction using specific primers, for instance as
described in US 4, 683, 202 or Saiki et al . , Science 239 (1988) ,
487 - 491.
3o The term nucleic acid construct may be synonymous with
the term expression cassette when the nucleic acid construct
contains all the control sequences required for expression of a
coding sequence of the present invention. The term "coding se-
quence" as defined herein is a sequence which is transcribed
35 into mRNA and translated into a polypeptide of the present
invention when placed under the control of the above mentioned
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control sequences. The boundaries of the coding sequence are
generally determined by a translation start codon ATG at the
5'-terminus and a translation stop codon at the 3'-terminus. A
coding sequence can include, but is not limited to, DNA, cDNA,
s and recombinant nucleic acid sequences.
The term "control sequences" is defined herein to include
all components which are necessary or advantageous for expres-
sion of the coding sequence of the nucleic acid sequence . Each
control sequence may be native or foreign to the nucleic acid
io sequence encoding the polypeptide. Such control sequences in-
clude, but are not limited to, a leader, a polyadenylation se-
quence, a propeptide sequence, a promoter, a signal sequence,
and a transcription terminator. At a minimum, the control se-
quences include a promoter, and transcriptional and transla-
i5 tional stop signals. The control sequences may be provided with
linkers for the purpose of introducing specific restriction si-
tes facilitating ligation of the control sequences with the
coding region of the nucleic acid sequence encoding a polypep-
tide.
2o The control sequence may be an appropriate promoter se-
quence, a nucleic acid sequence which is recognized by a host
cell for expression of the nucleic acid sequence. The promoter
sequence contains transcription and translation control se-
quences which mediate the expression of the polypeptide. The
25 promoter may be any nucleic acid sequence which shows tran-
scriptional activity in the host cell of choice and may be ob-
tained from genes encoding extracellular or intracellular poly-
peptides either homologous or heterologous to the host cell.
The control sequence may also be a suitable transcription
so terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably
linked to the 3' terminus of the nucleic acid sequence encoding
the polypeptide. Any terminator which is functional in the host
cell of choice may be used in the present invention.
35 The control sequence may also be a polyadenylation se-
quence, a sequence which is operably linked to the 3' terminus
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of the nucleic acid sequence and which, when transcribed, is
recognized by the host cell as a signal to add polyadenosine
residues to transcribed mRNA. Any polyadenylation sequence
which is functional in the host cell of choice may be used in
s the present invention.
The control sequence may also be a signal peptide-coding
region, which codes for an amino acid sequence linked to the
amino terminus of the polypeptide which can direct the ex-
pressed polypeptide into the cell's secretory pathway of the
io host cell. The 5' end of the coding sequence of the nucleic
acid sequence may inherently contain a signal peptide-coding
region naturally linked in translation reading frame with the
segment of the coding region which encodes the secreted poly-
peptide. Alternatively, the 5' end of the coding sequence may
i5 contain a signal peptide coding region which is foreign to that
portion of the coding sequence which encodes the secreted poly-
peptide. A foreign signal peptide-coding region may be required
where the coding sequence does not normally contain a signal
peptide-coding region. Alternatively, the foreign signal pep-
ao tide coding region may simply replace the natural signal pep-
tide coding region in order to obtain enhanced secretion rela-
tive to the natural signal peptide coding region normally asso-
ciated with the coding sequence. The signal peptide-coding re-
gion may be obtained from a glucoamylase or an amylase gene
is from an Aspergillus species, a lipase or proteinase gene from a
Rhizomucor species, the gene for the alpha-factor from Sac-
charomyces cerevisiae, an amylase or a protease gene from a Ba-
cillus species, or the calf preprochymosin gene. However, any
signal peptide coding region capable of directing the expressed
3o polypeptide into the secretory pathway of a host cell of choice
may be used in the present invention.
The control sequence may also be a propeptide coding re-
gion, which codes for an amino acid sequence positioned at the
amino terminus of a polypeptide. The resultant polypeptide is
35 known as a proenzyme or propolypeptide (or a zymogen in some
cases). A propolypeptide is generally inactive and can be con-
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verted to mature active polypeptide by catalytic or autocata-
lytic cleavage of the propeptide from the propolypeptide. The
propeptide coding region may be obtained from the Bacillus sub-
tilis alkaline protease gene (aprE), the Bacillus subtilis neu-
tral protease gene (nprT), the Saccharomyces cerevisiae alpha-
factor gene, or the Myceliophthora thermophilum laccase gene
(WO 95/33836).
The nucleic acid constructs of the present invention may
also comprise one or more nucleic acid sequences which encode
io one or more factors that are advantageous in the expression of
the polypeptide, e.g., an activator (e. g., a trans-acting fac-
tor), a chaperone, and a processing protease. Any factor that
is functional in the host cell of choice may be used in the
present invention. The nucleic acids encoding one or more of
is these factors are not necessarily in tandem with the nucleic
acid sequence encoding the polypeptide.
An activator is a protein which activates transcription
of a nucleic acid sequence encoding a polypeptide (Kudla et
al., 1990, EMBO Journal 9:1355-1364; Jarai and Buxton, 1994,
2o Current Genetics 26:2238-244; Verdier, 1990, Yeast 6:271-297).
The nucleic acid sequence encoding an activator may be obtained
from the genes encoding Bacillus stearothermophilus NprA
(nprA), Saccharomyces cerevisiae heme activator protein 1
(haply, Saccharomyces cerevisiae galactose metabolizing protein
25 4 (gal4), and Aspergillus nidulans ammonia regulation protein
(areA). For further examples, see Verdier, 1990, supra and
MacKenzie et al., 1993, Journal of General Microbiology
139:2295-2307.
A chaperone is a protein which assists another polypep
3o tide in folding properly (Hartl et al., 1994, TIBS 19:20-25;
Bergeron et al., 1994, TIBS 19:124-128; Demolder et al., 1994,
Journal of Biotechnology 32:179-189; Craig, 1993, Science
260:1902-1903; Gething and Sambrook, 1992, Nature 355:33-45;
Puig and Gilbert, 1994, Journal of Biological Chemistry
35 269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-
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11157; Robinson et al., 1994, Bio/Technology 1:381-384). The
nucleic acid sequence encoding a chaperone may be obtained from
the genes encoding Bacillus subtilis GroE proteins, Aspergillus
oryzae protein disulphide isomerase, Saccharomyces cerevisiae
s calnexin, Saccharomyces cerevisiae BiP/GRP78, and Saccharomyces
cerevisiae Hsp70. For further examples, see Gething and Sam-
brook, 1992, supra, and Hartl et al., 1994, supra.
A processing protease is a protease that cleaves a
propeptide to generate a mature biochemically active polypep
io tide (Enderlin and Ogrydziak, 1994, Yeast 10:67-79; Fuller et
al., 1989, Proceedings of the National Academy of Sciences USA
86:1434-1438; Julius et al., 1984, Cell 37:1075-1089; Julius et
al., 1983, Cell 32:839-852). The nucleic acid sequence encoding
a processing protease may be obtained from the genes encoding
i5 Aspergillus niger Kex2, Saccharomyces cerevisiae dipepti-
dylaminopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowia
lipolytica dibasic processing endoprotease (xpr6).
It may also be desirable to add regulatory sequences
which allow the regulation of the expression of the polypeptide
2o relative to the growth of the host cell. Examples of regula
tory systems are those which cause the expression of the gene
to be turned on or off in response to a chemical or physical
stimulus, including the presence of a regulatory compound.
Regulatory systems in prokaryotic systems would include the
z5 lac, tac, and trp operator systems . In yeast, the ADH2 system
or GAL1 system may be used. In filamentous fungi, the TAKA al-
pha-amylase promoter, Aspergillus niger glucoamylase promoter,
and the Aspergillus oryzae glucoamylase promoter may be used as
regulatory sequences. Other examples of regulatory sequences
3o are those which allow for gene amplification. In eukaryotic
systems, these include the dihydrofolate reductase gene which
is amplified in the presence of methotrexate, and the metal
lothionein genes which are amplified with heavy metals. In
these cases, the nucleic acid sequence encoding the polypeptide
35 would be placed in tandem with the regulatory sequence.
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Promoters
Examples of suitable promoters for directing the tran-
scription of the nucleic acid constructs of the present inven
tion, especially in a bacterial host cell, are the promoters
obtained from the E. coli lac operon, the Streptomyces coeli
color agarase gene (dagA), the Bacillus subtilis levansucrase
gene (sacB), the Bacillus subtilis alkaline protease gene, the
Bacillus licheniformis alpha-amylase gene (amyl), the Bacillus
io stearothermophilus maltogenic amylase gene (amyM), the Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus amy-
loliquefaciens BAN amylase gene, the Bacillus licheniformis pe-
nicillinase gene (penP), the Bacillus subtilis xylA and xylB
genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff
i5 et al., 1978, Proceedings of the National Academy of Sciences
USA 75:3727-3731), as well as the tac promoter (DeBoer et al.,
1983, Proceedings of the National Academy of Sciences USA
80:21-25) , or the Bacillus pumilus xylosidase gene, or by the
phage Lambda PR or PL promoters or the E. coli lac, ~ or tac
2o promoters. Further promoters are described in "Useful proteins
from recombinant bacteria" in Scientific American, 1980,
242:74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the tran
scription of the nucleic acid constructs of the present inven
25 tion in a filamentous fungal host cell are promoters obtained
from the genes encoding Aspergillus oryzae TAKA amylase, Rhi-
zomucor miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase, As-
pergillus niger or Aspergillus awamori glucoamylase (glaA),
3o Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus
nidulans acetamidase, Fusarium oxysporum trypsin-like protease
(as described in U.S. Patent No. 4,288,627, which is incorpo-
rated herein by reference), and hybrids thereof. Particularly
35 preferred promoters for use in filamentous fungal host cells
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are the TAKA amylase, NA2-tpi (a hybrid of the promoters from
the genes encoding Aspergillus niger neutral a-amylase and As-
pergillus oryzae triose phosphate isomerase), and glaA promot-
ers. Further suitable promoters for use in filamentous fungus
s host cells are the ADH3 promoter (McKnight et al . , The EMBO J.
4 (1985), 2093 - 2099) or the t~iA promoter.
Examples of suitable promoters for use in yeast host
cells include promoters from yeast glycolytic genes (Hitzeman
et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Ka-
io wasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol de-
hydrogenase genes (Young et al., in Genetic Engineering of Mi-
croorganisms for Chemicals (Hollaender et al, eds.), Plenum
Press, New York, 1982), or the TPI1 (US 4,599,311) or ADH2-4c
(Russell et al., Nature 304 (1983), 652 - 654) promoters.
i5 Further useful promoters are obtained from the Saccharo-
myces cerevisiae enolase (ENO-1) gene, the Saccharomyces cere-
visiae galactokinase gene (GAL1), the Saccharomyces cerevisiae
alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-
zo phosphoglycerate kinase gene. Other useful promoters for yeast
host cells are described by Romanos et al., 1992, Yeast 8:423-
488. In a mammalian host cell, useful promoters include viral
promoters such as those from Simian Virus 40 (5V40) , Rous sar-
coma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
Examples of suitable promoters for directing the tran
scription of the DNA encoding the polypeptide of the invention
in mammalian cells are the SV40 promoter (Subramani et al.,
Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein
gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814)
or the adenovirus 2 major late promoter.
