Proteinase inhibitor, precursor thereof and genetic sequences encoding same

The present invention relates generally to proteinase inhibitors, a precursor thereof and to genetic sequences encoding same. More particularly, the present invention relates to a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a type II serine proteinase inhibitor (PI) precursor from a plant wherein said precursor comprises at least three PI monomers and wherein at least one of said monomers has a chymotrypsin specific site and at least one other of said monomers has a trypsin specific site.

The present invention relates generally to proteinase inhibitors, a 
precursor thereof and to genetic sequences encoding same. 
Nucleotide and amino acid sequences are referred to herein by sequence 
identity numbers (SEQ ID NOs) which are defined after the bibliography. A 
general summary of the SEQ ID NOs is provided before the examples. 
Throughout this specification and the claims which follow, unless the 
context requires otherwise, the word "comprise", or variations such as 
"comprises" or "comprising", will be understood to imply the inclusion of 
a stated element or integer or group of elements or integers but not the 
exclusion of any other element or integer or group of elements or 
integers. 
Several members of the families Solanaceae and Fabaceae accumulate serine 
proteinase inhibitors in their storage organs and in leaves in response to 
wounding (Brown and Ryan, 1984; Richardson, 1977). The inhibitory 
activities of these proteins are directed against a wide range of 
proteinases of microbial and animal origin, but rarely against plant 
proteinases (Richardson, 1977). It is believed that these inhibitors are 
involved in protection of the plants against pathogens and predators. In 
potato tubers and legume seeds, the inhibitors can comprise 10% or more of 
the stored proteins (Richardson, 1977), while in leaves of tomato and 
potato (Green and Ryan, 1972), and alfalfa (Brown and Ryan, 1984), 
proteinase inhibitors can accumulate to levels of 2% of the soluble 
protein within 48 hours of insect attack, or other types of wounding 
(Brown & Ryan, 1984; Graham et al., 1986). High levels of these inhibitors 
(up to 50% of total soluble protein) are also present in unripe fruits of 
the wild tomato, Lycopersicon peruvianum (Pearce et al., 1988). 
There are two families of serine proteinase inhibitors in tomato and potato 
(Ryan, 1984). Type I inhibitors are small proteins (monomer Mr 8100) which 
inhibit chymotrypsin at a single reactive site (Melville and Ryan, 1970; 
Plunkett et al., 1982). Inhibitors of the type II family generally contain 
two reactive sites, one of which inhibits chymotrypsin and the other 
trypsin (Bryant et al., 1976; Plunkett et al., 1982). The type II 
inhibitors have a monomer Mr of 12,300 (Plunkett et al., 1982). Proteinase 
inhibitor I accumulates in etiolated tobacco (Nicotiana tabacum) leaves 
(Kuo et al., 1984), and elicitors from Phytophthora parasitica var. 
nicotianae were found to induce proteinase inhibitor I accumulation in 
tobacco cell suspension cultures (Rickauer et al., 1989). 
There is a need to identify other proteinase inhibitors and to investigate 
their potential use in the development of transgenic plants with enhanced 
protection against pathogens and predators. In accordance with the present 
invention, genetic sequences encoding a proteinase inhibitor precursor 
have been cloned. The precursor has multi-proteinase inhibitor domains and 
will be useful in developing a range of transgenic plants with enhanced 
proteinase inhibitor expression. Such plants will have enhanced protective 
properties against pathogens and predators. The genetic constructs of the 
present invention will also be useful in developing vaccines for ingestion 
by insects which are themselves predators or which act as hosts for plant 
pathogens. The recombinant precursor or monomeric inhibitors will also be 
useful in topical sprays and in assisting animals in feed digestion. 
Accordingly, one aspect of the present invention relates to a nucleic acid 
molecule comprising a sequence of nucleotides which encodes or is 
complementary to a sequence which encodes a type II serine proteinase 
inhibitor (PI) precursor from a plant wherein said precursor comprises at 
least three PI monomers and wherein at least one of said monomers has a 
chymotrypsin specific site and at least one other of said monomers has a 
trypsin specific site. 
The "nucleic acid molecule" of the present invention may be RNA or DNA (eg 
cDNA), single or double stranded and linear or covalently closed. The 
nucleic acid molecule may also be genomic DNA corresponding to the entire 
gene or a substantial portion thereof or to fragments or derivatives 
thereof. The nucleotide sequence may correspond to the naturally occurring 
nucleotide sequence of the genomic or cDNA clone or may contain single or 
multiple nucleotide substitutions, deletions and/or additions thereto. All 
such variants in the nucleic acid molecule either retain the ability to 
encode at least one monomer or active part thereof or are useful as 
hybridisation probes or polymerase chain reaction (PCR) primers for the 
same or similar genetic sequences in other sources. 
Preferably, the PI precursor comprises at least four, more preferably at 
least five and even more preferably at least six PI monomers. Still more 
preferably, the PI precursor further comprises a signal sequence. The PI 
precursor of the present invention preferably comprises amino acid 
sequences which are process sites for cleavage into individual monomers. 
The term "precursor" as used herein is not intended to place any limitation 
on the utility of the precursor molecule itself or a requirement that the 
molecule first be processed into monomers before PI activity is expressed. 
The precursor molecule has PI activity and the present invention is 
directed to the precursor and to the individual monomers of the precursor. 
Furthermore, the present invention extends to a nucleic acid molecule 
comprising a sequence of nucleotides which encodes or is complementary to 
a sequence which encodes a hybrid type II serine PI precursor wherein said 
precursor comprises at least two monomers from different PIs. The at least 
two monomers may be modified such as being unable to be processed into 
individual monomers or may retain the ability to be so processed. 
Preferably, at least one of said monomers has a chymotrypsin specific site 
and the other of said monomers has a trypsin specific site. Preferably 
there are at least three monomers, more preferably at least four monomers, 
still more preferably at least five monomers and yet still more preferably 
at least six monomers wherein at least two are from different PIs. In a 
most preferred embodiment, at least one of said monomers is a thionin. 
Such hybrid PI precursors and/or monomers thereof are particularly useful 
in generating molecules which are "multi-valent" in that they are active 
against a range of pathogens and predators such as both fungi and insects. 
Accordingly, reference herein to "PI precursor" includes reference to 
hybrid molecules. 
