Recombinant 21 kD cocoa protein and precursor

A 21 kD protein, and its 23 kD expression precursor, as the source of peptide flavor precursors in cocoa (Theobroma cacao) have been identified. Genes coding for them have been probed, identified and sequenced, and recombinant proteins have been synthesized.

This invention relates to proteins and nucleic acids derived from or 
otherwise related to cocoa. 
The beans of the cocoa plant (Theobroma cacao) are the raw material for 
cocoa, chocolate and natural cocoa and chocolate flavouring. As described 
by Rohan ("Processing of Raw Cocoa for the Market", FAO/UN (1963)), raw 
cocoa beans are extracted from the harvested cocoa pod, from which the 
placenta is normally removed, the beans are then "fermented" for a period 
of days, during which the beans are killed and a purple pigment is 
released from the cotyledons. During fermentation "unknown" compounds are 
formed which on roasting give rise to characteristic cocoa flavour. Robart 
suggests that polyphenols and theobromine are implicated in the flavour 
precursor formation. After fermentation, the beans are dried, during which 
time the characteristic brown pigment forms, and they are then stored and 
shipped. 
Biehl et al, 1982 investigated proteolysis during anaerobic cocoa seed 
incubation and identified 26 kD and 44 kD proteins which accumulated 
during seed ripening and degraded during germination. Biehl asserted that 
there were storage proteins and suggested that they may give rise to 
flavour-specific peptides. 
Biehl et al., 1985 again asserted that amino acids and peptides were 
important for flavours. 
Fritz et al, 1985 identified polypeptides of 20 kD and 28 kD appearing in 
the cytoplasmic fraction of cocoa seed extracts at about 100 days after 
pollination. It appears that the 20 kD protein is thought to have glyceryl 
acyltransferase activity. 
Pettipher et al., 1990 suggested that peptides are important for cocoa 
flavour and refers to 48 kD and 28 kD storage proteins. 
In spite of the uncertainties in the art, as summarised above, proteins 
apparently responsible for flavour production in cocoa beans have now been 
identified. Further, it has been discovered that, in spite of Fritz's 
caution that "cocoa seed mRNA levels are notably low compared to other 
plants" (loc. cit.), it is possible to apply the techniques of recombinant 
DNA techniques to the production of such proteins. 
According to a first aspect of the invention, there is provided a 23 kD 
protein of Th. cacao or a fragment thereof. 
The 23 kD protein may be processed in vivo to form a 21 kD polypeptide. 
According to a second aspect of the invention, there is provided a 21 kD 
protein of Th. cacao or a fragment thereof. 
The term "fragment" as used herein and as applied to proteins or peptides 
indicates a sufficient number of amino acid residues are present for the 
fragment to be useful. Typically, at least four, five, six or even at 
least 10 or 20 amino acids may be present in a fragment. Useful fragments 
include those which are the same as or similar or equivalent to those 
naturally produced during the fermentation phase of cocoa bean processing. 
It is believed that such fragments take part in Maillard reactions during 
roasting, to form at least some of the essential flavour components of 
cocoa. 
Proteins in accordance with the invention may be synthetic; they may be 
chemically synthesised or, preferably, produced by recombinant DNA 
techniques. Proteins produced by such techniques can therefore be termed 
"recombinant proteins". Recombinant proteins may be glycosylated or 
non-glycosylated; non-glycosylated proteins will result from prokaryotic 
expression systems. Theobroma cacao has two primary subspecies, Th. cacao 
cacao and Th. cacao sphaerocarpum. While proteins in accordance with the 
invention may be derived from these subspecies, the invention is not 
limited solely to these subspecies. For example, many cocoa varieties are 
hybrids between different species; an example of such a hybrid is the 
trinitario variety. 
The invention also relates to nucleic acid, particularly DNA, coding for 
the proteins referred to above (whether the primary translation products, 
the processed proteins or fragments). The invention therefore also 
provides, in further aspects: 
nucleic acid coding for a 23 kD protein of Th. cacao or for a fragment 
thereof; and 
nucleic acid coding for a 21 kD protein of Th. cacao or for a fragment 
thereof. 
Included in the invention is nucleic acid which is degenerate for the wild 
type protein and which codes for conservative or other non-deleterious 
mutants. Nucleic acid which hybridises to the wild type material is also 
included. 
Nucleic acid within the scope of the invention will generally be 
recombinant nucleic acid and may be in isolated form. Frequently, nucleic 
acid in accordance with the invention will be incorporated into a vector 
(whether an expression vector or otherwise) such as a plasmid. Suitable 
expression vectors will contain an appropriate promoter, depending on the 
intended expression host. For yeast, an appropriate promoter is the yeast 
pyruvate kinase (PK) promoter; for bacteria an appropriate promoter is a 
strong lambda promoter. 
Expression may be secreted or non-secreted. Secreted expression is 
preferred, particularly in eukaryotic expression systems; an appropriate 
signal sequence may be present for this purpose. Signal sequences derived 
from the expression host (such as that from the yeast alpha-factor in the 
case of yeast) may be more appropriate than native cocoa signal sequences. 
The invention further relates to host cells comprising nucleic acid as 
described above. Genetic manipulation may for preference take place in 
prokaryotes. Expression will for preference take place in a food-approved 
host. The yeast Saccharomyces cerevisiae is particularly preferred. 
The invention aim relates to processes for preparing nucleic acid and 
protein as described above by nucleic acid replication and expression, 
respectively. 
cDNA in accordance with the invention may be useful not only for obtaining 
protein expression but also for Restriction Fragment Length Polymorphism 
(RFLP) studies. In such studies, detectably labelled cDNA (eg 
radiolabelled) is prepared. DNA of a cultivar under analysis is then 
prepared and digested with restriction enzymes. Southern blotting with the 
labelled cDNA may then enable genetic correlations to be made between 
cultivars. Phenotypic correlations may then be deduced.

EXAMPLES 
Example 1 
Identification of the Major Seed Proteins 
It is not practicable to extract proteins directly from cocoa beans due to 
the high fat and polyphenol contents, and proteins were, therefore, 
extracted from acetone powders made as follows. Mature beans from cocoa of 
West African origin (Theobroma cacao amelonada) were lyophilised and 
ground roughly in a pestle and mortar. Lipids were extracted by Soxhlet 
extraction with diethyl ether for two periods of four hours, the beans 
being dried and further ground between extractions. Polyphenols and 
pigments were then removed by several extractions with 80% acetone, 0.1% 
thioglycollic acid. After extraction the resulting paste was dried under 
vacuum and ground to a fine powder. 
Total proteins were solubilised by grinding the powder with extraction 
buffer (0.05M sodium phosphate, pH 7.2; 0.01M 2-mercaptoethanoI; 1% SDS) 
in a hand-held homogeniser, at 5 mg/ml. The suspension was heated at 
95.degree. C. for 5 minutes, and centrifuged at 18K for 20 minutes to 
remove insoluble material. The resulting clear supernatant contained about 
1 mg/ml total protein. Electrophoresis of 25 .mu.l on an SDS-PAGE gel 
(Laemmli, 1970) gave three major bands, including one at 21 kD, comprising 
approximately 30% of the total proteins. The 21 kD protein is presumed to 
be the polypeptide subunit of a major storage protein. 
Characteristics of the Storage Polypeptide 
The solubility characteristics of the 21 kD polypeptide was roughly defined 
by one or two quick experiments. Dialysis of the polypeptide solution 
against SDS-free extraction buffer rendered some polypeptides insoluble, 
as judged by their ability to pass through a 0.22 micron membrane, whereas 
the 21 kD polypeptide remained soluble. Only the 21 kD polypeptide was 
extracted from the acetone powder by water and dilute buffers, showing 
that this protein could be classed as an albumin. 