An example of a suitable promoter for use in insect cells
is the polyhedrin promoter (US 4,745,051; Vasuvedan et al.,
FEBS Lett. 311, (1992) 7 - 11) , the P10 promoter (J.M. Vlak et
al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa
californica polyhedrosis virus basic protein promoter (EP 397
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16
485), the baculovirus immediate early gene 1 promoter (US
5,155,037; US 5,162,222), or the baculovirus 39K delayed-early
gene promoter (US 5,155,037; US 5,162,222).
s Terminators
Preferred terminators for filamentous fungal host cells
are obtained from the genes encoding Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Aspergillus niger alpha-glucosidase, and
io Fusarium oxysporum trypsin-like protease for fungal hosts) the
TPIl (Alber and Kawasaki, ~ cit.) or ADH3 (McKnight et al.,
op. cit.) terminators.
Preferred terminators for yeast host cells are obtained
from the genes encoding Saccharomyces cerevisiae enolase, Sac
is charomyces cerevisiae cytochrome C (CYC1), or Saccharomyces
cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other use-
ful terminators for yeast host cells are described by Romanos
et al., 1992, supra.
2o Polyadenylation Signals
Preferred polyadenylation sequences for filamentous fun
gal host cells are obtained from the genes encoding Aspergillus
oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergil
lus nidulans anthranilate synthase, and Aspergillus niger al
25 pha-glucosidase.
Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology
15:5983-5990.
Polyadenylation sequences are well known in the art for
3o mammalian host cells such as SV40 or the adenovirus 5 Elb re
gion.
Signal Sequences
An effective signal peptide-coding region for bacterial
35 host cells is the signal peptide-coding region obtained from
the maltogenic amylase gene from Bacillus NCIB 11837, the Ba
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cillus stearothermophilus alpha-amylase gene, the Bacillus
licheniformis subtilisin gene, the Bacillus licheniformis beta-
lactamase gene, the Bacillus stearothermophilus neutral prote-
ases genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA
s gene. Further signal peptides are described by Simonen and
Palva, 1993, Microbiological Reviews 57: 109-137.
An effective signal peptide coding region for filamentous
fungal host cells is the signal peptide coding region obtained
from Aspergillus oryzae TAKA amylase gene, Aspergillus niger
io neutral amylase gene, the Rhizomucor miehei aspartic proteinase
gene, the Humicola lanuginosa cellulase or lipase gene, or the
Rhizomucor miehei lipase or protease gene, Aspergillus sp. amy-
lase or glucoamylase, a gene encoding a Rhizomucor miehei li-
pase or protease. The signal peptide is preferably derived from
i5 a gene encoding A. oryzae TAKA amylase, A. niger neutral a-
amylase, A. niger acid-stable amylase, or A. niger glucoamy-
lase.
Useful signal peptides for yeast host cells are obtained
2o from the genes for Saccharomyces cerevisiae a-factor and Sac-
charomyces cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
For secretion from yeast cells, the secretory signal se
quence may encode any signal peptide which ensures efficient
25 direction of the expressed polypeptide into the secretory path
way of the cell. The signal peptide may be naturally occurring
signal peptide, or a functional part thereof, or it may be a
synthetic peptide. Suitable signal peptides have been found to
be the a-factor signal peptide, the signal peptide of mouse
3o salivary amylase (cf . O. Hagenbuchle et al . , Nature 289, 1981,
pp. 643-646), a modified carboxypeptidase signal peptide (cf.
L.A. Valls et al . , Cell 48, 1987, pp. 887-897) , the yeast BAR1
signal peptide (cf. WO 87/02670), or the yeast aspartic prote
ase 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast
35 6, 1990, pp. 127-137).
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For efficient secretion in yeast, a sequence encoding a
leader peptide may also be inserted downstream of the signal
sequence and upstream of the DNA sequence encoding the polypep-
tide. The function of the leader peptide is to allow the ex-
pressed polypeptide to be directed from the endoplasmic reticu-
lum to the Golgi apparatus and further to a secretory vesicle
for secretion into the culture medium (i.e. exportation of the
polypeptide across the cell wall or at least through the cellu-
lar membrane into the periplasmic space of the yeast cell). The
io leader peptide may be the yeast a-factor leader (the use of
which is described in e.g. US 4,546,082, EP 123 294, EP 123 544
and EP 163 529). Alternatively, the leader peptide may be a
synthetic leader peptide, which is to say a leader peptide not
found in nature. Synthetic leader peptides may, for instance,
i5 be constructed as described in WO 89/02463 or WO 92/11378.
For use in insect cells, the signal peptide may conven-
iently be derived from an insect gene (cf. WO 90/05783), such
as the lepidopteran Manduca sexta adipokinetic hormone precur-
sor signal peptide (cf. US 5,023,328).
Expression Vectors
The present invention also relates to recombinant expres
sion vectors comprising a nucleic acid sequence of the present
invention, a promoter, and transcriptional and translational
stop signals. The various nucleic acid and control sequences
described above may be joined together to produce a recombinant
expression vector which may include one or more convenient re-
striction sites to allow for insertion or substitution of the
3o nucleic acid sequence encoding the polypeptide at such sites.
Alternatively, the nucleic acid sequence of the present inven-
tion may be expressed by inserting the nucleic acid sequence or
a nucleic acid construct comprising the sequence into an appro-
priate vector for expression. In creating the expression vec-
tor, the coding sequence is located in the vector so that the
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19
coding sequence is operably linked with the appropriate control
sequences for expression, and possibly secretion.
The recombinant expression vector may be any vector
(e. g., a plasmid or virus) which can be conveniently subjected
s to recombinant DNA procedures and can bring about the expres
sion of the nucleic acid sequence. The choice of the vector
will typically depend on the compatibility of the vector with
the host cell into which the vector is to be introduced. The
vectors may be linear or closed circular plasmids. The vector
io may be an autonomously replicating vector, i.e., a vector which
exists as an extrachromosomal entity, the replication of which
is independent of chromosomal replication, e.g., a plasmid, an
extrachromosomal element, a mini-chromosome, or an artificial
chromosome. The vector may contain any means for assuring self-
i5 replication. Alternatively, the vector may be one which, when
introduced into the host cell, is integrated into the genome
and replicated together with the chromosomes) into which it
has been integrated. The vector system may be a single vector
or plasmid or two or more vectors or plasmids which together
zo contain the total DNA to be introduced into the genome of the
host cell, or a transposon.
The vectors of the present invention preferably contain
one or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
z5 which provides for biocide or viral resistance, resistance to
heavy metals, prototrophy to auxotrophs, and the like. Examples
of bacterial selectable markers are the dal genes from Bacillus
subtilis or Bacillus licheniformis, or markers which confer an-
tibiotic resistance such as ampicillin, kanamycin, chloram-
3o phenicol, tetracycline, neomycin, hygromycin or methotrexate
resistance. A frequently used mammalian marker is the dihydro-
folate reductase gene (DHFR). Suitable markers for yeast host
cells are ADE2 , HIS3 , LEU2 , LYS2 , MET3 , TRPl , and URA3 . A se-
lectable marker for use in a filamentous fungal host cell may
35 be selected from the group including, but not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
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(phosphinothricin acetyltransferase), hygB (hygromycin phos-
photransferase), niaD (nitrate reductase), pyre (orotidine-5'-
phosphate decarboxylase), sC (sulfate adenyltransferase), trpC
(anthranilate synthase), and glufosinate resistance markers, as
s well as equivalents from other species. Preferred for use in an
Aspergillus cell are the amdS and pyre markers of Aspergillus
nidulans or Aspergillus oryzae and the bar marker of Streptomy
ces hygroscopicus. Furthermore, selection may be accomplished
by co-transformation, e.g., as described in WO 91/17243, where
io the selectable marker is on a separate vector.
The vectors of the present invention preferably contain
an elements) that permits stable integration of the vector
into the host cell genome or autonomous replication of the vec-
tor in the cell independent of the genome of the cell.
is The vectors of the present invention may be integrated
into the host cell genome when introduced into a host cell.
For integration, the vector may rely on the nucleic acid se-
quence encoding the polypeptide or any other element of the
vector for stable integration of the vector into the genome by
2o homologous or non-homologous recombination. Alternatively, the
vector may contain additional nucleic acid sequences for di-
recting integration by homologous recombination into the genome
of the host cell. The additional nucleic acid sequences enable
the vector to be integrated into the host cell genome at a pre-
2s cise locations) in the chromosome(s). To increase the likeli-
hood of integration at a precise location, the integrational
elements should preferably contain a sufficient number of nu-
cleic acids, such as 100 to 1,500 base pairs, preferably 400 to
1, 500 base pairs, and most preferably 800 to 1, 500 base pairs,
3o which are highly homologous with the corresponding target se-
quence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homolo-
gous with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
3s encoding nucleic acid sequences . On the other hand, the vector
may be integrated into the genome of the host cell by non-
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21
homologous recombination. These nucleic acid sequences may be
any sequence that is homologous with a target sequence in the
genome of the host cell, and, furthermore, may be non-encoding
or encoding sequences.
s For autonomous replication, the vector may further com-
prise an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of bacte-
rial origins of replication are the origins of replication of
plasmids pBR322, pUCl9, pACYC177, pACYC184, pUB110, pE194,
io pTA1060, and pAMi3l. Examples of origin of replications for use
in a yeast host cell are the 2 micron origin of replication,
the combination of CEN6 and ARS4, and the combination of CEN3
and ARS1. The origin of replication may be one having a muta-
tion which makes its functioning temperature-sensitive in the
is host cell (see, e.g., Ehrlich, 1978, Proceedings of the Na-
tional Academy of Sciences USA 75:1433).
More than one copy of a nucleic acid sequence encoding a
polypeptide of the present invention may be inserted into the
host cell to amplify expression of the nucleic acid sequence.
zo Stable amplification of the nucleic acid sequence can be ob-
tained by integrating at least one additional copy of the se-
quence into the host cell genome using methods well known in
the art and selecting for transformants.
The procedures used to ligate the elements described
zs above to construct the recombinant expression vectors of the
present invention are well known to one skilled in the art
(see, e.g., Sambrook et al., 1989, supra).
Host Cells
3o The present invention also relates to recombinant host
cells, comprising a nucleic acid sequence of the invention,
which are advantageously used in the recombinant production of
the polypeptides. The term "host cell" encompasses any progeny
of a parent cell which is not identical to the parent cell due
3s to mutations that occur during replication.
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22
The cell is preferably transformed with a vector compris-
ing a nucleic acid sequence of the invention followed by inte-
gration of the vector into the host chromosome. "Transforma-
tion" means introducing a vector comprising a nucleic acid se-
quence of the present invention into a host cell so that the
vector is maintained as a chromosomal integrant or as a self-
replicating extra-chromosomal vector. Integration is generally
considered to be an advantage as the nucleic acid sequence is
more likely to be stably maintained in the cell. Integration of
io the vector into the host chromosome may occur by homologous or
non-homologous recombination as described above.