The present invention is exemplified by the isolation of the subject 
nucleic acid molecule from Nicotiana alata which has the following 
nucleotide sequence (SEQ ID NO. 1) and a corresponding amino acid sequence 
(SEQ ID NO. 3): 
AAG GCT TGT ACC TTA AAC 
Lys Ala Cys Thr Leu Asn 
- TGT GAT CCA AGA ATT GCC TAT 
GGA GTT TGC CCG CGT TCA GAA GAA 
AAG 
Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys 
- AAG AAT GAT CGG ATA TGC ACC 
AAC TGT TGC GCA GGC ACG AAG GGT 
TGT 
Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys 
- AAG TAC TTC AGT GAT GAT GGA 
ACT TTT GTT TGT GAA GGA GAG TCT 
GAT 
Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Cly Glu Ser Asp 
- CCT AGA AAT CCA AAG GCT TGT 
ACC TTA AAC TGT GAT CCA AGA ATT 
GCC 
Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala 
- TAT GGA GTT TGC CCG CGT TCA 
GAA GAA AAG AAG AAT GAT CGG ATA 
TGC 
Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys 
- ACC AAC TGT TGC GCA GGC ACG 
AAG GGT TGT AAG TAC TTC AGT GAT 
GAT 
Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser Asp Asp 
- GGA ACT TTT GTT TGT GAA GGA 
GAG TCT GAT CCT AGA AAT CCA AAG 
GCT 
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala 
- TGT CCT CGG AAT TGC GAT CCA 
AGA ATT GCC TAT GGG ATT TGC CCA 
CTT 
Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Tyr Gly Ile Cys Pro Leu 
- GCA GAA GAA AAG AAG AAT GAT 
CGG ATA TGC ACC AAC TGT TGC GCA 
GGC 
Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly 
- AAA AAG GGT TGT AAG TAC TTT 
AGT GAT GAT GGA ACT TTT GTT TGT 
GAA 
Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu 
- GGA GAG TCT GAT CCT AAA AAT 
CCA AAG GCC TGT CCT CGG AAT TGT 
GAT 
Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp 
- GGA AGA ATT GCC TAT GGG ATT 
TGC CCA CTT TCA GAA GAA AAG AAG 
AAT 
Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 
- GAT CGG ATA TGC ACC AAC TGC 
TGC GCA GGC AAA AAG GGT TGT AAG 
TAC 
Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 
- TTT AGT GAT GAT GGA ACT TTT 
GTT TGT GAA GGA GAG TCT GAT CCT 
AAA 
Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys 
- AAT CCA AAG GCT TGT CCT CGG 
AAT TGT GAT GGA AGA ATT GCC TAT 
GGG 
Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly 
- ATT TGC CCA CTT TCA GAA GAA 
AAG AAG AAT GAT CGG ATA TGC ACA 
AAC 
Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn 
- TGT TGC GCA GGC AAA AAG GGC 
TGT AAG TAC TTT AGT GAT GAT GGA 
ACT 
Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr 
- TTT GTT TGT GAA GGA GAG TCT 
GAT CCT AGA AAT CCA AAC GCC TGT 
CCT 
Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala Cys Pro 
- CCG AAT TGT GAT GGA AGA ATT 
GCC TAT GGA ATT TGC CCA CTT TCA 
GAA 
Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu 
- GAA AAG AAG AAT GAT CGG ATA 
TGC ACC AAT TGT TGC GCA GGC AAG 
AAG 
Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys 
- GGC TGT AAG TAC TTT AGT GAT 
GAT GGA ACT TTT ATT TGT GAA GGA 
GAA 
Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Glu 
- TCT GAA TAT GCC AGC AAA GTG 
GAT GAA TAT GTT GGT GAA GTG GAG 
AAT 
Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu Asn 
- GAT CTC CAG AAG TCT AAG GTT 
GCT GTT TCC 
Asp Leu Gln Lys Ser Lys Val Ala Val Ser 
This is done, however, with the understanding that the present invention 
extends to an equivalent or substantially similar nucleic acid molecule 
from any other plant. By "equivalent" and "substantially similar" is meant 
at the level of nucleotide sequence, amino acid sequence, antibody 
reactivity, monomer composition and/or processing of the precursor to 
produce monomers. For example, a nucleotide sequence having a percentage 
sequence similarity of at least 55%, such as about 60-65%, 70-75%, 80-85% 
and over 90% when compared to the sequence of SEQ ID NO. 1 would be 
considered "substantially similar" to the subject nucleic acid molecule 
provided that such a substantially similar sequence encodes a PI precursor 
having at least three monomers and preferably four, five or six monomers 
as hereinbefore described. 
In a particularly preferred embodiment, the nucleic acid molecule further 
encodes a signal sequence 5' to the open reading frame and/or a nucleotide 
sequence 3' of the coding region providing a full nucleotide sequence as 
follows (SEQ ID NO. 2): 
CGAGTAAGTA TGGCTGTTCA CAGAGTTAGT TTCCTTGCTC TCCTCCTCTT ATTTGGAATG 
- TCTCTGCTTG TAAGCAATGT GGAACATGCA GATGCC AAG GCT TGT ACC TTA AAC 
Lys Ala Cys Thr Leu Asn 
- TGT GAT CCA AGA ATT GCC TAT 
GGA GTT TGC CCG CGT TCA GAA GAA 
AAG 
Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys 
- AAG AAT GAT CGG ATA TGC ACC 
AAC TGT TGC GCA GGC ACG AAG GGT 
TGT 
Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys 
- AAG TAC TTC AGT GAT GAT GGA 
ACT TTT GTT TGT GAA GGA GAG TCT 
GAT 
Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp 
- CCT AGA AAT CCA AAG GCT TGT 
ACC TTA AAC TGT GAT CCA AGA ATT 
GCC 
Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala 
- TAT GGA GTT TGC CCG CGT TCA 
GAA GAA AAG AAG AAT GAT CGG ATA 
TGC 
Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys 
- ACC AAC TGT TGC GCA GGC ACG 
AAG GGT TGT AAG TAC TTC AGT GAT 
GAT 
Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser Asp Asp 
- GGA ACT TTT GTT TGT GAA GGA 
GAG TCT GAT CCT AGA AAT CCA AAG 
GCT 
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala 
- TGT CCT CGG AAT TGC GAT CCA 
AGA ATT GCC TAT GGG ATT TGC CCA 
CTT 
Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Tyr Gly Ile Cys Pro Leu 
- GCA GAA GAA AAG AAG AAT GAT 
CGG ATA TGC ACC AAC TGT TGC GCA 
GGC 
Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly 
- AAA AAG GGT TGT AAG TAC TTT 
AGT GAT GAT GGA ACT TTT GTT TGT 
GAA 
Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu 
- GGA GAG TCT GAT CCT AAA AAT 
CCA AAG GCC TGT CCT CGG AAT TGT 
GAT 
Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp 
- GGA AGA ATT GCC TAT GGG ATT 
TGC CCA CTT TCA GAA GAA AAG AAG 
AAT 
Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 
- GAT CGG ATA TGC ACC AAC TGC 
TGC GCA GGC AAA AAG GGT TGT AAG 
TAC 
Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 
- TTT AGT GAT GAT GGA ACT TTT 
GTT TGT GAA GGA GAG TCT GAT CCT 
AAA 
Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys 
- AAT CCA AAG GCT TGT CCT CGG 
AAT TGT GAT GGA AGA ATT GCC TAT 
GGG 
Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly 
- ATT TGC CCA CTT TCA GAA GAA 
AAG AAG AAT GAT CGG ATA TGC ACA 
AAC 
Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn 
- TGT TGC GCA GGC AAA AAG GGC 
TGT AAG TAC TTT AGT GAT GAT GGA 
ACT 
Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr 
- TTT GTT TGT GAA GGA GAG TCT 
GAT CCT AGA AAT CCA AAG GCC TGT 
CCT 
Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala Cys Pro 
- CGG AAT TGT GAT GGA AGA ATT 
GCC TAT GGA ATT TGC CCA CTT TCA 
GAA 
Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu 
- GAA AAG AAG AAT GAT CGG ATA 
TGC ACC AAT TGT TGC GCA GGC AAG 
AAG 
Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys 
- GGC TGT AAG TAC TTT AGT GAT 
GAT GGA ACT TTT ATT TGT GAA GGA 
GAA 
Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Glu 
- TCT GAA TAT GCC AGC AAA GTG 
GAT GAA TAT GTT GGT GAA GTG GAG 
AAT 
Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu Asn 
- GAT CTC CAG AAG TCT AAG GTT 
GCT GTT TCC TAAGTCCTAA CTAATAATAT 
Asp Leu Gln Lys Ser Lys Val Ala Val Ser 
- GTAGTCTATG TATGAAACAA AGGCATGCCA ATATGCTCTG TCTTGCCTGT AATCTGTAAT 
- ATGGTAGTGG AGCTTTTCCA 
CTGCCTGTTT AATAAGAAAT GGAGCACTAG 
TTTGTTTTAG 
- TTAAAAAAAA AAAAAAAAAA 
including substantially similar variants thereof. 