Purification of the major polypeptide 
The 21 kD polypeptide was purified by two rounds of gel filtration on a 
SUPEROSE-12 column of the PHARMACIA Fast Protein Liquid Chromatography 
system (FPLC), or by electroelution of bands after preparative electro- 
phoresis. (The words SUPEROSE and PHARMACIA are trade marks.) Concentrated 
protein extracts were made from 50 mg acetone powder per ml of extraction 
buffer, and 1-2 ml loaded onto 2 mm thick SDS-PAGE gels poured without a 
comb. After electrophoresis the gel was surface stained in aqueous 
Coomassie Blue, and the major bands cut out with a scalpel. Gel slices 
were electroeluted into dialysis bags in electrophoresis running buffer at 
15 V for 24 hours, and the dialysate dialysed further against 0.1% SDS. 
Samples could be concentrated by lyophilisation. 
Example 2 
Amino-acid Sequence Data from Protein 
Protein samples (about 10 .mu.g) were subjected to conventional N-terminal 
amino-acid sequencing. A 12 amino-acid sequence was obtained for the 21 kD 
protein, and this information was used to construct an oligonucleotide 
probe (Woods et at, 1982; Woods, 1984). 
Example 3 
Raising Antibodies to the 21 kD Polypeptide 
Polyclonal antibodies were prepared using the methodology of Catty and 
Raykundalia (1988). The serum was aliquoted into 1 ml fractions and stored 
at -20.degree. C. 
Characterising Antibodies to the 21 kD Polypeptide 
Serum was immediately characterised using the Ochterloney double-diffusion 
technique, whereby antigen and antibody are allowed to diffuse towards one 
another from wells cut in agarose in borate-saline buffer. Precipitin 
lines are formed where the two interact if the antibody `recognises` the 
antigen. This test showed that antibodies to the 21 kD protein antigen had 
been formed. 
The gamma-globulin fraction of the serum was partially purified by 
precipitation with 50% ammonium sulphate, solubilisation in 
phosphate-buffered saline (PBS) and chromatography on a DE 52 cellulose 
ion-exchange column as described by Hill, 1984. Fractions containing 
gamma-globulin were monitored at 280 nm (OD.sub.280 of 1.4 is equivalent 
to 1 mg/ml gamma-globulin) and stored at -20.degree. C. 
The effective titre of the antibodies was measured using an enzyme-linked 
immunosorbant assay (ELISA). The wells of a polystyrene microtitre plate 
were coated with antigen (10-1000 ng) overnight at 4.degree. C. in 
carbonate coating buffer. Wells were washed in PBS-Tween and the test 
gamma globulin added at concentrations of 10, 1 and 0.1 .mu.g/ml 
(approximately 1:100, 1:1000 and 1:10,000 dilutions). The diluent was 
PBS-Tween containing 2% polyvinyl pyrrolidone (PVP) and 0.2% BSA. Controls 
were preimmune serum from the same animal. Binding took place at 
37.degree. C. for 3-4 hours. The wells were washed as above and secondary 
antibody (goat anti-rabbit IgG conjugated to alkaline phosphatase) added 
at a concentration of 1 .mu.g/ml, using the same conditions as the primary 
antibody. The wells are again washed, and alkaline phosphatase substrate 
(p-nitrophenyl phosphate; 0.6 mg/ml in diethanoi-amine buffer pH 9.8) 
added. The yellow colour, indicating a positive reaction, was allowed to 
develop for 30 minutes and the reaction stopped with 3M NaOH. The colour 
is quantified at 405 nm. More detail of this method is given in Hill, 
1984. The method confirmed that the antibodies all had a high titre and 
could be used at 1 .mu.g/ml concentration. 
Example 4 
Isolation of Total RIVA from Immature Cocoa Beans 
The starting material for RNA which should contain a high proportion of 
mRNA specific for the storage proteins was immature cocoa beans, at about 
130 days after pollination. Previous work had suggested that synthesis of 
storage proteins was approaching its height by this date (Biehl et al, 
1982). The beans are roughly corrugated and pale pinkish-purple at this 
age. 
The initial requirement of the total RNA preparation from cocoa beans was 
that it should be free from contaminants, as judged by the UV spectrum, 
particularly in the far UV, where a deep trough at 230 nm (260 nm:230 nm 
ratio is approximately 2.0) is highly diagnostic of clean RNA, and is 
intact, as judged by agarose gel electrophoresis of heat-denatured 
samples, which should show clear rRNA bands. A prerequisite for obtaining 
intact RNA is scrupulous cleanliness and rigorous precautions against 
RNases, which are ubiquitous and extremely stable enzymes. Glassware is 
customarily baked at high temperatures, and solutions and apparatus 
treated with the RNase inhibitor diethyl pyrocarbonate (DEPC, 0.1%) before 
autoclaving. 
The most routine method for extraction of plant (and animal) RNA is 
extraction of the proteins with phenol/chloroform in the presence of SDS 
to disrupt protein-nucleic acid complexes, and inhibit the RNases which 
are abundant in plant material. Following phenol extraction the RNA is 
pelletted on a caesium chloride gradient before or after ethanol 
precipitation. This method produced more or less intact RNA, but it was 
heavily contaminated with dark brown pigment, probably oxidised 
polyphenols and tannins, which always co-purified with the RNA. High 
levels of polyphenols are a major problem in Theobroma tissues. 
A method was therefore adopted which avoided the use of phenol, and instead 
used the method of Hall et al. (1978) which involves breaking the tissue 
in hot SDS-borate buffer, digesting the proteins with proteinase K, and 
specifically precipitating the RNA with LiCl. This method gave high yields 
of reasonably clean, intact RNA. Contaminants continued to be a problem 
and the method was modified by introducing repeated LiCl precipitation 
steps, the precipitate being dissolved in water and clarified by 
microcentrifugation after each step. This resulted in RNA preparations 
with ideal spectra, which performed well in subsequent functional tests 
such as in vitro translation. 
Preparation of mRNA From Total RNA 
The mRNA fraction was separated from total RNA by affinity chromatography 
on a small (1 ml) oligo-dT column, the mRNA binding to the column by its 
poly A tail. The RNA (1-2 mg) was denatured by heating at 65.degree. C. 
and applied to the column in a high salt buffer. Poly A+ was eluted with 
low salt buffer, and collected by ethanol precipitation. The method is 
essentially that of Aviv and Leder (1972), modified by Maniatis et al 
(1982). From 1 mg of total RNA, approximately 10-20 .mu.g polyA+ RNA was 
obtained (1-2%). 
In vitro Translation of mRNA 
The ability of mRNA to support in vitro translation is a good indication of 
its cleanliness and intactness. Only mRNAs with an intact polyA tail (3' 
end) will be selected by the oligo-dT column, and only mRNAs which also 
have an intact 5' end (translational start) will translate efficiently. In 
vitro translation was carried out using RNA-depleted wheat-germ lysate 
(Amersham International), the de novo protein synthesis being monitored by 
the incorporation of [.sup.35 S]-methionine (Roberts and Paterson, 1973). 