The choice of a host cell will to a large extent depend
upon the gene encoding the polypeptide and its source. The host
cell may be a unicellular microorganism, e.g., a prokaryote, or
i5 a non-unicellular microorganism, e.g., a eukaryote. Useful uni-
cellular cells are bacterial cells such as gram positive bacte-
ria including, but not limited to, a Bacillus cell, e.g., Ba-
cillus alkalophilus, Bacillus amyloliquefaciens, Bacillus bre-
vis, Bacillus circulans, Bacillus coagulans, Bacillus lautus,
2o Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus stearothermophilus, Bacillus subtilis, and Bacillus
thuringiensis; or a Streptomyces cell, e.g., Streptomyces livi-
dans or Streptomyces murinus, or gram negative bacteria such as
E. coli and Pseudomonas sp. In a preferred embodiment, the bac-
z5 terial host cell is a Bacillus lentus, Bacillus licheniformis,
Bacillus stearothermophilus or Bacillus subtilis cell. The
transformation of a bacterial host cell may, for instance, be
effected by protoplast transformation (see, e.g., Chang and
Cohen, 1979, Molecular General Genetics 168:111-115), by using
3o competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81:823-829, or Dubnar and Davidoff-Abelson,
1971, Journal of Molecular Biology 56:209-221), by electropora
tion (see, e.g., Shigekawa and Dower, 1988, Biotechniques
6:742-751), or by conjugation (see, e.g., Koehler and Thorne,
35 1987, Journal of Bacteriology 169:5771-5278).
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23
The host cell may be a eukaryote, such as a mammalian
cell, an insect cell, a plant cell or a fungal cell.
Useful mammalian cells include Chinese hamster ovary
(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS
s cells, or any number of other immortalized cell lines avail
able, e.g., from the American Type Culture Collection.
Examples of suitable mammalian cell lines are the COS (ATCC CRL
1650 and 1651), BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL
10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods
io of transfecting mammalian cells and expressing DNA sequences
introduced in the cells are described in e.g. Kaufman and
Sharp, J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg,
J . Mol . Appl . Genet . 1 ( 1982 ) , 32 7 - 341; Loyter et al . , Proc .
Natl. Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell
is 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7
(1981), 603, Ausubel et al., Current Protocols in Molecular Bi
ology, John Wiley and Sons, Inc., N.Y., 1987, Hawley-Nelson et
al., Focus 15 (1993), 73; Ciccarone et al., Focus 15 (1993),
80; Graham and van der Eb, Virology 52 (1973), 456; and Neumann
2o et al., EMBO J. 1 (1982), 841 - 845.
In a preferred embodiment, the host cell is a fungal
cell. "Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
zs Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth
et al., 1995, supra, page 171) and all mitosporic fungi (Hawk-
sworth et al., 1995, supra). Representative groups of Ascomy-
cota include, e.g., Neurospora, Eupenicillium (=Penicillium),
3o Emericella (=Aspergillus), Eurotium (=Aspergillus), and the
true yeasts listed above. Examples of Basidiomycota include
mushrooms, rusts, and smuts. Representative groups of Chy-
tridiomycota include, e.g., Allomyces, Blastocladiella, Coelo-
momyces, and aquatic fungi. Representative groups of Oomycota
3s include, e.g., Saprolegniomycetous aquatic fungi (water molds)
such as Achlya. Examples of mitosporic fungi include Aspergil-
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24
lus, Penicillium, Candida, and Alternaria. Representative
groups of Zygomycota include, e.g., Rhizopus and Mucor.
In a preferred embodiment, the fungal host cell is a
yeast cell. "Yeast" as used herein includes ascosporogenous
s yeast (Endomycetales), basidiosporogenous yeast, and yeast be
longing to the Fungi Imperfecti (Blastomycetes). The ascosporo-
genous yeasts are divided into the families Spermophthoraceae
and Saccharomycetaceae. The latter is comprised of four sub-
families, Schizosaccharomycoideae (e. g., genus Schizosaccharo-
io myces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae
(e.g., genera Pichia, Kluyveromyces and Saccharomyces). The
basidiosporogenous yeasts include the genera Leucosporidim,
Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidi-
ella. Yeasts belonging to the Fungi Imperfecti are divided
i5 into two families, Sporobolomycetaceae (e. g., genera Sorobolo-
myces and Bullera) and Cryptococcaceae (e. g., genus Candida).
Since the classification of yeast may change in the future, for
the purposes of this invention, yeast shall be defined as de-
scribed in Biology and Activities of Yeast (Skinner, F.A.,
zo Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol.
Symposium Series No. 9, 1980. The biology of yeast and manipu-
lation of yeast genetics are well known in the art (see, e.g.,
Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J.,
and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts,
25 Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and
The Molecular Biology of the Yeast Saccharomyces, Strathern et
al., editors, 1981).
The yeast host cell may be selected from a cell of a spe
cies of Candida, Kluyveromyces, Saccharomyces, Schizosaccharo
3o myces, Candida, Pichia, Hansehula, or Yarrowia. In a preferred
embodiment, the yeast host cell is a Saccharomyces carlsbergen-
sis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Sac-
charomyces douglasii, Saccharomyces kluyveri, Saccharomyces
norbensis or Saccharomyces oviformis cell. Other useful yeast
35 host cells are a Kluyveromyces lactis Kluyveromyces fragilis
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Hansehula polymorpha, Pichia pastoris Yarrowia lipolytica,
Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose,
Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et
al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279
s and US 4,879,231).
In a preferred embodiment, the fungal host cell is a fil-
amentous fungal cell. "Filamentous fungi" include all filamen-
tous forms of the subdivision Eumycota and Oomycota (as defined
by Hawksworth et al., 1995, supra). The filamentous fungi are
io characterized by a vegetative mycelium composed of chitin, cel-
lulose, glucan, chitosan, mannan, and other complex polysaccha-
rides. Vegetative growth is by hyphal elongation and carbon
catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding
is of a unicellular thallus and carbon catabolism may be fermenta-
tive. In a more preferred embodiment, the filamentous fungal
host cell is a cell of a species of, but not limited to, Acre-
monium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,
Neurospora, Penicillium, Thielavia, Tolypocladium, and Tricho-
2o derma or a teleomorph or synonym thereof . In an even more pre-
ferred embodiment, the filamentous fungal host cell is an As-
pergillus cell. In another even more preferred embodiment, the
filamentous fungal host cell is an Acremonium cell. In another
even more preferred embodiment, the filamentous fungal host
z5 cell is a Fusarium cell. In another even more preferred embodi-
ment, the filamentous fungal host cell is a Humicola cell. In
another even more preferred embodiment, the filamentous fungal
host cell is a Mucor cell. In another even more preferred em-
bodiment, the filamentous fungal host cell is a Myceliophthora
3o cell. In another even more preferred embodiment, the filamen-
tous fungal host cell is a Neurospora cell. In another even
more preferred embodiment, the filamentous fungal host cell is
a Penicillium cell. In another even more preferred embodiment,
the filamentous fungal host cell is a Thielavia cell. In an-
other even more preferred embodiment, the filamentous fungal
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26
host cell is a Tolypocladium cell. In another even more pre-
ferred embodiment, the filamentous fungal host cell is a
Trichoderma cell. In a most preferred embodiment, the filamen-
tous fungal host cell is an Aspergillus awamori, Aspergillus
s foetidus, Aspergillus japonicas, Aspergillus niger, Aspergillus
nidulans or Aspergillus oryzae cell. In another most preferred
embodiment, the filamentous fungal host cell is a Fusarium cell
of the section Discolor (also known as the section Fusarium).
For example, the filamentous fungal parent cell may be a Fusa-
io rium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sulphureum, or Fusarium trichothecioides cell. In an-
ts other preferred embodiment, the filamentous fungal parent cell
is a Fusarium strain of the section Elegans, e.g., Fusarium ox-
ysporum. In another most preferred embodiment, the filamentous
fungal host cell is a Humicola insolens or Humicola lanuginosa
cell. In another most preferred embodiment, the filamentous
2o fungal host cell is a Mucor miehei cell. In another most pre-
ferred embodiment, the filamentous fungal host cell is a My-
celiophthora thermophilum cell. In another most preferred em-
bodiment, the filamentous fungal host cell is a Neurospora
crassa cell. In another most preferred embodiment, the filamen-
ts tous fungal host cell is a Penicillium purpurogenum cell. In
another most preferred embodiment, the filamentous fungal host
cell is a Thielavia terrestris cell or an Acremonium chry-
sogenum cell. In another most preferred embodiment, the Tricho-
derma cell is a Trichoderma harzianum, Trichoderma koningii,
3o Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma
viride cell. The use of Aspergillus spp. for the expression of
proteins is described in, e.g., EP 272 277, EP 230 023.
3s Transformation
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27
Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suit-
able procedures for transformation of Aspergillus host cells
s are described in EP 238 023 and Yelton et al., 1984, Proceed-
ings of the National Academy of Sciences USA 81:1470-1474. A
suitable method of transforming Fusarium species is described
by Malardier et al., 1989, Gene 78:147-156 or in co-pending US
Serial No. 08/269,449. Examples of other fungal cells are cells
io of filamentous fungi, e.g. Aspergillus spp., Neurospora spp.,
Fusarium spp. or Trichoderma spp., in particular strains of A.
oryzae, A. nidulans or A. niger. The use of Aspergillus spp.
for the expression of proteins is described in, e.g., EP 272277
or EP 230023. The transformation of F. oxysporum may for in-
i5 stance be carried out as described by Malardier et al., 1989,
Gene 78: 147-156. The transformation of Trichoderma spp. may be
performed as known in the art.
Yeast may be transformed using the procedures described
by Becker and Guarente, In Abelson, J.N. and Simon, M.I., edi
2o tors, Guide to Yeast Genetics and Molecular Biology, Methods in
Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New
York; Ito et al., 1983, Journal of Bacteriology 153:163; and
Hinnen et al., 1978, Proceedings of the National Academy of
Sciences USA 75:1920. Mammalian cells may be transformed by di-
25 rect uptake using the calcium phosphate precipitation method of
Graham and Van der Eb (1978, Virology 52:546).
Transformation of insect cells and production of het-
erologous polypeptides therein may be performed as described in
US 4,745,051; US 4,775,624; US 4,879,236; US 5,155,037; US
30 5,162,222; EP 397,485, all of which are incorporated herein by
reference. The insect cell line used as the host may suitably
be a Lepidoptera cell line, such as Spodoptera frugiperda cells
or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions
may suitably be as described in, for instance, WO 89/01029 or
35 WO 89/01028, or any of the aforementioned references.
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28
Transgenic animals
It is also within the scope of the present invention to
employ transgenic animal technology to produce the present
s polypeptide . A transgenic animal is one in whose genome a het
erologous DNA sequence has been introduced. In particular, the
polypeptide of the invention may be expressed in the mammary
glands of a non-human female mammal, in particular one which is
known to produce large quantities of milk. Examples of pre-
io ferred mammals are livestock animals such as goats, sheep and
cattle, although smaller mammals such as mice, rabbits or rats
may also be employed.
The DNA sequence encoding the present polypeptide may be
introduced into the animal by any one of the methods previously
i5 described for the purpose. For instance, to obtain expression
in a mammary gland, a transcription promoter from a milk pro-
tein gene is used. Milk protein genes include the genes encod-
ing casein (cf. US 5,304,489), beta-lactoglobulin, alpha-
lactalbumin and whey acidic protein. The currently preferred
2o promoter is the beta-lactoglobulin promoter (cf. Whitelaw et
al., Biochem J. 286, 1992, pp. 31-39).