Accordingly, a preferred embodiment of the present invention provides a 
nucleic acid molecule comprising a sequence of nucleotides as set forth in 
SEQ ID NO. 1 or 2 which encodes or is complementary to a sequence which 
encodes a type II serine PI precursor from Nicotiana alata or having at 
least 55% similarity to said precursor or at least one domain therein 
wherein said precursor comprises a signal peptide and at least five 
monomers and wherein one of said monomers has a chymotrypsin specific site 
and four of said monomers have trypsin specific sites. 
In still a more preferred embodiment, the nucleic acid molecule is a cDNA 
molecule and comprises a nucleotide sequence generally as set forth in SEQ 
ID NO. 1 or 2 or being substantially similar thereto as hereinbefore 
defined to the whole of said sequence or to a domain thereof. 
Another aspect of the present invention is directed to a nucleic acid 
molecule comprising a sequence of nucleotides which encodes or is 
complementary to a sequence which encodes a single type II serine PI 
having either a chymotrypsin specific site or a trypsin specific site and 
wherein said PI is a monomer of a precursor PI having at least three 
monomers of which at least one of said monomers has a chymotrypsin site 
and the other of said monomers has a trypsin site. Preferably, however, 
the precursor has four, five or six monomers and is as hereinbefore 
defined. 
In its most preferred embodiment, the plant is N. alata (Link et Otto) 
having self-incompatibility genotype S.sub.1 S.sub.3, S.sub.3 S.sub.3 or 
S.sub.6 S.sub.6, and the nucleic acid molecule is isolatable from or 
complementary to genetic sequences isolatable from stigmas and styles of 
mature plants. The corresponding mRNA is approximately 1.4 kb and the cDNA 
has six conserved domains wherein the first two domains are 100% identical 
and contain chymotrypsin-specific sites (Leu-Asn). The third, fourth and 
fifth domains share 95-98% identity and have sites specific for trypsin 
(Arg-Asn). A sixth domain which also has a trypsin specific site has less 
identity to the third, fourth and fifth domains (79-90%) due mainly to a 
divergent 3' sequence (see Table 1). The preferred PI inhibitor of the 
present invention has a molecular weight of approximately 42-45 kDa with 
an approximately 29 amino acid signal sequence. 
The N-terminal sequence of the monomeric PI is represented in each of the 
six repeated domains in the predicted sequence of the PI precursor 
protein. Thus, it is likely that the PI precursor protein is cleaved at 
six sites to produce seven peptides. Six of the seven peptides, peptides 
2, 3, 4, 5, 6 and 7 (FIG. 1, residues 25-82 [SEQ ID NO. 5], 83-140 [SEQ ID 
NO. 6], 141-198 [SEQ ID NO. 7], 199-256 [SEQ ID NO. 8], 257-314 [SEQ ID 
NO. 9] and 315-368 [SEQ ID NO. 9], respectively), would be in the same 
molecular weight range as the monomeric PI (about 6 kDa) and would have 
the same N-terminal sequence. Peptide 7 does not contain a consensus site 
for trypsin or chymotrypsin. Peptide 1 (residues 1-24 [SEQ ID NO. 4], FIG. 
1) is smaller than 6 kD, has a different N-terminus and was not detected 
in a purified monomeric PI preparation. It could be envisaged that peptide 
1 and peptide 7 would form a functional proteinase inhibitor with the 
inhibitory site on peptide 1 held in the correct conformation by 
disulphide bonds formed between the two peptides. 
Although not intending to limit the present invention to any one 
hypothesis, the PI precursor may be processed by a protease responsible, 
for example, for cleavage of an Asn-Asp linkage, to produce the bioactive 
monomers. More particularly, the protease sensitive sequence is R.sub.1 
-X.sub.1 -X.sub.2 -Asn-Asp-R.sub.2 where R.sub.1, R.sub.2, X.sub.1 and 
X.sub.2 are defined below. The discovery of such a sequence will enable 
the engineering of peptides and polypeptides capable of being processed in 
a plant by cleavage of the protease sensitive sequence. According to this 
aspect of the present invention there is provided a protease sensitive 
peptide comprising the amino acid sequence: 
EQU --X.sub.1 --X.sub.2 --Asn--Asp-- 
wherein X.sub.1 and X.sub.2 are any amino acid but are preferably both Lys 
residues. The protease sensitive peptide may also be represented as: 
EQU R.sub.1 --X.sub.1 --X.sub.2 --Asn--Asp--R.sub.2 
wherein X.sub.1 and X.sub.2 are preferably the same and are preferably both 
Lys residues and wherein R.sub.1 and R.sub.2 are the same or different, 
any D or L amino acid, a peptide, a polypeptide, a protein, or a non-amino 
acid moiety or molecule such as, but not limited to, an alkyl (eg methyl, 
ethyl), substituted alkyl , alkenyl, substituted alkenyl, acyl, dienyl, 
arylalkyl, arylalkenyl, aryl, substituted aryl, heterocyclic, substituted 
heterocyclic, cycloalkyl, substituted cycloalkyl, halo (e.g. Cl, Br, I, 
F), haloalkyl, nitro, hydroxy, thiol, sulfonyl, carboxy, alkoxy, aryloxy 
and alkylaryloxy group and the like as would be apparent to one skilled in 
the art. By alkyl, substitued alkyl, alkenyl and substituted alkenyl and 
the like is meant to encompass straight and branched molecules, lower 
(C.sub.1 -C.sub.6) and higher (more than C.sub.6) derivatives. The term 
"substituted" includes all the substituents set forth above. 
In its most preferred embodiment, the protease sensitive peptide is: 
EQU R.sub.1 --X.sub.1 --X.sub.2 --Asn--Asp--R.sub.2 
wherein R.sub.1 and R.sub.2 are the same or different and are peptides or 
polypeptides and wherein X.sub.1 and X.sub.2 are both Lys residues. 
Such a protease sensitive peptide can be placed between the same or 
different monomers so that upon expression in a suitable host or in vitro, 
the larger molecule can be processed to the peptides located between the 
protease sensitive peptides. 
The present invention also extends to a nucleic acid molecule comprising a 
sequence of nucleotides which encodes or is complementary to a sequence 
which encodes a protease sensitive peptide comprising the sequence: 
EQU --X.sub.1 --X.sub.2 --Asn--Asp-- 
wherein X.sub.1 and X.sub.2 are preferably the same and are most preferably 
both Lys residues. Such a nucleic acid molecule may be part of a larger 
nucleotide sequence encoding, for example, a precursor polypeptide capable 
of being processed via the protease sensitive sequence into individual 
peptides or monomers. 