Initially the ram of de novo synthesis was measured by the incorporation 
of [.sup.35 S]-methionine into TCA-precipitable material trapped on glass 
fibre filters (GFC, Whatman). The actual products of translation were 
investigated by running on SDS-PAGE, soaking the gel in fluor, drying the 
gel and autoradiography. The mRNA preparations translated efficiently and 
the products covered a wide range of molecular weights, showing that 
intact mRNAs for even the largest proteins had been obtained. None of the 
major translation products corresponded in size to the 21 kD polypeptide 
identified in mature beans, and it was apparent that considerable 
processing of the nascent polypeptide must occur to give the mature form. 
Example 5 
Identification of Precursor to the Mature Polypeptide by 
Immunoprecipitation 
Because the 21 kD storage polypeptides was not apparent amongst the 
translation products of mRNA from developing cocoa beans, the technique of 
immunoprecipitation, with specific antibodies raised to the 21 kD 
polypeptide, was used to identify the precursors from the translation 
mixture. This was done for two reasons: first to confirm that the 
appropriate mRNA was present before cloning, and second to gain 
information on the expected size of the encoding gene. 
Immunoprecipitation was by the method of Cuming et al, 1986. [.sup.35 
S]-labelled in vitro translation products were dissociated in SDS, and 
allowed to bind with specific antibody in PBS plus 1% BSA. The 
antibody-antigen mixture was then mixed with protein A-SEPHAROSE and 
incubated on ice to allow the IgG to bind to protein A. The slurry was 
poured into a disposable 1 ml syringe, and unbound proteins removed by 
washing with PBS+1% NONIDET P-40. The bound antibody was eluted with 1M 
acetic acid and the proteins precipitated with TCA. The antibody-antigen 
complex was dissociated in SDS, and subject to SDS-PAGE and fluorography, 
which reveals which labelled antigens have bound to the specific 
antibodies. 
The results showed that the anti-21 kD antibody precipitated a 23 kD 
precursor. The precursor size corresponded to a major band on the in vitro 
translation products. 
Example 6 
cDNA Synthesis From the mRNA Preparations 
cDNA synthesis was carried out using a kit from Amersham International. The 
first strand of the cDNA is synthesised by the enzyme reverse 
transcriptase, using the four nucleotide bases found in DNA (dATP, dTTP, 
dGTP, dCTP) and an oligo-dT primer. The second strand synthesis was by the 
method of Gubler and Hoffman (1983), whereby the RNA strand is nicked in 
many positions by RNase H, and the remaining fragments used to prime the 
replacement synthesis of a new DNA strand directed by the enzyme E. coli 
DNA polymerase I. Any 3' overhanging ends of DNA are filled in using the 
enzyme T4 polymerase. The whole process was monitored by adding a small 
proportion of [.sup.32 P]-dCTP into the initial nucleotide mixture, and 
measuring the percentage incorporation of label into DNA. Assuming that 
cold nucleotides are incorporated at the same rate, and that the four 
bases are incorporated equally, an estimate of the synthesis of cDNA can 
be obtained. From 1 .mu.g of mRNA approximately 140 ng of cDNA was 
synthesised. The products were analysed on an alkaline 1.4% agarose gel as 
described in the Amersham methods. Globin cDNA, synthesised as a control 
with the kit, was run on the same gel, which was dried down and 
autoradiographed. The cocoa cDNA had a range of molecular weights, with a 
substantial amount larger than the 600 bp of the globin cDNA. 
Example 7 
Cloning of cDNA into a Plasmid Vector by Homopolymer Tailing 
The method of cloning cDNA into a plasmid vector was to 3' tail the cDNA 
with dC residues using the enzyme terminal transferase (Boehringer 
Corporation Ltd), and anneal into a PstI-cut and 5' tailed plasmid 
(Maniatis et al, 1982 Eschenfeldt et al, 1987). The optimum length for the 
dC tail is 12-20 residues. The tailing reaction (conditions as described 
by the manufacturers) was tested with a 1.5 kb blunt-ended restriction 
fragment, taking samples at intervals, and monitoring the incorporation of 
a small amount of [.sup.32 P]-dCTP. A sample of cDNA (70 ng) was then 
tailed using the predetermined conditions. 
A dG-tailed plasmid vector (3+-oligo(dG)-tailed pUC9) was purchased from 
Pharmacia. 15 ng vector was annealed with 0.5-5 ng of cDNA at 58.degree. 
C. for 2 hours in annealing buffer: 5 mM, Tris-HCl pH 7.6; 1 mM EDTA, 75 
mM NaCl in a total volume of 50 .mu.l. The annealed mixture was 
transformed into E. coli RRI (Bethesda Research Laboratories), 
transformants being selected on L-agar+100 .mu.g/ml ampicillin. 
Approximately 200 transformants per ng of cDNA were obtained. 
Transformants were stored by growing in 100 .mu.l L-broth in the wells of 
microtitre plates, adding 100 .mu.l 80% glycerol, and storing at 
-20.degree. C. 
Some of the dC tailed cDNA was size selected by electrophoresing on a 0.8% 
agarose gel, cutting slits in the gel at positions corresponding to 0.5, 
1.0 and 1.5 kb, inserting DE81 paper and continuing electrophoresis until 
the cDNA had run onto the DE81 paper. The DNA was then eluted from the 
paper with high salt buffer, according to the method of Dretzen et al 
(1981). 
Example 8 
Construction of Oligonucleotide Probes for the 21 kD Gene 
The N-terminus of the 21 kD polypeptide (SEQ ID NO: 24), as determined in 
Example 2 above, was 
EQU Ala-Asn-Ser-Pro-Leu-Asp-Thr-Asp-Gly-Asp-Glu. 
From this the optimum region for synthesising a probe of 17 residues was as 
follows: 
##STR1## 
The 17-mer probe constructed is shown below the sequence (SEQ ID NO: 25): 
it is actually a mixture of 128 different 17-mers, one of which must be 
the actual coding sequence. Probe synthesis was carried out using an 
Applied Biosystems apparatus. 
The 21 kD probe was purified by electrophoresis on a 20% acrylamide gel, 
the bands being detected by UV shadowing, and eluted by dialysing against 
water. 
Example 9 
Use of Oligonucleotides to Probe cDNA Library 
The oligonucleotide probes were 5' end-labelled with gamma-[.sup.32 P]dATP 
and the enzyme polynucleotide kinase (Amersham International). The method 
was essentially that of Woods (1982, 1984), except that a smaller amount 
of isotope (15 .mu.Ci) was used to label about 40 ng probe, in 10 mM 
MgCl.sub.1, 100 mM Tris-HCl, pH 7.6; 20 mM 2-mereaptoethanol. 
The cDNA library was grown on GeneScreen (New England Nuclear) nylon 
membranes placed on the surface of L-agar+100 .mu.g/ml ampicillin plates. 
(The word GeneScreen is a trade mark.) Colonies were transferred from 
microtitre plates to the membranes using a 6.times.8 multi-pronged device, 
designed to fit into the wells of half the microtitre plate. Colonies were 
grown overnight at 37.degree. C., lysed in sodium hydroxide and bound to 
membranes as described by Woods (1982, 1984). After drying the membranes 
were washed extensively in 3.times.SSC/0.1% SDS at 65.degree. C., and 
hybridised to the labelled probe, using a HYBAID apparatus from Hybald 
Ltd, PO Box 82, Twickenham, Middlesex. (The word HYBAID is a trade mark.) 
Conditions for hybridisation were as described by Mason & Williams (1985), 
a T.sub.d being calculated for each oligonueleotide according to the 
formula: 
EQU T.sub.d =4.degree. C. per GC base pair+2.degree. C. per AT base pair. 
EQU At mixed positions the lowest value is taken. 