It is generally recognized in the art that DNA sequences
lacking introns are poorly expressed in transgenic animals in
comparison with those containing introns (cf. Brinster et al.,
z5 Proc. Natl. Acad. Sci. USA 85, 1988, pp. 836-840; Palmiter et
al., Proc. Natl. Acad. Sci. USA 88, 1991, pp. 478-482; Whitelaw
et al., Transgenic Res. 1, 1991, pp. 3-13; WO 89/01343; WO
91/02318). For expression in transgenic animals, it is there-
fore preferred, whenever possible, to use genomic sequences
3o containing all or some of the native introns of the gene encod-
ing the polypeptide of interest. It may also be preferred to
include at least some introns from, e.g. the beta-lactoglobulin
gene. One such region is a DNA segment which provides for in-
tron splicing and RNA polyadenylation from the 3' non-coding
35 region of the ovine beta-lactogloblin gene. when substituted
for the native 3' non-coding sequences of a gene, this segment
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29
may will enhance and stabilise expression levels of the poly-
peptide of interest. It may also be possible to replace the re-
gion surrounding the initiation codon of the polypeptide of in-
terest with corresponding sequences of a milk protein gene.
s Such replacement provides a putative tissue-specific initiation
environment to enhance expression.
For expression of the present polypeptide in transgenic
animals, a nucleotide sequence encoding the polypeptide is op-
erably linked to additional DNA sequences required for its ex-
io pression to produce expression units. Such additional sequences
include a promoter as indicated above, as well as sequences
providing for termination of transcription and polyadenylation
of mRNA. The expression unit further includes a DNA sequence
encoding a secretory signal sequence operably linked to the se-
i5 quence encoding the polypeptide. The secretory signal sequence
may be one native to the polypeptide or may be that of another
protein such as a milk protein (cf. von Heijne et al., Nucl.
Acids Res. 14, 1986, pp. 4683-4690; and US 4,873,316).
Construction of the expression unit for use in transgenic
2o animals may conveniently be done by inserting a DNA sequence
encoding the present polypeptide into a vector containing the
additional DNA sequences, although the expression unit may be
constructed by essentially any sequence of legations. It is
particularly convenient to provide a vector containing a DNA
as sequence encoding a milk protein and to replace the coding re-
gion for the milk protein with a DNA sequence coding for the
present polypeptide, thereby creating a fusion which includes
expression control sequences of the milk protein gene.
The expression unit is then introduced into fertilized
30 ova or early-stage embryos of the selected host species. Intro
duction of heterologous DNA may be carried out in a number of
ways, including microinjection (cf. US 4,873,191), retroviral
infection (cf. Jaenisch, Science 240, 1988, pp. 1468-1474) or
site-directed integration using embryonic stem cells (reviewed
35 by Bradley et al., Bio/Technology 10, 1992, pp. 534-539). The
ova are then implanted into the oviducts or uteri of pseudo-
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pregnant females and allowed to develop to term. Offspring car-
rying the introduced DNA in their germ line can pass the DNA on
to their progeny, allowing the development of transgenic herds.
General procedures for producing transgenic animals are
s known in the art, cf . for instance, Hogan et al . , Manipulating
the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Labo
ratory, 1986; Simons et al., Bio/Technology 6, 1988, pp. 179
183; Wall et al., Biol. Reprod. 32, 1985, pp. 645-651; Buhler
et al., Bio/Technology 8, 1990, pp. 140-143; Ebert et al.,
io Bio/Technology 6: 179-183, 1988; Krimpenfort et al.,
Bio/Technology 9: 844-847, 1991, Wall et al., J. Cell. Biochem.
49: 113-120, 1992; US 4,873,191, US 4,873,316; WO 88/00239, WO
90/05188; WO 92/11757 and GB 87/00458. Techniques for introduc-
ing heterologous DNA sequences into mammals and their germ
is cells were originally developed in the mouse. See, e.g. Gordon
et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980, Gordon
and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brin-
ster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl.
Acad. Sci. USA 82: 4438-4442, 1985; and Hogan et al. (ibid.).
zo These techniques were subsequently adapted for use with larger
animals, including livestock species (see e.g., WO 88/00239, WO
90/01588 and WO 92/11757; and Simons et al., Bio/Technology 6:
179-183, 1988). To summarize, in the most efficient route used
to date in the generation of transgenic mice or livestock, sev-
2s eral hundred linear molecules of the DNA of interest are in-
jected into one of the pro-nuclei of a fertilized egg according
to techniques which have become standard in the art . Inj ection
of DNA into the cytoplasm of a zygote can also be employed.
3o Transgenic plants
Production in transgenic plants may also be employed. It
has previously been described to introduce DNA sequences into
plants, which sequences code for protein products imparting to
the transformed plants certain desirable properties such as in-
3s creased resistance against pests, pathogens, herbicides or
stress conditions (cf. for instance EP 131 620, EP 205 518, EP
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31
270 355, WO 89/04371 or WO 90/02804), or an improved nutrient
value of the plant proteins (cf. for instance EP 90 033, EP 205
518 or WO 89/04371). Furthermore, WO 89/12386 discloses the
transformation of plant cells with a gene coding for levansu-
s crase or dextransucrase, regeneration of the plant (especially
a tomato plant) from the cell resulting in fruit products with
altered viscosity characteristics.
In the plant cell, the DNA sequence encoding the present
polypeptide is under the control of a regulatory sequence which
io directs the expression of the polypeptide from the DNA sequence
in plant cells and intact plants. The regulatory sequence may
be either endogenous or heterologous to the host plant cell.
The regulatory sequence may comprise a promoter capable
of directing the transcription of the DNA sequence encoding the
is polypeptide in plants . Examples of promoters which may be used
according to the invention are the 35s RNA promoter from cauli-
flower mosaic virus (CaMV) , the class I patatin gene B 33 pro-
moter, the ST-LS1 gene promoter, promoters conferring seed-
specific expression, e.g. the phaseolin promoter, or promoters
2o which are activated on wounding, such as the promoter of the
proteinase inhibitor II gene or the wunl or wun2 genes.
The promoter may be operably connected to an enhancer se-
quence, the purpose of which is to ensure increased transcrip-
tion of the DNA sequence encoding the polypeptide. Examples of
as useful enhancer sequences are enhancers from the 5'-upstream
region of the 35s RNA of CaMV, the 5'-upstream region of the
ST-LS1 gene, the 5'-upstream region of the Cab gene from wheat,
the 5'-upstream region of the 1'- and 2'-genes of the TR-DNA of
the Ti plasmid pTi ACH5, the 5'-upstream region of the octopine
3o synthase gene, the 5'-upstream region of the leghemoglobin ge-
ne, etc.
The regulatory sequence may also comprise a terminator
capable of terminating the transcription of the DNA sequence
encoding the polypeptide in plants. Examples of suitable termi-
35 nators are the terminator of the octopine synthase gene of the
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32
T-DNA of the Ti-plasmid pTiACH5 of Agrobacterium tumefaciens,
of the gene 7 of the T-DNA of the Ti plasmid pTiACH5, of the
nopaline synthase gene, of the 35s RNA-coding gene from CaMV or
from various plant genes, e.g. the ST-LS1 gene, the Cab gene
s from wheat, class I and class II patatin genes, etc.
The DNA sequence encoding the polypeptide may also be op-
erably connected to a DNA sequence encoding a leader peptide
capable of directing the transport of the expressed polypeptide
to a specific cellular compartment (e.g. vacuoles) or to ex-
io tracellular space. Examples of suitable leader peptides are the
leader peptide of proteinase inhibitor II from potato, the
leader peptide and an additional about 100 amino acid fragments
of patatin, or the transit peptide of various nucleus-encoded
proteins directed into chloroplasts (e. g. from the St-LS1 gene,
i5 SS-Rubisco genes, etc.) or into mitochondria (e.g. from the
ADP/ATP translocator).
Furthermore, the DNA sequence encoding the polypeptide
may be modified in the 5' non-translated region resulting in
enhanced translation of the sequence. Such modifications may,
2o for instance, result in removal of hairpin loops in RNA of the
5' non-translated region. Translation enhancement may be pro-
vided by suitably modifying the omega sequence of tobacco mo-
saic virus or the leaders of other plant viruses (e. g. BMV,
MSV) or of plant genes expressed at high levels (e.g. SS-
25 Rubisco, class I patatin or proteinase inhibitor II genes from
potato) .
The DNA sequence encoding the polypeptide may furthermore
be connected to a second DNA sequence encoding another polypep-
tide or a fragment thereof in such a way that expression of
3o said DNA sequences results in the production of a fusion pro-
tein. When the host cell is a potato plant cell, the second DNA
sequence may, for instance, encode patatin or a fragment the-
reof (such as a fragment of about 100 amino acids).
The plant in which the DNA sequence coding for the poly
3s peptide is introduced may suitably be a dicotyledonous plant,
examples of which are a tobacco, potato, tomato, or leguminous
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33
(e. g. bean, pea, soy, alfalfa) plant. It is, however, contem-
plated that monocotyledonous plants, e.g. cereals, may equally
well be transformed with the DNA sequence coding for the en-
zyme.
s Procedures for the genetic manipulation of mono-
cotyledonous and dicotyledonous plants are well known. In order
to construct foreign genes for their subsequent introduction
into higher plants, numerous cloning vectors are available
which generally contain a replication system for E. coli and a
io selectable/screenable marker system permitting the recognition
of transformed cells. These vectors include e.g. pBR322, the
pUC series, pACYC, M13 mp series etc. The foreign sequence may
be cloned into appropriate restriction sites. The recombinant
plasmid obtained in this way may subsequently be used for the
is transformation of E. coli. Transformed E. coli cells may be
grown in an appropriate medium, harvested and lysed. The chi-
meric plasmid may then be re-isolated and analyzed. Analysis of
the recombinant plasmid may be performed by a . g. determination
of the nucleotide sequence, restriction analysis, electrophore-
2o sis and other molecular-biochemical methods. After each manipu-
lation the sequence may be cleaved and ligated to another DNA
sequence. Each DNA sequence can be cloned on a separate plasmid
DNA. Depending on the way used for transferring the foreign DNA
into plant cells other DNA sequences might be of importance. In
zs case the Ti-plasmid or the Ri plasmid of Agrobacterium tumefa-
ciens or Agrobacterium rhizogenes, at least the right border of
the T-DNA may be used, and often both the right and the left
borders of the T-DNA of the Ri or Ti plasmid will be present
flanking the DNA sequence to be transferred into plant cells.
3o The use of the T-DNA for transferring foreign DNA into
plant cells has been described extensively in the prior litera-
ture (cf. Gasser and Fraley, 1989, Science 244, 1293 - 1299 and
references cited therein). After integration of the foreign DNA
into the plant genome, this sequence is fairly stable at the
3s original locus and is usually not lost in subsequent mitotic or
meiotic divisions. As a general rule, a selectable marker gene
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34
will be co-transferred in addition to the gene to be trans-
ferred, which marker renders the plant cell resistant to cer-
tain antibiotics, e.g. kanamycin, hygromycin, 6418 etc. This
marker permits the recognition of the transformed cells con-
s taming the DNA sequence to be transferred compared to non-
transformed cells.
Numerous techniques are available for the introduction of
DNA into a plant cell. Examples are the Agrobacterium mediated
transfer, the fusion of protoplasts with liposomes containing
io the respective DNA, microinjection of foreign DNA, electro-
poration etc. In case Agrobacterium mediated gene transfer is
employed, the DNA to be transferred has to be present in spe-
cial plasmids which are either of the intermediate type or the
binary type . Due to the presence of sequences homologous to T-
i5 DNA sequences, intermediate vectors may integrate into the Ri-
or Ti-plasmid by homologous recombination. The Ri- or Ti-
plasmid additionally contains the vir-region which is necessary
for the transfer of the foreign gene into plant cells. Interme-
diate vectors cannot replicate in Agrobacterium species and are
2o transferred into Agrobacterium by either direct transformation
or mobilization by means of helper plasmids (conjugation). (Cf.