The protease sensitive peptide of the present invention is particularly 
useful in generating poly and/or multi-valent "precursors" wherein each 
monomer is the same or different and directed to the same or different 
activities such as anti-viral, anti-bacterial, anti-fungal, anti-pathogen 
and/or anti-predator activity. 
Although not wishing to limit this aspect of the invention to any one 
hypothesis or proposed mechanism of action, it is believed that the 
protease acts adjacent the Asn residue as more particularly between the 
Asn-Asp residues. 
The present invention extends to an isolated type II serine PI precursor 
from a plant wherein said precursor comprises at least three PI monomers 
and wherein at least one of said monomers has a chymotrypsin specific site 
and at least one other of said monomers has a trypsin specific site. 
Preferably, the PI precursor has four, five or six monomers and is encoded 
by the nucleic acid molecule as hereinbefore described. The present 
invention also extends to the individual monomers comprising the 
precursor. The present invention also extends to a hybrid recombinant PI 
precursor molecule comprising at least two monomers from different Pls as 
hereinbefore described. 
The isolated PI or PI precursor may be in recombinant form and/or 
biologically pure. By "biologically pure" is meant a preparation of PI, PI 
precursor and/or any mixtures thereof having undergone at least one 
purification step including ammonium sulphate precipitation, Sephadex 
chromatography and/or affinity chromatography. Preferably, the preparation 
comprises at least 20% of the PI, PI precursor or mixture thereof as 
determined by weight, activity antibody, reactivity and/or amino acid 
content. Even more preferably, the preparation comprises 30-40%, 50-60% or 
at least 80-90% of PI, PI precursor or mixture thereof. 
The PI or its precursor may be naturally occurring or be a variant as 
encoded by the nucleic acid variants referred to above. It may also 
contain single or multiple substitutions, deletions and/or additions to 
its amino acid sequence or to non-proteinaceous components such as 
carbohydrate and/or lipid moieties. 
The recombinant and isolated PI, PI precursor and mixtures thereof are 
useful as laboratory reagents, in the generation of antibodies, in 
topically applied insecticides as well as orally ingested insecticides. 
The recombinant PI or PI precursor may be provided as an insecticide alone 
or in combination with one or more carriers or other insecticides such as 
the BT crystal protein. 
The PI of the present invention is considered to have a defensive role in 
organs of the plant, for example, the stigma, against the growth or 
infection by pests and pathogens such as fungi, bacteria and insects. 
There is a need, therefore, to develop genetic constructs which can be 
used to generate transgenic plants capable of expressing the PI precursor 
where this can be processed into monomers of a monomeric PI itself. 
Accordingly, another aspect of the present invention contemplates a genetic 
construct comprising a nucleic acid molecule comprising a sequence of 
nucleotides which encodes or is complementary to a sequence which encodes 
a type II serine PI precursor or monomer thereof from a plant wherein said 
precursor comprises at least three PI monomers and wherein at least one of 
said monomers has a chymotrypsin specific site and at least one of said 
other monomers has a trypsin specific site and said genetic sequence 
further comprises expression means to permit expression of said nucleic 
acid molecule, replication means to permit replication in a plant cell or, 
alternatively, integration means, to permit stable integration of said 
nucleic acid molecule into a plant cell genome. Preferably, the expression 
is regulated such as developmentally or in response to infection such as 
being regulated by an existing PI regulatory sequence. Preferably, the 
expression of the nucleic acid molecule is enhanced to thereby provide 
greater endogenous levels of PI relative to the levels in the naturally 
occurring plant. Alternatively, the PI precursor cDNA of the present 
invention is usable to obtain a promoter sequence which can then be used 
in the genetic construct or to cause its manipulation to thereby permit 
over-expression of the equivalent endogenous promoter. In another 
embodiment the PI precursor is a hybrid molecule as hereinbefore 
described. 
Yet another aspect of the present invention is directed to a transgenic 
plant carrying the genetic sequence and/or nucleic acid molecule as 
hereinbefore described and capable of producing elevated, enhanced or more 
rapidly produced levels of PI and/or PI precursor or hybrid PI precursor 
when required. Preferably, the plant is a crop plant or a tobacco plant 
but other plants are usable where the PI or PI precursor nucleic acid 
molecule is expressable in said plant. Where the transgenic plant produces 
PI precursor, the plant may or may not further process the precursor into 
monomers. Alternatively, the genetic sequence may be part of a viral or 
bacterial vector for transmission to an insect to thereby control 
pathogens in insects which would consequently interrupt the transmission 
of the pathogens to plants. 
In still yet another aspect of the present invention, there is provided 
antibodies to the PI precursor or one or more of its monomers. Antibodies 
may be monoclonal or polycional and are useful in screening for PI or PI 
precursor clones in an expression library or for purifying PI or PI 
precursor in a fermentation fluid, supernatant fluid or plant extract. 
The genetic constructs of the present invention can also be used to 
populate the gut of insects to act against the insect itself or any plant 
pathogens therein or to incorporate into the gut of animals to facilitate 
the digestion of plant material.

EXAMPLE 1 
1. MATERIALS AND METHODS 
Plant Material 
Nicotiana alata (Link et Otto) plants of self-incompatibility genotype 
S.sub.1 S.sub.3, S.sub.3 S.sub.3 and S.sub.6 S.sub.6 were maintained under 
standard glasshouse conditions as previously described (Anderson et al., 
1989). Organs were collected directly into liquid Nitrogen to avoid 
induction of a wound response and stored at -70.degree. until required. To 
study the effect of wounding on gene expression, leaves were wounded by 
crushing across the mid-vein with a dialysis clip. Leaves were collected 4 
and 24 hours after wounding. 
Identification and Sequencing of a cDNA Clone Encoding PI 
Polyadenylated RNA was prepared fron stigmas and styles, isolated from 
mature flowers of N. alata (genotype S.sub.3 S.sub.3), and used to 
construct a cDNA library in Lambda gt10 (Anderson et al., 1989). Single 
stranded .sup.32 P-labelled cDNA was prepared from mRNA from stigmas and 
styles of N. alata (genotype S.sub.3 S.sub.3 and S.sub.6 S.sub.6) and used 
to screen the library for highly expressed clones which were not 
S-genotype specific (Anderson et al., 1989). Plaques which hybridised 
strongly to cDNA probes from both S-genotypes were selected and assembled 
into groups on the basis of cross-hybridisation. The longest clone of each 
group was subcloned into M13mp18 and pGEM 3zf +, and sequenced using an 
Applied Biosystems Model 373A automated sequencer. Both dye primer and dye 
terminator cycle sequencing chemistries were performed according to 
standard Applied Biosystems protocols. Consensus sequences were generated 
using SeqEd.TM. sequence editing software (Applied Biosystems). The 
GenBank database was searched for sequences homologous to these clones. 
Because of the high degree of sequence similarity between the six domains 
of the N. alata PI clone, sequencing primers were made to non-repeated 3' 
sequences (nucleotides 1117-1137, 1188-1203 and 1247-1267), and to a 5' 
sequence before the start of the repetitive regions (nucleotides 74-98). 