Hybridisation was carried out at T.sub.d -5.degree. C. Washing was in 
6.times.SSC, 0.1% SDS initially at room temperature in the HYBAID 
apparatus, then at the hybridisation temperature (T.sub.d -5.degree. C.) 
for some hours, and finally at T.sub.d for exactly 2 minutes. Membranes 
were autoradiographed onto FUJI X-ray film, with intensifying screens at 
-70.degree. C. (The word FUJI is a trade mark.) After 24 -48 hours 
positive colonies stood out as intense spots against a low background. 
Example 10 
Analysis of Positive Clones for the 21 kD Polypeptide 
Several positive clones were obtained with the 21 kD probe, and most of 
these contained an insert of 0.9 kb when digested with PstI (the original 
vector PstI site is re-created by the dG/dC tailing procedure). The 
inserts had the same restriction pattern, and are easily large enough to 
encode the 23 kD precursor, and it therefore seemed likely that they 
represented full-length clones. A map of the inset is shown in FIG. 1. 
The 0.9 kb PstI fragment was purified away from the vector by agarose gel 
electrophoresis onto DE81 paper (Dretzen et al, 1981), and about 500 ng 
was nick-translated using the Amersham nick-translation kit. The resulting 
probe was -4.times.10.sup.7 cpm and 10.sup.6 cpm were used for the 
subsequent probing of the cDNA library, using the hybridisation method 
described by Wahl and Berger (1987). The conditions of 50% formamide and 
42.degree. C. were used. Several more incomplete positive clones were 
obtained, which were used in subsequent sequencing. 
Example 11 
Sequencing the Cloned Inserts 
The sequencing strategy was to clone the inserts, and where appropriate 
subclones thereof, into the multiple cloning site of the plasmids 
pTZ18R/pTZ19R (Pharmacia). These plasmids are based on the better-known 
vectors pUC18/19 (Norrander et al, 1983), but contain a single-stranded 
origin of replication from the filamentous phage fl. When superinfected 
with phages in the same group, the plasmid is induced to undergo 
single-stranded replication, and the single-strands are packaged as phages 
extruded into the medium. DNA can be prepared from these `phages` using 
established methods for M13 phages (Miller, 1987), and used for sequencing 
by the method of Sanger (1977) using the reverse sequencing primer. The 
superinfecting phage used is a derivative of M13 termed M13K07, which 
replicates poorly and so does not compete well with the plasmid, and 
contains a selectable kanamycin-resistance marker. Detailed methods for 
preparing single-strands from the pTZ plasmids and helper phages are 
supplied by Pharmacia. DNA sequence was compiled and analysed using the 
Staden package of programs (Staden, 1986), on a PRIME 9955 computer. (The 
word PRIME is a trade mark.) 
Example 12 
Features of the 21 kD cDNA, and Deduced Amino-acid Sequence of the 23 kD 
Precursor 
The DNA sequence of the 21 kD cDNA, and the presumed amino-acid sequence of 
the encoded 23 kD precursor is shown in FIG. 2. The cDNA is 917 bases, 
excluding the 3' poly A tail. The ATG start codon is at position 21, 
followed by an open reading frame of 221 codons, ending with a stop codon 
at position 684. This is followed by a 233-base untranslated region, which 
is relatively AT-rich (60%) and has several stop codons in all three 
frames. There are two polyadenylation signals (AATAAA) at positions 753 
and 887 (Proudfoot and Brownlee, 1976). At position 99 the sequence 
corresponding to the oligonucleotide probe is found, and at 167 the Cla 
site found experimentally. 
The presumed 23 kD precursor polypeptide comprises 221 amino-acids and a 
molecular weight of 24003. The mature N-terminus is found at position 27, 
and the first 26 residues are highly hydrophobic, characteristic of a 
signal sequence recognised by the proteins responsible for translocating 
newly- synthesised proteins across membranes in the process of 
compartmentalisation (Kreil, 1981). The mature protein has 195 residues 
and a molecular weight of 21223, in good agreement with that deduced from 
polyacrylamide gels. The amino-acid composition of the mature protein is 
typical of a soluble protein with 24% charged residues and about 20% 
hydrophobic residues. 
Homologies Between the 21 kD Protein and Other Known Proteins 
Searching the protein identification resource (PIR) databank (National 
Biomedical Research Foundation, Washington D.C.) using the sequence 
matching program FASTP (Lipman and Pearson, 1985), showed a high degree of 
homology between the 21 kD protein and Kunitz-type protease and 
.alpha.-amylase inhibitors found in large amounts in the seeds of several 
species, particularly legumes and cereals. Examples, shown in FIG. 3, 
include the barley .alpha.-amylase/subtilisin inhibitor, B-ASI (Svendsen 
et al. 1986), wheat .alpha.-amylase/subtilisin inhibitor, W-ASI (Maeda, 
1986), winged bean (Pscophocarpus tetragonolobus) chymotrypsin inhibitor, 
W-CI (Shibata et al. 1988), winged bean trypsin inhibitor, W-TI (Yamamoto 
et al. 1983), soybean trypsin inhibitor, S-TI (Koide and Ikenaka, 1973b), 
Erythrina latissima trypsin inhibitor, E-TI (Joubert et al. 1985). 
All the Kunitz-type inhibitors are of a similar size and align along their 
entire length. Thus the 21 kD protein must belong to this general class. 
Example 13 
Expression of the 23 kD and 21 kD Polypeptides in E. coli 
The DNA encoding the 23 kD and 21 kD polypeptides (ie. with and without the 
hydrophobic signal peptide) was subcloned into the E. coli expression 
vector, pJLA502 (Schauder et al, 1987) marketed by Medac GmbH, Postfach 
303629, D-7000, Hamburg 36 (see FIG. 4). The vector contains the strong 
lambda promoters, P.sub.L and P.sub.R, and the leader sequence and 
ribosome binding site of the very efficiently translated E. coli gene, 
atpE. It also contains a temperature-sensitive cI repressor, and so 
expression is repressed at 30.degree. C. and activated at 42.degree. C. 
The vector has an NcoI site (containing an ATG codon: CCATGG) correctly 
placed with respect to the ribosome binding site, and foreign coding 
sequences must be spliced in at this point. The 23 kD coding sequence does 
not have an NcoI site at the initial ATG, so one was introduced by in 
vitro mutagenesis. 
In vitro mutagenesis was carried out using a kit marketed by Amersham 
International, which used the method of Eckstein and co-workers (Taylor et 
al, 1985). After annealing the mutagenic primer (SEQ ID NO: 26) to 
single-stranded DNA the second strand synthesis incorporates 
alpha-thio-dCTP in place of dCTP. After extension and ligation to form 
closed circles, the plasmid is digested with NciI, an enzyme which cannot 
nick DNA containing thio-dC. Thus only the original strand is nicked, and 
subsequently digested with exonuclease III. The original strand is then 
resynthesised, primed by the remaining DNA fragments and complementing the 
mutated position in the original strand. Plasmids are then transformed 
into E. coli and checked by plasmid mini preparations. 
An NcoI site was introduced into the 23 kD cDNA in plasmid pMS101 (in the 
vector pTZ19R, so that single-stranded DNA could readily be produced) 
using the mutagenic primer: 5' ACTTAACCATG GAGACC 3', to create the 
plasmid pMS106. The primer was chosen to avoid extensive hybridisation 
elsewhere in the plasmid. 
The 23 kD coding region was cloned into the E. coli expression vector 
pJLA502 on an NcoI-EcoI fragment (pMS107). The coding region was then 
cloned back into pTZ19 on a XhoI (upstream of the NcoI) -EcoRI fragment. 