Gasser and Fraley, op. cit. and references cited therein).
Binary vectors may replicate in both Agrobacterium spe
cies and E. coli. They may contain a selectable marker and a
25 poly-linker region which to the left and right contains the
border sequences of the T-DNA of Agrobacterium rhizogenes or
Agrobacterium tumefaciens. Such vectors may be transformed di-
rectly into Agrobacterium species. The Agrobacterium cell serv-
ing as the host cell has to contain a vir-region on another
3o plasmid. Additional T-DNA sequences may also be contained in
the Agrobacterium cell.
The Agrobacterium cell containing the DNA sequences to be
transferred into plant cells either on a binary vector or in
the form of a co-integrate between the intermediate vector and
35 the T-DNA region may then be used for transforming plant cells.
Usually either multicellular explants (e. g. leaf discs, stem
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segments, roots), single cells (protoplasts) or cell suspen-
sions are co-cultivated with Agrobacterium cells containing the
DNA sequence to be transferred into plant cells. The plant
cells treated with the Agrobacterium cells are then selected
s for the co-transferred resistance marker (e.g. kanamycin) and
subsequently regenerated to intact plants. These regenerated
plants will then be tested for the presence of the DNA se-
quences to be transferred.
If the DNA is transferred by e.g. electroporation or mi
io croinjection, no special requirements are needed to effect
transformation. Simple plasmids e.g. of the pUC series may be
used to transform plant cells. Regenerated transgenic plants
may be grown normally in a greenhouse or under other condi
tions. They should display a new phenotype (e.g. production of
i5 new proteins) due to the transfer of the foreign gene(s). The
transgenic plants may be crossed with other plants which may
either be wild-type or transgenic plants transformed with the
same or another DNA sequence. Seeds obtained from transgenic
plants should be tested to assure that the new genetic trait is
2o inherited in a stable Mendelian fashion.
See also Hiatt, Nature 344: 469-479, 1990; Edelbaum et
al., J. Interferon Res. 12: 449-453, 1992; Sijmons et al.,
Bio/Technology 8: 217-221, 1990: and EP 255 378.
25 Methods of Production
The transformed or transfected host cells described above
are cultured in a suitable nutrient medium under conditions
permitting the production of the desired molecules, after which
these are recovered from the cells, or the culture broth.
3o The medium used to culture the cells may be any conven-
tional medium suitable for growing the host cells, such as
minimal or complex media containing appropriate supplements.
Suitable media are available from commercial suppliers or may
be prepared according to published recipes (e. g. in catalogues
35 of the American Type Culture Collection). The media are pre-
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36
pared using procedures known in the art (see, e.g., references
for bacteria and yeast; Bennett, J.W. and LaSure, L., editors,
More Gene Manipulations in Fungi, Academic Press, CA, 1991).
If the molecules are secreted into the nutrient medium,
s they can be recovered directly from the medium. If they are not
secreted, they can recovered from cell lysates. The molecules
are recovered from the culture medium by conventional proce
dures including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
io components of the supernatant or filtrate by means of a salt,
e.g. ammonium sulphate, purification by a variety of chroma-
tographic procedures, e.g. ion exchange chromatography, gelfil-
tration chromatography, affinity chromatography, or the like,
dependent on the type of molecule in question.
i5 The molecules of interest may be detected using methods
known in the art that are specific for the molecules. These de-
tection methods may include use of specific antibodies, forma-
tion of a product, or disappearance of a substrate. For exam-
ple, an enzyme assay may be used to determine the activity of
2o the molecule. Procedures for determining various kinds of ac-
tivity are known in the art.
The molecules of the present invention may be purified by
a variety of procedures known in the art including, but not
limited to, chromatography (e.g., ion exchange, affinity, hy-
25 drophobic, chromatofocusing, and size exclusion), electropho-
retic procedures (e. g., preparative isoelectric focusing (IEF),
differential solubility (e. g., ammonium sulfate precipitation),
or extraction (see, e.g., Protein Purification, J.C. Janson and
Lars Ryden, editors, VCH Publishers, New York, 1989).
Detailed description of the invention
When cell cultures rather than purified substances are used
in assays, a complication arises: Cells, cell particles, growth
media or matter secreted by the cells can have an effect on the
treatment (e. g., heat treatment) of the substance in question,
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37
on the assay itself or on the detection of the assay signal.
The present invention provides solutions to these problems.
As it is not feasible to handle individual clones sepa
rately, it also becomes necessary with respect to the assay to
s level out differences in growth, gene expression and other dif
fering characteristics of individual clones, in order to handle
many clones simultaneously in an automated manner.
The present invention solves these problems, by defining
conditions of growth so as to achieve a uniform distribution of
io growth and gene expression in identical clones in the wells of
the same microtiter plate (MTP), or by developing a method for
doing HTS-assays that are independent of molecule concentra-
tions within a broad range. Solutions to these problems include
accurate and simple detection of the concentration of the ac-
i5 tive molecules, and development of automated assays that incor-
porate this information in the protocol during the assay, or
that produce raw-data that can be corrected by use of this in-
formation after the assay has been performed.
Altogether, the solutions provided by this invention
2o makes microtiter plate based high throughput screening of sub-
stances produced by cell cultures a practical and reliable
method for the identification of novel substances with inter-
esting properties, from large libraries of molecules.
A first preferred embodiment of the invention relates to
25 a method for high throughput screening (HTS) of a large popula
tion of host cells for production of a molecule of interest,
the method comprising the steps of:
a) arranging the host cells in a spatial array so each posi-
tion in the spatial array is occupied by one cell,
3o b) cultivating the host cells under growth conditions suit-
able for HTS,
c) assaying each array position for production of the mole-
cule of interest, and
d) selecting the cells from those positions where the mole-
35 cule was produced, as determined in step c).
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A method of the first aspect can be performed in many
ways, the spatial array of step a) can take on any physical
form whatsoever, that enables the assaying of several samples
s at once, without one sample contaminating another. Examples of
preferred spatial arrays are different kinds of microtiter-
plates with any number of wells, such as 96 or 384, and of any
kind of material, as well as positions in a High Performance
Liquid Chromatography (HPLC) autosampler device. Any kind of
io physical arrangement which allows the unambiguous identifica
tion of the samples by a number or a position in the array.
Even samples placed as drops on a surface in a specific re
corded pattern, the surface being of a solid material or of
more complex nature such as a textile or a tissue, e.g. cotton,
i5 wool, paper, or cellulose.
A preferred embodiment relates to a method of the first
aspect, wherein the spatial array of point a) is a microtiter
plate, a solid surface or a textile surface.
A way of carrying out step c) of the first aspect of the
2o invention could be to take a sample from each position of the
spatial array, e.g. from a supernatant or cell, and transfer
this to another spatial array for further testing or assaying
for production of the molecule of interest (see example 2). The
second spatial array may be identical to the first one used in
z5 the specific method, but may also be of any other kind that
fulfils the above mentioned criteria, such as a microtiter
plate, a solid surface, a textile, any material etc.
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein after step b) a sample is transferred
3o from each position in the spatial array to a position in a sec
ond spatial array which is then used onwards in the method,
preferably the second array is a microtiter plate, more pref-
erably a solid surface, and most preferably a textile surface.
It is a difficult task to make protein libraries in fila
35 mentous fungi. One problem is that the different morphology and
growth rate of different clones give rise to large and small
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39
clones that tend to grow out of their position in the spatial
array. Assaying fungal transformants of different sizes is also
a difficult task due to differences in amounts of produced
molecules. The present invention solves this by growing the in-
s dividual clones/cells within microenvironments in each position
of the spatial array (see example 9). Such microenvironments
are initially sterile beads or balls of any material that al-
lows growth of the clone, preferably beads comprising agarose,
alginate, polysaccharide, carbohydrate, alginate, carrageenan,
io chitosan, cellulose, pectin, dextran or polyacrylamide, all al-
lowing diffusion to/from each micro-environment.
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein each position in the spatial array is
occupied by a bead comprising one cell, preferably the bead is
is an agarose-bead.
The assaying of the first aspect of the invention for
production of the molecule of interest can be done in a great
number of ways. As indicated, some kinds of spatial arrays like
microtiter plates, can sometimes be assayed directly, or sam-
zo ples can be taken from each position and transferred to another
spatial array for assaying. The assay as such can be based on a
number of techniques well known in the art such as antibody
binding, ELISA and many others; common to these assays is that
a measurement is taken of a detectable property e.g. fluores-
2s cence, luminescence, (3-galactosidase or another enzymatic ac-
tivity etc. The method of the invention can be performed with
any number of these assays, and consequently the molecules as-
sayed for in the first aspect step c) can be obtained in a num-
ber of ways, depending on the host cell construct. The molecule
30 of interest may be secreted by the host cell into the super-
natant, lysis of the host cells may release the molecule or the
molecule may remain intracellular.
A preferred embodiment relates to a method of the first aspect,
wherein the molecule of interest in the first aspect step c) is
3s assayed in either whole broth, supernatant of cells that se
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Crete the molecule, a lysate of cells that produce the mole-
cule, or is assayed while still inside cells that produce the
molecule.
As described above, any number of host cells can be used
s to perform the method of the invention.
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the host cells are of mammalian ori-
gin, preferably hybridoma cells.
More preferably the host cells are of microbial origin,
io preferably bacterial or fungal.
Even more preferably the host cells are chosen from the
group consisting of Escherichia coli, Bacillus subtilis, Bacil
lus licheniformis, Bacillus clausii, Aspergillus niger, Asper
gillus oryzae, Aspergillus nidulans, and Saccharomyces cere
i5 visiae.
The molecule of interest in the first aspect of the in-
vention can be any molecule of biological origin, non-limiting
examples of which are peptides, polypeptides, proteins, en-
zo zymes, post-translationally modified polypeptides such as
lipopeptides or glycosylated peptides, antimicrobial peptides
or molecules, primary or secondary metabolites such as algi-
nates, small organic molecules, molecules having pharmaceutical
properties etc.
z5 Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the molecule of interest is chosen
from the group consisting of polypeptides, small peptides,
lipopeptides, antimicrobials, small molecules and pharmaceuti-
cal molecules.
3o Another preferred embodiment relates to a method of the
first aspect, wherein the molecule is an enzyme, preferably
chosen from the group consisting of amylase, amyloglucosidase,
carbohydrase, carboxypeptidase, cellulase, glucoamylase, glu-
cose oxidase, glucosidase, haloperoxidase, hemicellulase, in-
35 vertase, isomerase, laccase, lipase, lyase, mannosidase, oxi-
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41
dase, pectinolytic enzyme, peroxidase, phytase, phenoloxidase,
polyphenoloxidase, protease, and transglutaminase.
An obstacle when working with more than one clone is that
different clones produce different levels of different vari
ants, which means that a quantitative comparison of e.g. mole
cule yield between the clones is difficult. The invention pro-
vides a way to overcome this obstacle, by growing the clones
under conditions that ensure minimal variation in molecule con-
centration between the clones. Non-limiting examples of growth
io conditions that must be kept essentially equal over the entire
spatial array, or similar in each position are temperature,
component concentrations in solution (equalize the evaporation
from each position, see example 1), amount of molecule produced
in each position (see example 9) etc.
is Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the growth conditions of the first
aspect step b) ensure minimal variation in the molecule concen-
tration between all positions in the spatial array.