In addition, the pNA-PI-2 insert was restricted with endonuclease HaeIII, 
which cut at nucleotides 622 and 970 to produce three fragments. The 
fragments were subcloned into pGEM7zf+ and sequenced in both directions, 
using the M13 forward and reverse primers. The repetitive nature of the 
pNA-PI-2 insert rendered it unstable in both phagemid and plasmid vectors 
when cultures were grown longer than 6 hours. 
RNA Gel Blot Analysis 
Total RNA was isolated and separated on a 1.2% w/v agarose/formaldehyde gel 
as previous described (Anderson et al., 1989). The RNA was transferred to 
Hybond-N (Amersham) and probed with the insert from pNA-PI-2 labelled with 
.sup.32 P using random hexanucleotides (1.times.10.sup.8 cpm .mu.g.sup.-1 
; 1.times.10.sup.7 cpm ml.sup.-1)(Feinberg and Vogelstein, 1983). 
Prehybridisation and hybridisation, at 68.degree. C., were as described by 
Anderson et al. (1989). The filters were washed in 2.times.SSC, 0.1% w/v 
SDS or 0.2.times.SSC, 1% w/v SDS at 68.degree. C. 
In Situ Hybridisation 
In situ hybridisation was performed as described by Cornish et al., 1987. 
The probe was prepared by labelling the insert from pNA-PI-2 (100 ng) to a 
specific activity of 10.sup.8 cpm .mu.g.sup.-1 by random hexanucleotide 
priming (Feinberg and Vogelstein, 1983). The labelled probe was 
precipitated, and resuspended in hybridisation buffer (50 .mu.l), and 5 
.mu.l was applied to the sections. The sections were covered with 
coverslips, and incubated overnight at 40.degree. C. in a closed box 
containing 50% v/v formamide. After incubation, sections were washed 
sequentially in 4.times.SSC at room temperature, 2.times.SSC at room 
temperature, and 1.times.SSC at 40.degree. C. for 40 min. The slides were 
dried and exposed directly to X-ray film (Cronex MRF 32, Dupont) at room 
temperature, overnight. Hybridised sections were counterstained with 
0.025% w/v toluidine blue in H.sub.2 O, and mounted in Eukitt (Carl Zeiss, 
Freilburg, FRG). Autoradiographs were transposed over sections to give the 
composites shown. 
DNA Gel Blot Analysis 
Genomic DNA was isolated from young leaves of N. alata by the procedure of 
Bernatzky and Tanksley (1986). DNA (10 .mu.g) was digested to completion 
with the restriction endonucleases EcoRI or HindIII, separated by 
electrophoresis on a 0.9% w/v agarose gel, and transferred to Hybond-N 
(Amersham) by wet blotting in 20.times.SSC. Filters were probed and washed 
as described for RNA blot analysis. 
Preparation of Protein Extracts 
Soluble proteins were extracted from plant material by freezing the tissue 
in liquid N.sub.2, and grinding to a fine powder in a mortar and pestle. 
The powdered tissue was extracted in a buffer consisting of 100 mM 
Tris-HCl, pH 8.5, 10 mM EDTA, 2 mM CaCl.sub.2, 14 .mu.M 
.beta.-mercaptoethanol. Insoluble material was removed by centrifugation 
at 10,000 g for 15 min. Protein concentrations were estimated by the 
method of Bradford (1976) with Bovine Serum Albumin (BSA) as a standard. 
Proteinase Inhibition Assays 
Protein extracts and purified protein were assayed for inhibitory activity 
against trypsin and chyrnotrypsin as described by Rickauer et al. (1989). 
Inhibitory activity was measured against 1 pg of trypsin (TPCK-treated; 
Sigma) or 3 .mu.g of chymotrypsin (TLCK-treated; Sigma). The rate of 
hydrolysis of synthetic substrates N-.alpha.-P-tosyl-L-arginine methyl 
ester (TAME) and N-benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin and 
chymotrypsin, respectively, were taken as the uninhibited activity of the 
enzymes. Inhibitory activity of the extract was expressed as the 
percentage of control protease activity remaining after the protease had 
been pre-incubated with the extract. The PI peptides from stigma, PI 
precursor and Asp-N processed peptides were assayed for inhibitory 
activity as described by Christeller et al. (1989). 
Purification of the N. alata PI Protein 
Stigmas (1000; 10 g) were ground to a fine powder in liquid N.sub.2, and 
extracted in buffer (100 mM Tris-HCl, pH8.5, 10 mM EDTA, 2 mM CaCl.sub.2, 
14 .mu.M .beta.-mercaptoethanol, 4 ml/g tissue). To concentrate the 
extract prior to the first purification step, gel filtration, the 
inhibitory activity was precipitated with 80% w/v ammonium sulphate, the 
concentration required to precipitate all the proteinase inhibitory 
activity. 
The ammonium sulphate pellet was resuspended in 5 ml of 0.15 M KCl , 10 mM 
Tris-HCl, pH 8.1, and loaded onto a Sephadex G-50 column (2 cm.times.100 
cm) equilibrated with the same buffer. The fractions (10 ml) eluted from 
this column and containing proteinase inhibitory activity were pooled and 
applied to an affinity column of Chymotrypsin-Sepharose CL4B [100 mg 
TLCK-treated .alpha.-chymotrypsin (Sigma) cross-linked to 15 ml Sepharose 
CL4B (Pharmacia) by manufacturers instructions]. The column was washed 
with 10 volumes of 0.15M KCl/10 mM Tris-HCl, pH 8.1, prior to elution of 
bound proteins with 7 m urea, pH 3 (5 ml fractions). The eluate was 
neutralised immediately with 200 .mu.l 1M Tris-HCl pH 8, and dialyzed 
extensively against deionised H.sub.2 O. 
Amino Acid Sequence Analysis 
Purified PI protein was chromatographed on a reverse phase HPLC microbore 
column prior to automated Edman degradation on a gas phase sequencer (Mau 
et al., 1986). Phenylthiohydantoin (PTH) amino acids were analysed by HPLC 
as described by Grego et al. (1985). 
Production of a Polyclonal Antiserum to the N. alata PI 
The purified proteinase inhibitor (FIG. 6c, lane 3) was conjugated to a 
carrier protein, keyhole limpet haemocyanin (KLH) (Sigma), using 
glutaraldehyde, as follows. 1 mg of PI protein was dissolved in 1.5 ml 
H.sub.2 O, and mixed with 0.3 mg KLH in 0.5 ml of 0.4M phosphate buffer, 
pH7.5. 1 ml of 20 mM glutaraldehyde was added dropwise over 5 min, with 
stirring at room temperature. The mixture was stirred for 30 min at room 
temperature, 0.25 ml of glycine was added, and the mixture was stirred for 
a further 30 min. The conjugated protein was then dialyzed extensively 
against normal saline (0.8% w/v NaCl). The equivalent of 100 .mu.g of PI 
protein was used for each injection. Freund's complete adjuvant was used 
for the first injection, and incomplete adjuvant for two subsequent 
booster injections. The IgG fraction of the antiserum was separated on 
Protein A Sepharose (Pharmacia) according to manufacturer's instructions. 