This creates a pTZ-23 kD plasmid (pMS108) which has eliminated the poly G 
region, likely to disrupt transcription between the T7 promoter in the 
vector and the coding region. In vitro transcription, using T7 RNA 
polymerase, produced abundant RNA which translated in a wheat germ system 
to give a 23 kD protein. This proves that a functional gene, capable of 
producing a protein of the anticipated size, is present on the plasmid. 
The hydrophobic sequel sequence was deleted from plasmid pMS108 using a 
mutagenic primer (SEQ ID NO: 27) designed to bind either side of the 
proposed deletion: 
EQU 5' TGGAGACTGCCATGGCAAACTCTCCTGTG 3' 
The resulting plasmid, pMS111, had retained an NcoI site at the ATG start, 
and the 21 kD coding region was subcloned into pJLA502 on an NcoI-BamHI 
fragment (pMS113). 
The two expression vectors were transformed into E. coli UT580. The 
transformed strains were grown in L-broth+ampicillin (100 .mu.g/ml) at 
30.degree. C. until log phase (OD.sub.610 =0.5) and the temperature was 
then shifted to 42.degree. C. and samples taken at intervals. Samples were 
dissociated by boiling in SDS loading buffer, and run on SDS-PAGE gels. 
The proteins were electroblotted onto nitrocellulose membranes (Towbin et 
al, 1979) and Western blotting carried out using the anti-21 kD antibody 
prepared in Example 3 above (at 2 .mu.g/ml) and as a secondary antibody, 
goat anti-rabbit -IgG conjugated to alkaline phosphatase (Scott et al, 
1988). 
For the vector pMS107 the antibody detected specific protein of molecular 
weight about 23 kD, but there were also smaller bands, including one at 21 
kD suggesting that E. coli was partially cleaving the hydrophobic signal. 
The largest mount of protein was seen after 18 hours, and was the 
equivalent of at least 1-2 mg/l. Controls containing only the vector gave 
no immuno-detectable proteins. For the vector pMS113 a similar result was 
obtained, except that only the 21 kD protein was seen: there was no 
evidence of higher expression in the absence of the signal sequence. 
However transforming the vectors into the protease-deficient strain CAG629 
(Dr C. A. Gross) resulted in a much higher level of expression in both 
cases, in the order of 5-10 mg/l. 
Example 14 
Expression of the 21/23 kD Polypeptides in Yeast (Saccharomyces cerevisiae) 
Two yeast expression vectors were used, both based on a yeast-E. coli 
shuffle vector containing yeast and E. coli origins of replication, and 
suitable selectable markers (ampicillin-resistance for E. coli and leucine 
auxotrophy for yeast). Both vectors contain the yeast pyruvate kinase (PK) 
promoter and leader sequence and have a HindIII cloning site downstream of 
the promoter. One vector, A, is designed for internal expression, and the 
other, B, for secreted expression, having a portion of the signal sequence 
of the yeast mating alpha-factor downstream of the promoter, with a 
HindIII site within it to create fusion proteins with incoming coding 
sequences. The vectors are illustrated in FIG. 5. 
To use the vectors effectively it is desirable to introduce the foreign 
coding region such that for vector A, the region from the HindIII cloning 
site to the ATG start is the same as the yeast PK gene, and for vector B, 
the remainder of the alpha-factor signal, including the lysine at the 
cleavage point. In practice this situation was achieved by synthesising 
two sets of HindIII-NcoI linkers to breach the gap between the HindIII 
cloning site in the vector and the NcoI at the ATG start of the coding 
sequence. For vector B, when the coding sequence is to be spliced to the 
yeast alpha-factor signal, the coding region of the 21 kD polypeptide (ie. 
with the cocoa signal sequence removed) was used. The constructs are 
illustrated in FIG. 6. For ease of construction of the yeast vectors, 
HindIII-NcoI linkers were first cloned into the appropriate pTZ plasmids, 
and HindIII-BamIII fragments containing linkers plus coding region cloned 
into the yeast vector. 
The yeast expression plasmids were transferred into yeast spheroplasts 
using the method of Johnston (1988). The transformation host was the 
LEU.sup.- strain AH22, and transformants were selected on leucine-minus 
minimal medium. LEU.sup.+ transformants were streaked to single colonies, 
which were grown in 50 ml YEPD medium (Johnston, 1988) at 28.degree. C. 
for testing the extent and distribution of foreign protein. Cells were 
harvested from cultures in preweighed tubes in a bench-top centrifuge, and 
washed in 10 ml lysis buffer (200 mm Tris, pH 8.1; 10% glycerol). The cell 
medium was reserved and concentrated 10-25.times. in an AMICON mini 
concentrator. (The word AMICON is a trade mark.) The washed cells were 
weighed and resuspended in lysis buffer plus protease inhibitors (1 mM 
phenyl methyl sulphonyl fluoride (PMSF); 1 .mu.g/ml aprotinin; 0.5 
.mu.g/ml leupeptin) at a concentration of 1 g/mi. 1 volume acid-washed 
glass-beads was added and the cells broken by vortexing for 8 minutes in 
total, in 1 minute bursts, with 1 minute intervals on ice. After checking 
under the microscope for cell breakage, the mixture was centrifuged at 
7000 rpm for 3 minutes to pellet the glass buds. The supernatant was 
removed to a pre-chilled centrifuge tube, and centrifuged for 1 hour at 
20,000 rpm. (Small samples can be centrifuged in a microcentrifuge in the 
cold.) The supernatant constitutes the soluble fraction. The pellet was 
resuspended in 1 ml lysis buffer plus 10% SDS and 1% mercaptoethanol and 
heated at 90.degree. C. for 10 minutes. After centrifuging for 15 minutes 
in a microcentrifuge the supernatant constitutes the particulate fraction. 
Samples of each fraction and the concentrated medium were examined by 
Western blotting. Plasmid pMS116, designed for internal expression, 
produced both 23 kD and 21 kD polypeptides in the soluble fraction of the 
cell lysate, and in the medium considerable amounts (2-5 mg/l) of the 21 
kD polypeptide. Thus the yeast is recognising the cocoa signal sequence 
and transporting the protein across the membrane, cleaving the signal 
during the process. The cleavage site appears to be correct, judging by 
the size of the final protein. 
Plasmid pMS117, designed for secreted expression, gave a rather similar 
result with rather more 21 kD polypeptide in the medium. No evidence of 
the uncleaved polypeptide with the yeast alpha-factor signal still 
attached was found, either in the soluble or particulate fraction. 
Example 15 
Scale-up of Production of the 21 kD Protein in a 5 L Fermenter 
To assess the productivity of the 21 kD protein from yeast AH22 containing 
the plasmid pMS117 under scale-up conditions the strain was grown in a 5 L 
bioreactor manufactured by Life Technologies Inc. Like the small-scale 
growth experiments the medium used was YEPD, and the inoculum was 10 ml of 
a late log phase culture (OD.sub.600 4.0). The aeration rate was 2 L/min 
and the stirring speed 350 rpm, and to control the foaming caused by these 
aeration and stirring speeds 10 ml safflower oil was added. The cells were 
just entering log phase after 10 hours and by 15 hours the log phase was 
over with the disappearance of the glucose and accumulation of ethanol. 
However growth continued until the harvesting point at 60 hours, with the 
concomitant oxidation of the ethanol. The final biomass was 28 g/L wet 
weight, 7.3 g/L dry weight. Western blotting of the medium showed that 21 
kD protein was exported to the medium slowly at first, but accumulated 
rapidly in late stationary phase rising to approximately 20-30 mg/L at the 
time of harvesting. 