An obstacle to achieve efficient HTS is how to obtain an
2o accurate determination of the amount of specific active mole
cule produced by each individual clone. One solution is to con
figure an assay in such a way that it is insensitive to the
concentration of the molecule (as exemplified in example 2).
Another solution is that when the concentration of the specific
25 molecule in a position of the spatial array has been deter-
mined, this information can be used to determine the specific
activity from the total activity determined in that position;
alternatively, the information can be used to adjust the input-
amount of the molecule into the activity assay.
3o Non-limiting examples of how the concentration of the
specific molecule may be determined are: a) Total molecule
measurement. In cases where the molecule of interest is se-
creted and makes up a large proportion of the culture super-
natant, a total molecule determination will provide a reason-
35 able estimate for the amount of specific molecule; b) Specific
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42
molecule measurements. The concentration of e.g. a specific
molecule such as an enzyme may be determined by any of the fol-
lowing methods (see examples):
a.Active site titration of enzymes using a specific enzyme
s inhibitor.
b.By using a fluorescently labelled molecule that binds spe
cifically to the molecule of interest, and measure the
change in fluorescence polarization as the labelled mole
cule is added to the culture supernatant containing the
io molecule of interest,
c. By ELISA measurements using antibodies against the spe-
cific protein, including competitive ELISA,
d. Co-expression of green fluorescent protein. By transcrip
tional (or translational) fusion of a detectable protein
i5 (e.g., Green Fluorescent Protein, GFP) and measurement of
fluorescense in the enzyme/protein solution,
e.Tagging of the enzyme/protein in question, followed by la-
belling and subsequent detection (e. g. with fluorescent
probe or NMR imaging agent) of the tag.
2o f. Alternatively, the amount of enzyme/protein input into the
assay may be controlled (kept constant) by the inclusion
of a purification step, e.g. Ni-NTA purification of His-
tagged proteins or binding of enzyme/protein to specific
antibody.
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the assay of the first aspect step c)
is done under conditions where the assay is essentially inde-
pendent of the concentration of the molecule.
3o Preferably the molecule is an enzyme, and the enzyme con-
centration is in a range of preferably 98% below to 400 % above
the average concentration of enzyme employed in the assay, more
preferably 50% below to 200% above the average concentration of
enzyme employed in the assay.
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Another preferred embodiment relates to a method of the
first aspect, wherein the assay done in the first aspect step
c) is followed by the additional steps of:
a) determining the concentration of the molecule of in-
s terest for each position; and
b) adjusting the concentration of molecules in each posi-
tion to essentially the same level and then performing
the assay of the first aspect step c); or
c) performing the assay in the first aspect step c) and
io then correcting the data obtained with regard to the
concentration of the molecule.
Preferably the molecule of interest is an enzyme, and the
concentration in step a) of the preceding embodiment is indi-
rectly determined by a method chosen from the group consisting
is of active site titration using an enzyme inhibitor, measurement
of fluorescence polarization using a fluorescently labelled en-
zyme inhibitor, measurement of labelled anti-enzyme antibodies,
and measurement of a marker protein co-expressed with the en-
zyme.
2o An additional preferred embodiment relates to a method of
the first aspect, wherein the cultivation of the first aspect
step b) is followed by an additional step of recovering from
each position essentially the same amount of molecule and using
these recovered amounts in the assay of the first aspect step
as c) .
Preferably the molecule of the previous embodiment is a
polypeptide, and the recovery is achieved by fusing a polyhis-
tidine-tag to the polypeptide and recovering this construct on
a Ni-NTA solid support.
3o when a screening method is used, the choice of assay de-
pends on the molecule of interest and on the host cell con-
struct. Many ways are known in the art to assay or monitor the
production of a molecule, some in vivo others in vitro. Usually
such assays are based on the determination of a chemical or
35 physical event, which may be directly indicative or indirectly
through a cascade of events resulting in e.g. a change of col-
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44
our in the host cell or in the surrounding medium, emission of
light, production of detectable secondary molecules like pro-
teins or enzymes, binding of detectable molecules, cell death,
cell lysis, cell growth inhibition or promotion (see examples).
s Non-limiting examples of events are:
1. Cleavage or other modification of a substrate in solution.
2. Cleavage or other modification of a substrate (e.g. a
stain), covalently or non-covalently attached to textile or
other type of solid material, e.g. ceramics, metal surfaces,
io plastic or other polymer.
3. Cleavage or other modification of substrate cross-
linked/immobilized to bacterial cellulose or other type of
polymer surface .
4. Cleavage or other modification of non-stained textile
i5 swatches (e. g., cellulose swatch).
5. Interaction with live cells, resulting in lysis, growth in-
hibition or growth promotion of these cells.
6. Interaction with another molecule, or group of molecules,
leading to a binding event.
ao These events may be detected by e.9. radioactivity, fluo-
rescence, fluorescense polarization, luminiscence, NMR, mass
spectrometry, absorbance etc. The substance assayed for may be
derived from for example cell culture supernatants, lyzed cells
or live cells.
25 Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the assay used in the first aspect
step c) allows the determination of a chemical or physical
event in a live cell, preferably lysis, growth inhibition,
growth promotion, production of a protein or another molecule
3o by the cell.
Another preferred embodiment relates to a method of the
first aspect, wherein the assay used in the first aspect step
c) allows the determination of an event in which the molecule
of interest interacts with another molecule, preferably leading
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to a binding event or a chemical modification of the other
molecule.
Yet another preferred embodiment relates to a method of
the first aspect, wherein the assay used in the first aspect
s step c) allows the determination of the cleavage, isomeriza
tion, ligation or other modification of a substrate.
Preferably the substrate of the preceding embodiment is a
polymer, more preferably chosen from the group consisting of
polypeptides, celluloses, polysaccharides, and starches.
io Also, preferably the substrate of the two preceding em-
bodiments is immobilized to the surface of a solid material,
preferably textile or ceramics.
More preferably the substrate is a solid surface, most
preferably a cellulose swatch.
is The substrate of the four preceding embodiments is pref-
erably labelled with a detectable probe.
Preferably the probe of the preceding embodiment is fluo-
rescent, more preferably it is fluorescein-5-isothiocyanate
(FITC) or dichlorotriazino-5-aminofluorescein (DTAF).
2o An industrially very interesting area of research, is the
optimization of enzymes to be used in washing applications,
consequently the method of the invention is particularly inter-
esting in this area of enzyme research. The assay of the first
aspect step c) can be carried out under conditions of high pH
is and elevated temperatures by adding detergent, powder or fluid,
to the assay and controlling the temperature as well as the
shaking.
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the assay of the first aspect step c)
3o is performed in a solution comprising a detergent and where the
molecule of interest is an enzyme.
As describe above, a number of assays well known in the
art can be used with the present invention, these could be
based on any detectable or measurable property that can be as
35 sociated with production of the molecule of interest.
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46
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the assay used in the first aspect
step c) involves detection of a signal chosen from the group
consisting of fluorescence, fluorescence polarization, absorb-
s ance, radioactivity, nuclear magnetic resonance (NMR) and
luminiscence.
It was mentioned above how varying concentrations of the
molecule in the positions of the spatial array could impair the
method of the invention, however not only concentration but
io also total amount can vary, and measures can be taken to equal-
ize total amount produced of the molecule of interest (see ex-
amples 4, 5, 7, and 9).
Accordingly a preferred embodiment relates to a method of
the first aspect, wherein the growth conditions of the first
i5 aspect step b), have been optimized to give minimal variation
in the amount produced of the molecule of interest in each po-
sition of the spatial array.
A preferred embodiment relates to a method of the first
aspect, wherein the growth conditions are chosen so as to mimic
2o the conditions under full-scale fermentations, preferably high
ly dilute media are chosen.
A method for screening a DNA library for DNA of interest
according to the second aspect of the invention. An essential
element in this method is a step of using the method of the
25 first aspect as described herein.
In the art, ways of generating and producing DNA librar-
ies from natural sources are well known, but besides natural
DNA sequences, a number of ways are also known in which to gen-
erate very large populations of diverse artificial DNA se-
3o quences starting from one or more natural sequences, e.g. shuf-
fling or directed evolution (WO 98/42832; US 5,965,408; WO
98/01581; WO 97/07205; WO 95/22625; US 5,093,257).
A preferred embodiment relates to a method of the second
aspect, wherein the DNA library is generated from a natural DNA
35 sequence by DNA shuffling or directed evolution.
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Preferably the host cells of the second aspect step a)
are of microbial origin.
A recombinant vector according to the third aspect com
prising DNA isolated by a method as defined in the second as
s pect of the invention.
A recombinant host cell according to the fourth aspect
comprising DNA isolated by a method as described in the second
aspect or the vector according to the third aspect.
Preferably the cell according to the fourth aspect is of
io microbial origin, even more preferably of Bacillus or Aspergil
lus origin, and most preferably chosen from the group consist
ing of: Escherichia coli, Bacillus subtilis, Bacillus licheni
formis, Bacillus clausii, Aspergillus niger, Aspergillus
oryzae, Aspergillus nidulans, and Saccharomyces cerevisiae.
i5 A transgenic animal according to the fifth aspect con-
taining and expressing the DNA of interest according to the
second aspect.
A transgenic plant according to the sixth aspect
containing and expressing the DNA of interest according to the
2o second aspect.
The industrial interest in the method of the invention is
of course spurred by the potential of producing the molecule of
interest according to the aspects of the invention. Hence the
invention relates to the ways known in the art of producing
as such molecules as described in more detail above.
A method of producing a molecule of interest according to
the seventh aspect, which method comprises cultivating a cell
according to the fourth aspect in suitable culture medium under
conditions permitting expression of the DNA of interest and
so recovering the resulting molecule from the culture medium.
A method of producing a molecule of interest according to
the eight aspect, which method comprises recovering the
molecule from any part or secrete of/from the transgenic animal
according to the fifth aspect.
35 A method of producing a molecule of interest according to
the final aspect, which method comprises growing a cell of a
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48
transgenic plant according to the sixth aspect, and recovering
the molecule from the resulting plant.
Example 1
s Minimization of evaporation of liquids from microtiter-
plates by the use of a culture box.
A culture box was designed in order to minimize evapora-
tion of liquids from miniaturized microtiter plate fermenta-
tions and analytical methods at temperatures from 30-45°C, or
io even 4-99°C. The box is designed with a shallow base to hold wa-
ter and absorbent paper. A tray is secured to a base that holds
stacks of microtiter plates between stainless steel rods. An
airtight lid fitted with a silicon gasket is placed over the
base/tray and the lid is secured with clamps on all 4 sides.
i5 Rubber bands are placed around each set of 4 rods that are used
to secure the plates into position, such that when the box is
placed in an incubator shaker the entire device moves as a sin-
gle unit.
Use of the culture box eliminates or minimizes many of
2o the problems associated with miniaturized analytical and fer
mentation methods in microtiter plates. By limiting evapora
tion, the growth and expression differences across the plate,
particularly the differences between clones in the centre and
at the edges are minimized. It allows a more uniform growth in
as static or shaken cultures, as well as allows adequate oxygena-
tion of cultures by rapid plate shaking (up to 400 RPM) for
several days. The use of this box allows for a wide variety of
experimental conditions to be performed in microtiter plates,
e.g. , very low volumes (2 - 20 ml in a 96 well plate) and very
3o dilute solutions (media) may be used without significant loss
of volume.
Example 2
Wash performance assay for detergent a.-amylase.