Protein Gel Blot Analysis 
Protein extracts were electrophoresed in 15% w/v SDS-polyacrylamide gels 
(Laemmli, 1970) and transferred to nitrocellulose in 25 mM Tris-HCl, 192 
mM glycine, 20% v/v methanol, using a BioRad Trans-Blot.RTM.Semi-dry 
electrophoretic transfer cell (12 V, 20 min). Loading and protein transfer 
were checked by staining the proteins on the membranes with Ponceau S 
(Harlow and Lane, 1988). Membranes were blocked in 3% w/v bovine serum 
albumin for 1 h, and incubated with the anti-PI antibody (2 .mu.g/ml in 1% 
w/v BSA, Tris Buffered Saline) overnight at room temperature. Bound 
antibodywas detected using biotinylated donkey anti-rabbit IgG (1/500 
dilution, Amersham) and the Amersham Biotin-Streptavidin system according 
to procedures recommended by the manufacturer. 
Proteolysis of the PI Precursor by Endoproteinase Asp-N 
Affinity-purified PI precursor (1.25 mg) was incubated at 37.degree. C. 
with endoproteinase Asp-N (2 .mu.g) in 100 mM NH.sub.4 HCO.sub.3, pH 8.5 
in a total volume of 1 ml for 48 h. Reaction products were separated by 
reversed-phase HPLC using an analytical Brownlee RP-300 Aquapore column 
(C8, 7 .mu.m, 4.6.times.100 mm). The column was equilibrated in 0.1% v/v 
TFA and peptides were eluted with the following program: 0-25%B (60% v/v 
acetonitrile in 0.089% v/v TFA) applied over 5 min, followed by a gradient 
of 25-42%B over the next 40 min, and ending with a gradient of 42-100%B 
over 5 minutes. The flow rate was 1.0 ml/min and peptides were detected by 
absorbance at 215 nm. Each peak was collected manually and freeze dried. 
Concentration was estimated by response obtained with each peak on the UV 
detector at 215 nm. 
2. CLONING OF PI PRECURSOR GENE 
Isolation and Characterisation of the PI cDNA Clone 
A cDNA library, prepared from mRNA isolated from the stigmas and styles of 
mature flowers of N. alata was screened for clones of highly expressed 
genes which were not associated with self-incompatibility genotype. Clones 
encoding a protein with some sequence identity to the type II proteinase 
inhibitors from potato and tomato (Thornburg et al., 1987; Graham et al., 
1985) were selected. The largest clone, NA-PI-2, is 1360 base pairs long 
with an open reading frame of 1191 nucleotides. The nucleic acid sequence 
(SEQ ID NO. 2) and the predicted amino acid sequence (SEQ ID NO. 3) of the 
N. alata clone, NA-PI-2 is shown in FIG. 1. There are no potential 
N-glycosylation sites. 
Surprisingly, the N. alata cDNA clone encodes a protein with six repeated 
domains that have high, but not perfect, sequence identity (FIG. 1). Each 
of these domains contains a potential reactive site which is highlighted 
in FIG. 1. The residues at the putative reactives sites of the N. alata PI 
are consistent with the inhibitor having two sites which would 
specifically inhibit chymotrypsin (Leu5-Asn6, Leu63-Asn64) and four sites 
specific for trypsin (Arg121-Asn122, Arg179-Asn180, Arg237-Asn238 and 
Arg295-Asn296). 
To ensure that the repeat structure of NA-PI-2 was not due to a cloning 
artifact, three additional cDNA clones were sequenced, and found to be 
identical to NA-PI-2. 
Table 1 is a comparison of the percentage amino acid identity of the six 
domains of the PI precursor. 
Temporal and Spatial Expression of the PI mRNA 
Total RNA, isolated from various tissues of N. alata, was probed with the 
PI cDNA clone in the RNA gel blot analyses shown in FIG. 2. Two 
hybridising messages of 1.0 and 1.4 kb were present in total RNA isolated 
from styles (including stigmas). Only the larger message, which was 
predominant in this tissue, is of sufficient size to encode the cDNA clone 
NA-PI-2 (1.4 kb). The smaller message is not detected with the cDNA probe 
at higher stringency. An homologous message of approximately 1.4 kb was 
also present in RNA isolated from the styles of N. tabacum and N. 
sylvestris (FIG. 2). 
In the other floral organs (except pollen), both messages were detectable 
at low levels, however, the smaller RNA species appeared more abundant. 
There was no hybridisation to pollen RNA. No hybridising species were 
evident in leaf RNA, but two species, 1.0 and 1.4 kb were detected 24 
hours after mechanical wounding. The smaller message (1.0 kb) was more 
abundant in this case. 
In situ hybridisation of radiolabelled N. alata PI cDNA to longitudinal 
sections of styles from immature (1 cm long) buds is shown in FIG. 3. RNA 
homologous to the cDNA clone bound strongly to cells of the stigma and 
weakly to vascular bundles. No hybridisation was detected in the cortical 
tissue, transmitting tract tissue, or epidermis of the style. The same 
pattern of hybridisation was observed in mature receptive flowers. Control 
sections treated with ribonuclease A prior to hybridisation were not 
labelled. 
Genomic DNA Blot Analysis 
The cDNA clone NA-PI-2, was used as a probe on the DNA gel blot shown in 
FIG. 4 which contained genomic DNA, digested with either EcoRI or HindIII. 
EcoRI produced two hybridising fragments (11 kb and 7.8 kb) and HindIII 
produced three large hybridising fragments (16.6, 13.5 and 10.5 kb). 
Distribution of PI Activity in Various Tissues of N. alata 
The inhibition of trypsin and chymotrypsin by crude extracts of various 
organs of N. alata is shown in FIG. 5. Stigma extract was the most 
effective inhibitor of both trypsin and chymotrypsin. The stigma extracts 
had up to eight times more inhibitory activity than sepal extracts, and 
more than 20 times more activity than extracts from styles, petals, leaves 
and wounded leaves. 
Purification of PI from N. alata Stigmas 
Stigmas of N. alata were extracted in buffer and the inhibitory activity 
was concentrated by precipitation with 80% w/v ammonium sulphate. The 
precipitate was redissolved and fractionated by gel filtration on Sephadex 
G-50. Most of the protein in the extract eluted early in the profile 
illustrated in FIGS. 6a and 6b, relative to the proteinase inhibitor. 
Fractions with proteinase inhibitor activity were pooled and applied to an 
affinity column of chymotrypsin-Sepharose. The PI activity co-eluted with 
a protein of about 6 kD, which appeared to migrate as a single band on the 
20% SDS-polyacrylamide gel shown in FIG. 6c. The purity of the PI at 
various stages of purification was assessed by SDS-PAGE (FIG. 6c). The 
purified inhibitor represented approximately 50% of the inhibitory 
activity present in the crude extract. 
Amino Acid Sequence of the N-terminus of the 6 kD PI Protein 
The N-terminal amino acid sequence DRICTNCCAG(T/K)KG (SEQ ID NO. 11; SEQ ID 
NO. 12, respectively) was obtained from the purified PI protein. This 
sequence of amino acids corresponds to six regions in the deduced sequence 
of the cDNA clone, starting at positions 25, 83, 141, 199, 257 and 315 in 
FIG. 1. At position 11 of the N-terminal sequence, both threonine and 
lysine were detected. 