At the end of the experiment yeast cells were removed from the medium by 
cross-flow filtration through a 0.2 .mu.m membrane, and the protein (or 
macromolecular) constituents in the medium were concentrated by cross-flow 
filtration through an ultra filtration membrane with a molecular weight 
cut-off of 10 kD. The crossflow filtration apparatus was manufactured by 
Sartorius GmbH, Goettingen, Germany. The 21 kD protein can be further 
crudely purified by precipitation with 80% ammonium sulphate, followed by 
redissolving in water and dialysis. 
Some enhancement of the yield was obtained by a batch feed process whereby 
the glucose levels were topped up to 2% from a concentrated solution as 
soon as the glucose levels had dropped below 0.1%. Four such additions 
were made at 16, 23, 34 and 37 hours, and growth continued until 58 hours. 
Improved yields of the 21 kD protein were obtained, up to 50 mg/L by the 
end of the experiment. 
Example 16 
Expression of the 23 kD/21 kD Protein in Hansenula polymorpha 
The methylotrophic yeast Hansenula polymorpha offers a number of advantages 
over Saccharamyces cerevisiae as a host for the expression of heterologous 
proteins (EP-A-0173378 and Sudbery et al, 1988). The yeast will grow on 
methanol as sole carbon source, and under these conditions the enzyme 
methanol oxidase (MOX) can represent up to 40% of the total cell protein. 
Thus the MOX promoter is a very powerful one that can be used in a vector 
to drive the synthesis of heterologous proteins, and it is effective even 
as a single copy. This gives the potential to use stable integrated 
vectors. Hansenula can also grow on rich carbon sources such as glucose, 
in which case the MOX promoter is completely repressed. This means that 
cells containing the heterologous gene can be grown to a high density on 
glucose, and induced to produce the foreign protein by allowing the 
glucose to run out and adding methanol. 
Constructs (pMY10 and pMY9) containing a 21 kD or 23 kD gene sandwiched 
between a MOX promoter and MOX terminator were made in the yeast episomal 
plasmid YEp13. Both contained a yeast secretion signal from invertase 
spliced to the cocoa gene coding region, as illustrated in FIG. 7. These 
constructs were transformed into Hansenula and both secreted the 21/23 kD 
protein into the medium under inducing conditions, although pMY10, 
containing the yeast signal but not the plant signal, was the most 
effective. 
The Hansenula construct pMY10 has also been grown under scale-up conditions 
in a fermenter, and biomass yields of 45 g/L dry weight were obtained 
after induction with methanol. After induction the 21 kD protein was found 
in the medium in increasing mounts up to 50 mg/L. 
E. coli Strains 
______________________________________ 
RR1 F.sup.- v.sub.B.sup.- M.sub.B ara-14 proA2 leuB6 lacY1 galK2 
vpsL20 (str.sup.I) 
xyl-5 mtl-1 supE44 
CAG629 lac.sub.am typ.sub.am pho.sub.am htp.sup.R.sub.am mal rpsL lon 
supC.sub.ts 
UT580 (lac-pro) supE thi hsdD5 .vertline. F'tra D36 proA .sup.+ B.sup.+ 
lacI.sup.q lacZ 
M15 
______________________________________ 
References 
Aviv, H., and Leder, P. Proc. Natl. Acad. Sci. USA 69, 1408-1412 (1972). 
Purification of biologically active globin mRNA by chromatography on oligo 
dT cellulose 
Biehl, B., Wewetzer, C., and Passern, D. J. Sci. Food Agric. 33, 1291-1304 
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Biehl, B., Brunner E., Passern, D., Quesnel, V. C. and Adomako, D. J. Sci. 
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Catty., D. and Raykundalia, C. Production and Quality control of Polyclonal 
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Non-coding region sequences in enkayotic messenger RNA. 
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polymorpha as a novel yeast system for the expression of heterologous 
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4350-4534 (1979). Electrophoretic transfer of proteins from polyacrylamide 
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Screening of cDNA Libraries. 
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R. Proc. Natl. Acad. Sci. USA 79, 5561 (1982). 
Yamamoto, M., Hara, S. and Ikenaka, T. J. Biochem. (Tokyo) 94, 849-863 
(1983). Amino-acid sequences of two trypsin inhibitors from winged bean 
seeds (Psophocarpus tetragonolobus). 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 28 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 917 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 21..686 
(D) OTHER INFORMATION: /product="DNA CODING SEQUENCE FOR 
21kD PROTEIN AND DEDUCED AMINO ACIDS" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
CAGCAACATTTCACTTAACCATGAAGACCGCAACAGCCGTAGTTTTACTC50 
MetLysThrAlaThrAlaValValLeuLeu 
1510 
CTCTTCGCCTTCACATCAAAATCATATTTCTTTGGGGTAGCNAACGCT98 
LeuPheAlaPheThrSerLysSerTyrPhePheGlyValAlaAsnAla 
152025 
GCAAACTCTCCTGTGCTTGACACTGATGGTGATGAGCTCCAAACTGGG146 
AlaAsnSerProValLeuAspThrAspGlyAspGluLeuGlnThrGly 
303540 
GTTCAATATTACGTCTTGTCATCGATATCGGGTGCTGGGGGTGGAGGG194 
ValGlnTyrTyrValLeuSerSerIleSerGlyAlaGlyGlyGlyGly 
455055 
CTAGCCCTAGGAAGGGCTACAGGTCAAAGCTGCCCAGAAATTGTTGTC242 
LeuAlaLeuGlyArgAlaThrGlyGlnSerCysProGluIleValVal 
606570 
CAAAGACGATCCGACCTTGACAATGGTACTCCTGTAATCTTTTCAAAT290 
GlnArgArgSerAspLeuAspAsnGlyThrProValIlePheSerAsn 
75808590 
GCGGATAGCAAAGATGATGTTGTCCGCGTATCTACTGATGTAAACATA338 
AlaAspSerLysAspAspValValArgValSerThrAspValAsnIle 
95100105 
GAGTTCGTTCCCATCAGAGACAGACTCTGCTCAACGTCAACTGTGTGG386 
GluPheValProIleArgAspArgLeuCysSerThrSerThrValTrp 
110115120 
AGGCTTGACAATTATGACAACTCGGCAGGCAAATGGTGGGTGACAACT434 
ArgLeuAspAsnTyrAspAsnSerAlaGlyLysTrpTrpValThrThr 
125130135 
GATGGGGTTAAAGGTGAACCTGGTCCTAACACTTTGTGCAGTTGGTTT482 
AspGlyValLysGlyGluProGlyProAsnThrLeuCysSerTrpPhe 
140145150 
AAGATTGAGAAGGCCGGAGTACTCGGTTACAAATTCAGGTTCTGTCCT530 
LysIleGluLysAlaGlyValLeuGlyTyrLysPheArgPheCysPro 
155160165170 
TCTGTCTGTGATTCGTGCACAACTTTATGCAGCGATATTGGAAGACAT578 
SerValCysAspSerCysThrThrLeuCysSerAspIleGlyArgHis 
175180185 
TCAGATGATGATGGACAAATACGTTTGGCTCTCAGTGACAATGAATGG626 