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Coating of swatches with starch
Unmodified starch from a natural source is mixed with
small amounts of fluorescently labelled starch and coated on a
solid phase. The natural starch source can be flour derived
s from e.g. potato, corn, or rice. Especially rice flour has been
observed to provide a good correlation to higher scale wash
trials. The solid phase may be twill, as twill has been found
to provide good correlation to larger scale washes and has good
handling properties. The overall starch concentration as well
io as the ratio between labelled and unlabeled starch can be var
ied over a wide range, but we have found 50 (w/v) unmodified
starch and 0.025% (w/v) fluorescin 5-isothiocyanate (FITC) la
belled potato starch (50-300 glucose units per FITC molecule)
to provide the best compromise between sensitivity and response
i5 level (FITC was obtained from Sigma).
The starches are suspended in water and boiled for 10
min, alternatively jet-cooked for 5 min at 105°C and 2 bar. Af-
ter cooling, a selected textile is soaked with the cooked sus-
pension, excess slurry removed by rolling, and the textile is
2o dried either overnight at ambient temperature or for a few min-
utes at high temperature, e.g. 70°C, at high air velocity, e.g.
12 m/s. We have found that automation of this coating procedure
to significantly dampen coating variation. By use of a coating
machine to continuously coat several hundred meters at a time,
as subsequently performed assays with repeated dose-response
curves exhibit considerably less variation compared to non-
continuous coatings.
Growth of Bacillus cells secreting a-amylase variants
3o Bacillus cells secreting a-amylase variants are grown in
2xYT and 6 ~g/ml Chloramphenicol for three days at 37°C and 240
rpm, in a culture box (see example 1).
High throughput screen for detergent a,-amylases with better
35 wash performance
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Dry starch coated textile is punched into wells of a
polystyrene microtiter plate, preferentially of the 96-well
format. Assays are performed by first applying a detergent so-
lution, e.g. 150.1, to each well. A range of detergents can be
s used such as liquid detergent or powder detergent dissolved in
water. water hardness is controlled by the addition of a de-
sired amount of calcium and magnesium ions. The assay itself is
insensitive to water hardness over a wide range, e.g. 0-30°dH.
If a detergent that contains enzymes is used, these enzymes can
io be inactivated by e.g. heating the detergent to 85°C for 5 min
prior to the assay. Furthermore, the detergent can be centri
fuged and/or filtered before use to minimise particulate mat
ter. However, no assay interference due to un-removed particles
has been observed with detergents used in the range of dosages
i5 recommended by the manufacturers.
The intrinsic pH of detergents can be enforced with buff-
ering capacity by adding e.g. glycine or 3-[cyclohexylamino]-1-
propane-sulfonic acid (CAPS). This is an important quality en-
hancing measure, since the high throughput screen uses culture
2o supernatants as the source of expressed enzyme. Growth media
normally contain buffer components to ensure the optimal growth
pH, which is rarely equal to the high pH (often pH 9-11) pre-
sent in dissolved detergents.
Culture medium from above is added immediately after dis
25 pensing the detergent. We prefer to use a small amount of en
zyme solution compared to detergent/buffer, in order to mini
mise artefacts caused by e.g. a rich growth medium. For exam
ple, we routinely apply 15 ~1 of enzyme solution to 150 ~,l de
tergent.
3o Following addition of detergent and enzyme (culture me-
dium), the simulated "wash" takes place. Application of me-
chanic motion such as vigorous shaking (e. g., 240 rpm) at this
point strongly promotes the effect of tested enzymes, thus cre-
ating a good dynamic range for ranking candidates. Incubation
3s times at this step from 5 min to 2 h have been tested. When
screening for amylases at low-temperature conditions, we have
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51
found 15 min "washing" to provide a good window of difference
between "good" or "bad" candidates.
Heating can be applied during incubation to simulate the
heating of water during machine washing used in many parts of
s the world. For example, simulation of European washing condi
tions could include heating up to 40 or 60°C. This heating can
be introduced gradually by for example placing the ambient tem-
perature microtiter tray in a shaking incubator set to the ap-
propriate temperature. By this approach, a heat-up profile is
io generated. Alternatively, if constant high temperature is de-
sired, heated buffers can be added or insulated tubings and
thermostated surroundings can be employed.
The assay responses can be read by transferring the wash
solution to another microtiter plate and measure released fluo
i5 rescence in this solution. Alternatively, the wash solution can
be completely removed and the response measured as residual
fluorescence of the swatch.
By using the conditions described above, the two ap
proaches generate almost exact mirror image data, meaning that
ao a high degree of released fluorescence in a well is reflected
by a low degree of residual fluorescence on the corresponding
swatch and vice versa. We prefer the last method, as this only
requires one well per assay, thus ensuring minimal amounts of
operations leading to maximal throughput in e.g. a robotic set
z s up .
The growth conditions used ensure a reasonably uniform
growth of identical clones in different wells of the plate.
However, large differences in expression level between differ-
ent variants still exist. V~Ie have found that amylase variants
3o with different performance in the final application release
different amounts of fluorescent stain from the swatch in the
wash performance assay, even when the enzyme is used in satu-
rating concentrations. Therefore, the screen has been set up
using very high concentrations of enzyme. Under these condi-
35 tions the stain removal efficiency depends only minimally on
the enzyme concentration within a broad range; consequently,
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52
differences in expression levels between variants have little
effect on the performance of the individual variant in the high
throughput screening assay.
when applying the assay to libraries of amylase producing
s clones, hits are defined as wells of the plate wherein the
measurements exceed a given assay response relative to a se
lected benchmark amylase.
Results.
io Correlation with the more application-relevant mini-wash test.
Orange coloured rice starch textile was shaken vigorously
in 60 mL Asia-Pacific model detergent containing an amylase va-
riant, and the reflectance measured. Table 1 below shows the
correlation between the performance of three different amylases
i5 ( 1, 2 , and 3 ) in the swatch assay ( the High Throughput Screen
ing set-up) and the conventional mini-wash test. The improve
ment factor in a given assay is given by the performance of the
variant amylase divided by the performance of a given benchmark
amylase. Obviously, the improved amylase (amylase 3) performs
zo better in both the swatch assay and the mini-wash.
Amylase 1 Amylase 2 Amylase 3
Swatch assay 1.0 0.4 1.1
Mini-wash 1.0 0.3 1.3
Table 1: Relative residual fluorescence intensity on washed
swathes compared to relative reflectance of orange coloured
as rice starch textile in mini wash. Application dosage of 0.2
~,g/ml amylase was used for the mini wash, while approx. 2 ~g/ml
final dosage was used in the swatch assay (the average concen-
tration from culture supernatants in the High Throughput As-
say) .
Example 3
HTS screening assay for anti-fungal activity
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An anti-fungal agent can either inhibit the outgrowth of
spores, vegetative cells, or both. To identify an anti-fungal
agent inhibiting vegetative cells, 50 ~1 samples of a liquid
culture of a tester strain, e.g. Botrytis cinerae, are distrib-
s uted into each well of a microtiter plate; 50 ~l of samples of
sterile culture supernatants from cultures of bacterial cells,
e.g. Bacillus, to be screened for secretion of anti-fungal ac-
tivities (e. g. variants of an antimicrobial peptide or enzyme),
are then added to each well of the microtiter plate. Option-
io ally, instead of sterilizing the culture supernatants, a tissue
culture insert (e. g., Nunc TC Insert) may be inserted, to pre-
vent contact between the secreting cells and cells of the
tester strain. The insert may contain a membrane non-permeable
to proteins or other macromolecular components, which allows
is the passage of for example small antimicrobial peptides. The
microtiter plate is incubated at 30°C for 2-5 days whereafter
anti-fungal activity is analysed by optical density measure
ments in each well at a suitable wavelength. Low optical den
sity indicates the presence of an anti-fungal activity in the
2o well .
Example 4
Use of very dilute media for HTS screening of enzyme-secreting
fungi for improved yield.
25 In order to mimic the nutrient limiting conditions of
fermentor tanks used for production of for example enzymes, mi-
crotiter plate-based screens for improved yield are done in di-
lute growth media. The dilution ensures that the nutrients,
rather than the oxygen, become the growth limiting factors.
3o This allows a simplification in the handling of the assay since
the dilute substrate can be added directly to the cultures, be-
cause the effects of the media on the assay will be very weak.
35 Example 5
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Use of very dilute media for HTS screening of enzyme-secreting
fungi for improved expression
General HTS set up for yield mutant screening.
s Mutant libraries of Aspergillus oryzae or Aspergillus
niger strains are screened by HTS to identify mutants having
increased expression, i.e. improved yield of an enzyme. Better
yield mutants expressing for example peroxidase, laccase,
lipase, glucose oxidase, amylase, xylanase, phytase, and
io aminopeptidase have been identified this way.
For 96-well plate screens, very dilute medium containing
100 - 300 mg/1 carbon-source, ensures carbon-limitation without
oxygen limitation.
Primary 96-well plate screens involve the dilution of
i5 spores from distinct mutant pools into fermentation substrate
so that one spore in average is inoculated per well when 1001
of medium is dispensed into each well. After inoculation, the
plates are incubated for 3-4 days at 30-34°C under static con
ditions in a culture box (see example 1). The wells are then
2o assayed for enzyme activity, for example by the addition of a
substrate solution directly to the growth wells.
Mutants of interest are isolated and single colonies
transferred to agar slants. Spores from agar slants are inocu-
lated into 96-well plates with approx. 103 spores per well and
25 fermented under static conditions for 4 days at 34°C to retest
isolates. Subsequently, selected isolates are tested in shake
flask fermentations.
Isolation of mutants having increased Shearzyme~ expression
3o An Aspergillus oryzae transformant MT2181 expressing re-
combinant Shearzyme~, was mutated by using UV-irradiation. The
MT2181 mutant library was fermented in SH-CDY substrate (pH
6.5) in 96 wells plates as described above. The SH-CDY sub-
strate contained pr litre : 0 . 3 g MgS04, 7H20, 0 . 3 g KzS04, 5 . 0 g
35 KHzP04, 0.1 ml tracemetal solution, 0.013 g urea, 0.010 g yeast
extract, and 0.1 g maltose. The tracemetal solution contains pr
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WO 01/32844 PCT/DK00/00568
litre: 13.8 g FeS04, 7H20, 8.5 g MnS04, HZO, 14.3 g ZnS04, 7 H20,
CuS04, 5 HZO, NiCl2, 6 HZO, 3 g citric acid.
Cultures were then assayed for increased yield, i.e. in-
s creased Shearzyme~ activity. Shearzymeo, which is an endoxy-
lanase, could be quantitated by using a microtiter plate assay
and p-nitrophenyl-(3-D-xylopyranoside (Sigma N-2132) as sub-
strate, since the enzyme possesses some exo activity, and yel-
low p-nitrophenol was generated. A 100 mg/ml p-nitrophenyl-(3-D-
io xylopyranoside stock solution was prepared in DMSO. Subse
quently, working substrate was prepared by diluting the stock
0
10 folds in 0.1 M phosphate buffer (pH 6.0). Standard Shearzyme
(BioFeed~ (S), Novo Nordisk A/S, Bagsvaerd, Denmark) was prepared
so as to contain 20 FXU/ml in 0.1 M phosphate buffer, pH 6Ø
i5 The standard was stored at -20°C until use. Shearzyme~ stock was
diluted appropriately to obtain a standard series ranging from
0.1 to 5.0 FXU/ml just before use. 25 ~,1 samples were dispensed
to wells in 96-well plates followed by 120 ~.l of substrate by
using a robotic set up. Using a plate reader, the absorbance at
zo 405 nm was recorded as the difference of two readings taken at
approximately 4 minutes intervals. Shearzyme~ units/ml (FXU/ml)
were calculated relative to the Shearzyme~ standards.