This is consistent with the purified inhibitor comprising a mixture of six 
peptides beginning with the sequences underlined in FIG. 1, as the first 
two peptides contain threonine at this position, while the other four 
peptides have lysine at this position. The position of these peptides 
relative to the six repeated domains in the predicted precursor protein is 
illustrated in FIG. 7. Five of the six predicted 6 kD peptides, contain a 
reactive site for either chymotrypsin or trypsin (FIG. 1 and 7). The sixth 
potential peptide is four amino-acids shorter than the other five peptides 
(fifty eight amino-acids) and may not be active, as it does not contain an 
inhibitory site. The peptide from the N-terminus (x in FIG. 7) has a 
potential chymotpsin reactive site but is much shorter (24 amino acids). 
Distribution of the PI Protein in N. alata 
A polyclonal antiserum was raised to the purified PI protein conjugated to 
keyhole limpet haemocyanin. The antibody reacted strongly with the 
purified 6 kD PI protein in immunoblot analyses and bound only to a 6 kD 
and a 32 kD protein, which appears as a doublet, in total stigma and style 
extracts from mature flowers. FIG. 8 is an immunoblot containing protein 
extracts of stigmas from flowers at different stages of development (1 cm 
long buds to mature flowers) probed with the anti-PI antiserum. Larger 
cross reacting proteins of approximately 18 kD, and 42 kD were detected in 
buds from 1 cm to 5 cm in length in addition to the 6 kD and the 32 kD 
protein. The 18 kD and 42 kD proteins decreased in concentration with 
maturity, while the 6 kD protein reached a peak concentration just before 
anthesis. The concentration of the 32 kD protein remained relatively 
constant during flower maturation. 
TABLE 1 
______________________________________ 
N. alata PI 
1 2 3 4 5 6 
______________________________________ 
N. alata 
1 100 88 88 90 79 
2 88 88 90 79 
3 97 95 86 
4 98 90 
5 90 
6 
______________________________________ 
EXAMPLE 2 
PURIFICATION AND IDENTIFICATION OF PI MONOMERS 
1. MATERIALS AND METHODS 
Separation of the 6 kD PI Species by Reversed Phase Chromatography 
Stigmas (21,000) were ground and extracted as described for purification of 
the PI protein. After gel filtration on a Sephadex G-50 gel filtration 
column (5 cm.times.800 cm, 3000 stigmas per separation) the peptides were 
lyophilized and applied to a Brownlee RP-300 C8 Reversed-phase column, 
10.times.250 mm, on a Beckman HPLC system Gold, and eluted with 0.1% v/v 
Trifluoroacetic acid (TFA) and an acetonitrile gradient (0-10% over 5 
mins, 10-25% over 40 mins and 25-60% over 10 mins), at 5 ml/min. Peak 
fractions, designated fraction 1, 2, 3 and 4 were collected and freeze 
dried. 
Electrospray Mass Spectrometry 
On line mass spectrometric analysis of HPLC eluates was performed by 
application of 20 pmoles of each PI preparation (fraction 1, 2, 3 & 4) in 
2 .mu.l of water onto a Brownlee RP-300 C8 reversed-phase column 
(150.times.0.20 mm internal diameter fused-silica capillary column) on a 
modified Hewlett-Packard model HP1090L liquid chromatograph and elution 
with a linear gradient of acetonitrile (0.05% v/v TFA to 0.045% v/v 
TFA/60% v/v acetonitrile in 30 min.) at a flow rate of 1 .mu.l/min and a 
column temperature of 25.degree. C. The eluant was monitored at 215 nm 
using a Spectral Physics forward optics scanning detector with a 6-mm 
pathlength U-shaped axial beam capillary flow cell (LC Packings, 
Netherlands). Mass spectra were acquired on a Finnigan-Mat triple 
quadrupole mass spectrometer (modelTSQ-700, San Jose, Calif.) equipped 
with an electrospray ionisation (ESI) source (Analytica, Branford, Conn.). 
The electrospray needle was operated in positive ion mode at a voltage 
differential of -4 kV. The sheath liquid was 2-methoxyethanol delivered at 
1 .mu.l/min via a syringe drive (Harvard Apparatus, South Natick, Mass.). 
The nitrogen drying gas conditions were as follows: heater temperature, 
275.degree. C.; pressure, 15 psi; flow rate, .about.15 stdL/min. The 
nitrogen sheath gas was supplied at 33 psi. Gaseous nitrogen was obtained 
from a boiling liquid nitrogen source. Peptides were introduced into the 
ESI source at 1.0 .mu.l/min by on-line capillary RP-HPLC as described 
above. Spectra were acquired scanning from m/z 400 to 2000 at a rate of 3 
sec. Data collection and reduction were performed on a Dec5100 computer 
using Finnigan BIOMASS.TM. software. 
2. RESULTS 
Separation and Identification of the Individual 6 kD PI Species from 
N.alata Stigmas 
The five of the six peptides of about 6 kD that were predicted to be 
present in the purified 6 kD PI preparation have been separated from each 
other by reversed-phase HPLC chromatography. Four peaks were obtained 
(FIG. 9a) and the peptides within each peak were identified by 
electrospray mass spectrometry (Table 2). The peptides have been 
designated C1, T1, T2, T3 and T4 according to their position in the PI 
precursor and the presence of a chymotrypsin or trypsin reactive site 
(FIG. 9b). The first HPLC peak (FIG. 9a) corresponds to the chymotrypsin 
inhibitor C1, the second peak is composed of a mixture of T2 and T3 
(identical to each other) and T4 that differs from T2 and T3 by one 
amino-acid at position 32. The third peak contains the peptide T1 and the 
fourth peak is composed of a nixture of T1, T2/T3 and T4 (Table 2). 
The site of processing has not been precisely determined, but is likely to 
be located between the aspartate (N) and asparagine (D) residues in the 
sequence outlined in FIG. 10. Proteases with specific requirements for 
asparagine residues have been isolated from vacuoles from immature soybean 
seeds and pumpkin cotyledons (Scott et al., 1992, Hara-Nishimura et al., 
1991). This is consistent with the immunogold localization of the PI in 
the vacuoles of the papillae and the underlying secretory cells in the 
stigma of N.alata (Atkinson, 1992). In the case of the N.alata PI, 
processing analogous to that of peptide hormones is also possible because 
each of the possible 6 kD peptides are flanked by dibasic residues 
(Lys-Lys, position-2 & -3 in FIG. 10). However, a system like this has not 
been described in plants, and it is more likely that the dibasic residues 
contribute to the predicted hydrophilic loops that present the processing 
site on the surface of the molecule. 
The data from the mass spectrometric analysis shows that once the initial 
cleavage has occurred the new carboxy terminus is trimmed back (FIG. 10). 
The EEKKN sequence (SEQ ID NO. 14) is removed completely but the trimming 
is not precise, sometimes an additional amino acid is removed. Steric 
hindrance probably prevents further trimming. Occasionally the aspartate 
is also removed from the N-terminus. 