SerAspAspAspGlyGlnIleArgLeuAlaLeuSerAspAsnGluTrp 
190195200 
GCATGGATGTTTAAGAAAGCAAGTAAGACAATAAAACAAGTTGTTAAC674 
AlaTrpMetPheLysLysAlaSerLysThrIleLysGlnValValAsn 
205210215 
GCGAACGATTAATTTTAAGTTTAATGTACGAAGTGTACGTCCAAAGCAG723 
AlaAsnAsp 
220 
CAATACTAGCCGGTCGTTACTTTCCACTAAATAAAAGTTAAGTATGTGGTTCCCAGCCCA783 
GTGTTGTAATGCTATGCCTATGTAGTCAGTGTCTTGTTTGAGGGTGGAGATGCTTAAAGG843 
GTGTGTCTTCACAGTCCCAGCTTCGTAGTCTTTCAGCTTTATGAATAAATGATTTGCCTC903 
TTGCCTCTTTTATT917 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 221 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
MetLysThrAlaThrAlaValValLeuLeuLeuPheAlaPheThrSer 
151015 
LysSerTyrPhePheGlyValAlaAsnAlaAlaAsnSerProValLeu 
202530 
AspThrAspGlyAspGluLeuGlnThrGlyValGlnTyrTyrValLeu 
354045 
SerSerIleSerGlyAlaGlyGlyGlyGlyLeuAlaLeuGlyArgAla 
505560 
ThrGlyGlnSerCysProGluIleValValGlnArgArgSerAspLeu 
65707580 
AspAsnGlyThrProValIlePheSerAsnAlaAspSerLysAspAsp 
859095 
ValValArgValSerThrAspValAsnIleGluPheValProIleArg 
100105110 
AspArgLeuCysSerThrSerThrValTrpArgLeuAspAsnTyrAsp 
115120125 
AsnSerAlaGlyLysTrpTrpValThrThrAspGlyValLysGlyGlu 
130135140 
ProGlyProAsnThrLeuCysSerTrpPheLysIleGluLysAlaGly 
145150155160 
ValLeuGlyTyrLysPheArgPheCysProSerValCysAspSerCys 
165170175 
ThrThrLeuCysSerAspIleGlyArgHisSerAspAspAspGlyGln 
180185190 
IleArgLeuAlaLeuSerAspAsnGluTrpAlaTrpMetPheLysLys 
195200205 
AlaSerLysThrIleLysGlnValValAsnAlaAsnAsp 
210215220 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 192 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Theobroma cacao 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..192 
(D) OTHER INFORMATION: /note= "21kD protein from T. cacao" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
AlaAsnSerProValLeuAspThrAspGlyAspGluLeuGlnThrGly 
151015 
ValGlnTyrTyrValLeuSerSerIleSerGlyAlaGlyGlyGlyGly 
202530 
LeuAlaLeuGlyArgAlaThrGlyGlnSerCysProGluIleValVal 
354045 
GlnArgArgSerAspLeuAspAsnGlyThrProValIlePheSerAsn 
505560 
AlaAspSerLysAspAspValValArgValSerThrAspValAsnIle 
65707580 
GluPheValProIleArgAspArgLeuCysSerThrSerThrValTrp 
859095 
ArgLeuAspAsnTyrAspAsnSerAlaGlyLysTrpTrpValThrThr 
100105110 
AspGlyValLysGlyGluProGlyProAsnThrLeuCysSerTrpPhe 
115120125 
LysIleGluLysAlaGlyValLeuGlyTyrLysPheArgPheCysPro 
130135140 
SerValCysAspSerCysThrThrLeuCysSerAspIleGlyArgHis 
145150155160 
SerAspAspAspGlyGlnIleArgLeuAlaLeuSerAspAsnGluTrp 
165170175 
AlaTrpMetPheLysLysAlaSerLysThrIleLysGlnValValAsn 
180185190 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: BARLEY 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..181 
(D) OTHER INFORMATION: /note= "ALPLHA-AMYLASE-SUBTILISIN 
INHIBITOR" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
AlaAspProProProValHisAspThrAspGlyHisGluLeuArgAla 
151015 
AspAlaAsnTyrTyrValLeuSerAlaAsnArgAlaHisGlyGlyGly 
202530 
LeuThrMetAlaProGlyHisGlyArgHisCysProLeuPheValSer 
354045 
GlnAspProAsnGlyGlnHisAspGlyPheProValArgIleThrPro 
505560 
TyrGlyValAlaProSerAspLysIleIleArgLeuSerThrAspVal 
65707580 
ArgIleSerPheArgAlaTyrThrThrCysLeuGlnSerThrGluTrp 
859095 
HisIleAspSerGluLeuAlaAlaGlyArgArgHisValIleThrGly 
100105110 
ProValLysAspProSerProSerGlyArgGluAsnAlaPheArgIle 
115120125 
GluLysTyrSerGlyAlaGluValHisGluTyrLysLeuMetSerCys 
130135140 
GlyAspTrpCysGlnAspLeuGlyValPheArgAspLeuLysGlyGly 
145150155160 
AlaTrpPheLeuGlyAlaThrGluProTyrHisValValValPheLys 
165170175 
LysAlaProProAla 
180 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 180 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: WHEAT 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..180 
(D) OTHER INFORMATION: /note= "ALPHA-AMYLASE-SUBTILISIN 
INHIBITOR" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
AspProProProValHisAspThrAspGlyAsnGluLeuArgAlaAsp 
151015 
AlaAsnTyrTyrValLeuProAlaAsnArgAlaHisGlyGlyGlyLeu 
202530 
ThrMetAlaProGlyHisGlyArgArgCysProLeuPheValSerGln 
354045 
GluAlaAspGlyGlnArgAspGlyLeuProValArgIleAlaProHis 
505560 
GlyGlyAlaProSerAspLysIleIleArgLeuSerThrAspValArg 
65707580 
IleSerPheArgAlaTyrThrThrCysValGlnSerThrGluTrpHis 
859095 
IleAspSerGluLeuValSerGlyArgArgHisValIleThrGlyPro 
100105110 
ValArgAspProSerProSerGlyArgGluAsnAlaPheArgIleGlu 
115120125 
LysTyrSerGlyAlaGluValHisGluTyrLysLeuAlaSerCysGly 
130135140 
AspSerCysGlnAspLeuGlyValPheArgAspLeuLysGlyGlyAla 
145150155160 
TrpPheLeuGlyAlaThrGluProTyrHisValValValPheLysLys 
165170175 
AlaProProAla 
180 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 182 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: WINGED BEAN 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..182 
(D) OTHER INFORMATION: /note= "CHYMOTRYPSIN INHIBITOR" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
AspAspAspLeuValAspAlaGluGlyAsnLeuValGluAsnGlyGly 
151015 
ThrTyrTyrLeuLeuProHisIleTrpAlaHisGlyGlyGlyIleGlu 
202530 
ThrAlaLysThrGlyAsnGluProCysProLeuThrValValArgSer 
354045 
ProAsnGluValSerLysGlyGluProIleArgIleSerSerGlnPhe 
505560 
LeuSerLeuPheIleProArgGlySerLeuValAlaLeuGlyPheAla 
65707580 
AsnProProSerCysAlaAlaSerProTrpTrpThrValValAspSer 
859095 
ProGlnGlyProAlaValLysLeuSerGlnGlnLysLeuProGluLys 
100105110 
AspIleLeuValPheLysPheGluLysValSerHisSerAsnIleHis 
115120125 
ValTyrLysLeuLeuTyrCysGlnHisAspGluGluAspValLysCys 
130135140 
AspGlnTyrIleGlyIleHisArgAspArgAsnGlyAsnArgArgLeu 
145150155160 
ValValThrGluGluAsnProLeuGluLeuValLeuLysAlaLysSer 
165170175 
GluThrAlaSerSerHis 
180 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 171 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: WINGED BEAN 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..171 
(D) OTHER INFORMATION: /note= "TRYPSIN INHIBITOR" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
GluProLeuLeuAspSerGluGlyGluLeuValArgAsnGlyGlyThr 
151015 
TyrTyrLeuLeuProAspArgTrpAlaLeuGlyGlyGlyIleGluAla 
202530 
AlaAlaThrGlyThrGluThrCysProLeuThrValValArgSerPro 
354045 
AsnGluValSerValGlyGluProLeuArgIleSerSerGlnLeuArg 
505560 
SerGlyPheIleProAspTyrSerLeuValArgIleGlyPheAlaAsn 
65707580 
ProProLysCysAlaProSerProTrpTrpThrValValGluAspGln 
859095 
ProGlnGlnProSerValLysLeuSerGluLeuLysSerThrLysPhe 
100105110 
AspTyrLeuPheLysPheGluLysValThrSerLysPheSerSerTyr 
115120125 