In the 96-well primary screen followed by the re-test in
z5 96-well plates, 9 mutants from the "MT2181 mutant library" were
selected. These mutants produced higher yields of Shearzyme~
than the control strain Aspergillus oryzae MT2181. The 4 Shear-
zyme~ producing mutants with highest yields were evaluated in
shake flasks. The results obtained are shown in table 2 below
3o where the Shearzyme~ yield of A, oryzae MT2181 as a control is
normalized to 100.
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96-well re-test Shake flask
Strain # (FXU/ml) (FXU/ml)
MT2181-3-14 148 118
MT2181-5-16 140 121
MT2181-6-Ol 126 130
MT2181-7-23 130 125
Table 2. Shearzyme expression by Mw~l~l inutanzs
As shown in table 2, the 4 selected mutants produce ap
proximately 20-30 % more Shearzyme~ than the control strain A.
s oryzae MT2181, when fermented in shake flasks.
Example 6
Screening for cellulase activity using Dichlorotriazino-5-
aminofluorescein (DTAF) labelled cotton Swatches.
io
Preparation of Cotton swatches
Style 400 cotton (Center for Test Materials, PO Box 120,
3130 AC Vlowdingen, Holland) was cut to appropriate sizes. The
cotton swatches were swollen in a 0.1 N NaOH solution for 24
is hrs. prior to the labelling in order to enhance the accessibil-
ity of the hydroxylic groups to the probe. DTAF dissolved in
0.1 N NaOH was added to the swollen swatches, and the reaction
mixture was allowed to react for 24 hrs. at room temperature in
the dark on a celloshaker-board. The labelled swatches were
2o washed several times using 0.1 N NaOH, water, MeOH, and finally
water again. After the last wash-cycle no probe could be found
in the supernatant. The swatches were finally dried. The la
belled cotton swatches have a clear yellow colour. Further the
DTAF-probe makes the swatches highly fluorescent at the 485/518
2s (excitation/emission) wavelength bands.
Similar fluorescence measurements were obtained whether
cotton was labelled in swatches of 5-11 g or whether it had
been cut into swatches of approx. 3 mg, suitable for subsequent
microtiter plate analysis.
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Specific release of fluorescence by incubation of DTAF labelled
cotton swatches with purified cellulases
Labelled swatches were distributed in each well of a 96
well microtiter plate, washed in deionized water, and incubated
with 50 ~,1 cellulase enzyme solution and 150 ~1 buffer for 2h,
at 40°C, 400 rpm. The microtiter plates were centrifuged at 2800
rpm and fluorescence intensity (485/518 nm) was measured on 150
~1 of the supernatant, using a spectrophotometer. The cellu-
io lases used were endoglucanase (EG) v, EGV-core, EG I, and CBH
II from Humicola insolens (prepared as described in M.
Schiilein, J. Biotechnology, vol 57, pp. 71-81, 1997).
Table 3 shows that EG V performs much better per endocel
lulase activity unit than EG I, EG V-Core or CBH II. This is
i5 consistent with the performance of these enzymes in multi-wash
experiments to determine their effect. Hence, DTAF-swatches can
be used to assay cellulases and the results correlate with the
performance of the cellulase in performing a full-scale wash
experiments.
2o Further, the signal-to-noise ratio is improved by pre-
washing the cotton twenty times in a laundry machine, using ei-
ther water or a model detergent, before labelling. Also, the
signal-to-noise ratio is improved by increasing the number of
labelling cycles.
cellulase EG V
units/ml CBH II EG I EG V core
0,0 24,8 29,0 32,6 29,6
0,9 27,2 33,6 37,7 29,7
2,8 24,5 39,2 37,0 29,7
8,0 21,6 39,4 49,3 32,8
25,0 23,8 50,7 70,2 36,4
75, 0 1 36, 1 57, 6 1 112, 1 61,
9 8 91
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Table 3. Performance of 4 cellulases in the swatch assay ex-
pressed as fluorescence intensity in arbitrary units (example
6) .
s High throughput screening for cellulase activities in cell cul-
ture supernatants
Recombinant H.insolens cellulase VI (W09901544) was ex-
pressed in yeast cells (S.cerevisiae, YNG318) using standard
methodologies (Sambrook et al.: "Molecular Cloning. A Laboratory
io Manual", Cold Spring Harbor, NY,1989). Transformed cells were
grown in SC-ura- media for 3 days at 30°C. 100~.L culture super-
natant was transferred to an assay plate containing labelled
cellulose swatches as described above. After an appropriate
time of incubation the well supernatants were transferred to an
is assay plate, and the fluorescence was measured. A control ex
periment was performed in 0,2M tris buffer at pH 7 employing
100 ~1 supernatant from yeast cultures expressing either recom
binant active cellulase EGVI, or an inactive variant of EGVI.
Clones expressing active cellulases gave rise to higher levels
20 of fluorescence than clones expressing the inactive variant:
EGVI(active): 47.8 +/- 1.1 (n=6)
EGVI(inactive): 77.7 +/- 1.9 (n=6)
25 The DTAF labelled cotton swatches can also be used in a
cell culture-based screen for active cellulases in a high-
throughput format: Yeast cells expressing cellulases are sus-
pended in minimal media, and aliquoted into 96 well plates at
an average of about 30 wells with growth per 96 well plate (to
30 obtain predominantly single cell isolates). After 3 days of
growth at 30°C, 100~L culture supernatant is transferred to an
assay plate containing cellulose swatches as described above.
Optionally, a well insert (e.g., Nunc TC Insert) may be in-
serted, to separate yeast cells and the cellulose swatch. The
35 insert should contain a membrane permeable to the cellulase en-
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59
zyme but not to the cells. After an appropriate time of incuba
tion, the well supernatants (without any prior centrifugation)
are transferred to an empty assay plate, and the fluorescence
is measured for each well. Clones expressing active cellulases
s give rise to higher levels of fluorescence.
Example 7
His-tagging/purification of proteases to achieve equal protein
input from differentially expressing clones.
io The DNA sequence encoding the protease Savinase~ (Novo
Nordisk A/S, Denmark) is translationally fused to a sequence
encoding a His6 tag and libraries of Savinase~-His6 variants are
produced and introduced into Bacillus. After standard growth, a
limited number of Savinase~ enzymes of each variant (about 100
i5 of what is secreted by Bacillus carrying the wildtype Savinase
gene) are immobilized in the wells of Ni-NTA microtiter plates.
The unbound fraction including cells and excess Savinase~ is re-
moved, the plate washed once or twice in a buffer containing 5-
20 mM Imidazole. The His-tagged Savinase~ variants are released
zo from the solid support by the addition of 250 mM Imidazole, and
aliquots of the supernatants from each well are used as input
in a wash performance assay as described in the previous exam-
ples.
z5 Example 8
Determination of Savinase~ concentration by fluorescence polari-
zation measurements.
The CI-2 protease inhibitor is labelled by standard means
with a fluorescent probe. After growth as described above, the
3o amount of a Savinase~ variant in a given well may be measured
directly in the wells by addition of fluorescence-labelled in-
hibitor which upon binding to the protease changes rotational
speed. The rotational speed may be monitored by fluorescence
polarization analysis or by other means which monitors diffu-
35 sion (e.g. fluorescence correlation spectroscopy). Since the
amount of Savinase~ variant may vary significantly between the
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individual wells, the fluorescently labelled CI-2 inhibitor is
added in two or three steps of defined amounts and the fluores-
cence polarisation is measured after each CI-2 addition. The
determined concentration of Savinase~ variant in the individual
s well may be used to adjust the input volume from this well into
the activity assay, or can be used to correct the obtained ac-
tivity data, in order to determine the specific activity of the
Savinase~ variant of that well.
io Example 9
High throughput screening in filamentous fungi
It is a difficult task to make protein libraries in fila-
mentous fungi. One problem is the different morphology and
growth rate of different clones. This gives rise to large and
i5 small clones, as well as clones that might be outcompeted by
other clones. Assaying fungal transformants of different sizes
is also a difficult task due to differences in amounts of ex-
pressed enzymes. Another problem is the handling of all the
clones on the plate.
2o These problems can be overcome by growing the individual
clones in alginate balls with a high molecular weight (HMW)
dextran as carbon source, and then using the~balls as the sour
ce of clonal cell cultures in a microtiter plate . The HMW dex
tran lets the slow-growing transformants outgrow, without cross
z5 contamination from faster growing transformants.
A Polymerase chain reaction, using pAHL (carrying a Li-
pase gene) as the template and 2 pmol/ml of each primer: oligo
115120: (GCTTTGTGCAGGGTAAATC), and oligo 134532: (GAGCAATAT-
CAGGCCGCGCACG) is run under the following conditions: 94°C, 5
3o min.; 30 cycles of (94°C, 30 sec.; 50°C, 30 sec.;
72°C, 1 min.),
and 72°C, 5 min. and a commercial Taq polymerase, such as Am-
pliTaq, (Perkin-Elmer Corp., Branchburg NJ, USA). For example,
the PCR conditions 'may result in a high rate of error, and
therefore a library of Lipase mutants are generated.
35 Protoplasts of the filamentous fungi strain JaL250 is
transformed using 2~.g of pENI1298, which had been digested with
CA 02388982 2002-04-24
WO 01/32844 PCT/DK00/00568
61
Ball and SgrAl to remove most of the lipase encoding sequence,
and 5~,g of the above PCR product . The vector and the PCR frag-
ment are allowed to recombine in vivo as described in WO
97/07205.
s The protoplasts are washed twice with ST (0.6M sorbitol,
100mM TRIS pH7.0) in order to remove CaCl2. The protoplasts are
then re-suspended in an alginate solution (1.5% alginate, 2 0
high molecular weight dextran (5-40*106 kd), 1.2 M sorbitol, 10
mM TRIS pH7.5) . Using a pump, the suspension is pumped through
io a small nozzle where small suspension droplets are made. These
drops fall down (15 cm) into a shake flask (500m1) containing
0.2M CaCl2, 1.2 M sorbitol, 10 mM TRIS pH7.5. Droplets of an al-
ginate solution (typically 1-2 %) turn into hard balls when
they encounter a CaClz solution (such as a 0.2 M solution).
i5 Small alginate balls of the size 2.5 mm in diameter are gener-
ated by this procedure. The protoplast suspension should be
made so that approximately 1 out of 5 balls contains a trans-
formed protoplast, in order to avoid multiple clones in the
same ball.
zo The protoplast-containing alginate balls are grown over-
night at 30°C degrees in STC (10 mM CaCl2, 1.2 M sorbitol, 10 mM
TRIS pH7.5) in order to regenerate the cell wall. After a cou-
ple of washes in sterile water to remove sorbitol, the balls
are transferred to 1* Vogel media and grown 2-3 days at 30°C de-
25 grees. The sole carbon source is the dextran in the balls. This
prevents cross-contamination from ball to ball, and allows
slow-growing transformants to gain reasonable biomass.
The alginate balls are transferred to a microtiter plate;
one ball into each microtiter well. Liquid growth media is
3o added to the wells and after a period of growth incubation, the
growth media is analyzed for lipase activity under the desired
conditions.