LysLeuLysTyrCysAlaLysArgAspThrCysLysAspIleGlyIle 
130135140 
TyrArgAspGlnGlyTyrAlaArgLeuValValThrAspGluAsnPro 
145150155160 
LeuValValIlePheLysLysValGluSerSer 
165170 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 181 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: SOYBEAN 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..181 
(D) OTHER INFORMATION: /note= "TRYPSIN INHIBITOR" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
AspPheValLeuAspAsnGluGlyAsnProLeuGluAsnGlyGlyThr 
151015 
TyrTyrIleLeuSerAspIleThrAlaPheGlyGlyIleArgAlaAla 
202530 
ProThrGlyAsnGluArgCysProLeuThrValValGlnSerArgAsn 
354045 
GluLeuAspLysGlyIleGlyThrIleIleSerSerProTyrArgIle 
505560 
ArgPheIleAlaGluGlyHisProLeuSerLeuLysPheAspSerPhe 
65707580 
AlaValIleMetLeuCysValGlyIleProThrGluTrpSerValVal 
859095 
GluAspLeuProGluGlyProAlaValLysIleGlyGluAsnLysAsp 
100105110 
AlaMetAspGlyTrpPheArgLeuGluArgValSerAspAspGluPhe 
115120125 
AsnAsnTyrLysLeuValPheCysProGlnGlnAlaGluAspAspLys 
130135140 
CysGlyAspIleGlyIleSerIleAspHisAspAspGlyThrArgArg 
145150155160 
LeuValValSerLysAsnLysProLeuValValGlnPheGlnLysLeu 
165170175 
AspLysGluSerLeu 
180 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 172 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Erythrina latissima 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..172 
(D) OTHER INFORMATION: /note= "TRYPSIN INHIBITOR" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
ValLeuLeuAspGlyAsnGlyGluValValGlnAsnGlyGlyThrTyr 
151015 
TyrLeuLeuProGlnValTrpAlaGlnGlyGlyGlyValGlnLeuAla 
202530 
LysThrGlyGluGluThrCysProLeuThrValValGlnSerProAsn 
354045 
GluLeuSerAspGlyLysProIleArgIleGluSerArgLeuArgSer 
505560 
ThrPheIleProAspAspAspGluValArgIleGlyPheAlaTyrAla 
65707580 
ProLysCysAlaProSerProTrpTrpThrValValGluAspGluGln 
859095 
GluGlyLeuSerValLysLeuSerGluAspGluSerThrGlnPheAsp 
100105110 
TyrProPheLysPheGluGlnValSerAspLysLeuHisSerTyrLys 
115120125 
LeuLeuTyrCysGluGlyLysHisGluLysCysAlaSerIleGlyIle 
130135140 
AsnArgAspGlnLysGlyTyrArgArgLeuValValThrGluAspAsn 
145150155160 
ProLeuThrValValLeuLysLysAspGluSerSer 
165170 
(2) INFORMATION FOR SEQ ID NO: 10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: YEAST PYRUVATE KINASE GENE 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 41..49 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 
TTTACAAGACACCAATCAAAACAAATAAAACATCATCACAATGTCTAGA49 
MetSerArg 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 
MetSerArg 
1 
(2) INFORMATION FOR SEQ ID NO: 12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: YEAST PYRUVATE KINASE GENE 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 41..49 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 6..11 
(D) OTHER INFORMATION: /function="Hin cloning site 
introduced" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 
TTTACAAGCTTCCAATCAAAACAAATAAAACATCATCACAATGTCTAGA49 
MetSerArg 
1 
(2) INFORMATION FOR SEQ ID NO: 13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
MetSerArg 
1 
(2) INFORMATION FOR SEQ ID NO: 14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..33 
(D) OTHER INFORMATION: /product="Hin-Nco linker" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 
AGCTTCCAATCAAAACAAATAAAACATCATCAC33 
(2) INFORMATION FOR SEQ ID NO: 15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..33 
(D) OTHER INFORMATION: /product="Hin-Nco linker" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 
CATGGTGATGATGTTTTATTTGTTTTGATTGGA33 
(2) INFORMATION FOR SEQ ID NO: 16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 41..49 
(D) OTHER INFORMATION: /product="21kD EXPRESSION VECTOR A" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 
TTTACAAGCTTCCAATCAAAACAAATAAAACATCATCACCATGGAGACC49 
MetGluThr 
1 
(2) INFORMATION FOR SEQ ID NO: 17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 
MetGluThr 
1 
(2) INFORMATION FOR SEQ ID NO: 18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: YEAST ALPHA-FACTOR SIGNAL SEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..27 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 
GAAGGGGTAAGCTTGGATAAAAGAGAG27 
GluGlyValSerLeuAspLysArgGlu 
15 
(2) INFORMATION FOR SEQ ID NO: 19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 
GluGlyValSerLeuAspLysArgGlu 
15 
(2) INFORMATION FOR SEQ ID NO: 20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /product="Hin-Nco linker" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 
AGCTTGGATAAAAGAGC17 
(2) INFORMATION FOR SEQ ID NO: 21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /product="Hin-Nco linker" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 
CATGGCTCTTTTATCCA17 
(2) INFORMATION FOR SEQ ID NO: 22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..36 
(D) OTHER INFORMATION: /product="IN-PHASE FUSION OF 21kD 
CODING REGION 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 
GAAGGGGTAAGCTTGGATAAAAGAGCCATGGCAAAC36 
GluGlyValSerLeuAspLysArgAlaMetAlaAsn 
1510 
(2) INFORMATION FOR SEQ ID NO: 23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 
GluGlyValSerLeuAspLysArgAlaMetAlaAsn 
1510 
(2) INFORMATION FOR SEQ ID NO: 24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 
AlaAsnSerProLeuAspThrAspGlyAspGlu 
1510 
(2) INFORMATION FOR SEQ ID NO: 25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /product="N at position 3 = C or T 
N at position 6 = C, T, A or G 
N at position 9 = C or T 
N at position 12 = C, T, A or G 
N at position 15 = C or T" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 
GANACNGANGGNGANGA17 
(2) INFORMATION FOR SEQ ID NO: 26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /product="mutagenic primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 
ACTTAACCATGGAGACC17 
(2) INFORMATION FOR SEQ ID NO: 27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 1..29 
(D) OTHER INFORMATION: /product="mutagenic primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 
TGGAGACTGCCATGGCAAACTCTCCTGTG29 
(2) INFORMATION FOR SEQ ID NO: 28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 
GGAGACTGCCATGG14 
__________________________________________________________________________