EXAMPLE 3 
Production of PI Precursor in Insect Cell (Sf9) Culture Using a Recombinant 
Baculovirus Vector. 
cDNA encoding the PI precursor (FIG. 1) was inserted into the Eco R1 site 
of the plasmid vector pVL 1392, which is the sane as pVL941 (Lucknow and 
Summers, 1989) except that a multiple cloning site was inserted at the 
BamH1 site. The plasmid designated pRH11, contains the PI cDNA in the 
correct orientation with respect to the direction of transcription 
directed by the polyhedrin promoter. Recombinant baculovirus was obtained 
by co-transfection of Spodoptera frugiperda cells with baculovirus DNA and 
pRH11. The recombinant viruses, produced by homologous recombination, were 
plaque purified and amplified prior to infection of insect cells for 
protein production. All procedures for production of recombinant 
baculovirus, titration of the virus and maintenance and infection of the 
Sf9 cells were obtained from King and Posse (1992). For production of the 
PI precursor, monolayers of Sf9 cells in large flasks (175 cm.sup.2) were 
infected at the time of confluence with an inoculum of high-titre 
recombinant virus at a multiplicity of infection of 5-10 pfu/cell. Culture 
fluid was collected after 4 days of infection, clarified by centrifugation 
and the PI precursor was purified by application to a 
Chymotrypsin-Sepharose affinity column as described for the 6 kD PI 
species from stigmas. PI precursor eluted from the column in 7M urea, pH3 
was neutralized immediately with 1M Tris-HCl buffer pH8, dialysed 
extensively against Milli-Q water, concentrated 20-50 fold by 
ultrafiltration using a Diaflow YM10 filter and stored frozen at 
-20.degree. C. 
The cDNA clone encoding the PI precursor was engineered into a baculovuirus 
vector for the production of the precursor from infected insect cells. The 
insect cells produced a 42 kD protein that cross reacted with the 
antibodies raised to the 6 kD PI peptides from stigma and bound to the 
chymotrypsin affinity column. This 42 kD protein was identical in size to 
the 42 kD precursor produced in the immature stigmas of N.alata (FIG. 11) 
and had the N-terminal sequence LysAlaCysThrLeuAsn (SEQ ID NO. 13) 
demonstrating that the signal sequence had been processed correctly by the 
insect cells (FIG. 1). Based on these results, the 42 kD protein produced 
in the baculovirus expression system will now be referred to as the PI 
precursor. The 42 kD PI precursor had inhibitory activity against 
chymotrypsin but no inhibitory activity against trypsin (FIG. 13). 
Processing of the PI precursor by the endoproteinase AspN led to the 
production of stable peptides of about 6 kD that were partially purified 
by reversed phase HPLC (FIG. 12). These peptides have equivalent 
inhibitory activity against trypsin and chymotrypsin as the 6 kD peptides 
isolated from stigma, indicating that processing of the precursor is 
required to activate the trypsin inhibitory activity but not all the 
chymotrypsin activity. Since AspN cleaves specifically adjacent to 
Aspartate residues (between Asn-1 and Asp1 in FIG. 10) and has no trimming 
activity, the peptides produced in vitrowill be similiar to those produced 
in stigmas except for the presence of the sequence EEKKN (SEQ ID NO. 14) 
at the C-terminus. This provides further evidence that precise processing 
of the N-and C-termini is not required to obtain an active 6 kD PI 
peptide. Asp-N1 is more efficient at inhibiting chymotrypsin than trypsin 
and is thus likely to be predominantly a C1 analogue (FIG. 9b). Asp-N2 is 
a more efficient trypsin inhibitor and probably contains the T1-T4 
analogues. 
EXAMPLE 4 
Effect of PIs on Protease Activity in Unfractionated Gut Extacts from 
Various Insects 
Activity of PIs on gut proteases was measured using the procedure of 
Christeller et al., (1992) as follows. An aliquot of 1 uM of inhibitor 
(0-10 .mu.l, at least 5-fold excess over proteases present in the gut) was 
mixed with 150 .mu.l of 10 mM CAPS buffer, pH 10, and preincubated with 
each insect gut extract (0-15 .mu.l ), for 20 min at 30.degree. C. The 
reaction was started by the addition of 50 .mu.l of .sup.14 C -labelled 
casein substrate (400 .mu.g protein, specific activity 25,000-75,000 dpm 
mg.sup.-1) and continued for 30 min at 30.degree. C. until 50 .mu.l of 
cold 30% (w/v) TCA was added to terminate the reaction. After incubation 
on ice for 30 min, undigested protein was pelleted by centrifugation at 
20.degree. C. for 5 min at 10,000 g. The supernatant was removed, mixed 
with scintillation fluid and the radioactivity measured. Assays were 
performed at pH 10 except for L.sericata and C.rufifacies when 10 mM 
Tris-HCl , pH 8.0 was used. 
Table 3 shows the inhibitory activity of the pooled 6 kD PI peptides (C1, 
T1, T2/T3, T4), the mixture of trypsin inhibitors T2/T3 and T4, and the 
chymotrypsin inhibitor C1 against the proteases in the gut of various 
members of the Lepidoptera, Coleoptera, Orthoptera and Diptera. In most 
cases, the pooled peptides and the trypsin inhibitors had an equivalent 
effect against the gut proteases with the degree of inhibition ranging 
from 37-79% depending on the insect tested. The inhibitors had negligible 
effect on the gut proteases of the potato tuber moth, P.opercullela. The 
chymotrypsin inhibitor C1 also affected the activity of the proteases but 
was less effective than the trypsin inhibitors in five cases (W.cervinata, 
L.serricata, C.zealandica, P.octo, sugar cane grub). 
The experimental details are described in the legend to FIG. 14. The N. 
alata PI was more effective than Soybean Bowman-Birk inhibitor in reducing 
cricket weight. It has shown that there is a good correlation between the 
ability of a proteinase inhibitor to inhibit the enzymes of the insect 
midgut and its effectiveness in retarding the growth of insects in insect 
feeding trials (Christeller et al., 1992). FIG. 14 shows that the pooled 
PIs that inhibited the gut proteases of the black field cricket 
(T.commodus) by 70% in the in vitro assay retarded the growth of the 
crickets by 30% in a feeding trial conducted over a 10 week period. The 
correlation between in vitro assays and feeding trials has been confirmed 
recently by Johnston and collegues (1993) working on growth and 
development of Helicoverpa armigera. 
TABLE 2 
______________________________________ 
HPLC retention time 
molecular 
peak (min) weight assigned peptide* 
______________________________________ 
1 15.5 5731.5 C1 
5644.4 C1 minus Ser.sub.53 
5616.4 C1 minus Asp.sub.1 & 
Ser.sub.53 
55.29.3 C1 minus Asp.sub.1 
2 20.5 5700.5 T2/T3 
5728.5 T4 
5585.4 T2/T3 minus Asp.sub.1 
5613.5 T4 minus Asp.sub.1 
3 22.5 5725.5 T1 
5610.5 T1 minus Asp.sub.1 
4 24 5654.4 T1 minus Ala.sub.53 
5641.4 T4 minus Ser.sub.53 
5613.4 T2/T3 minus Ser.sub.53 
5539.4 T1 minus Asp.sub.1 & 
Ala.sub.53 
5498.4 T2/T3 minus Asp.sub.1 & 
Ser.sub.53 
5526.4 T4 minus Asp.sub.1 & 
Ser.sub.53 
______________________________________ 
*See FIG. 9 for designation of C1 and T1-T4. 
TABLE 3 
______________________________________ 
Effect of Nicotiana alata proteinase inhibitors and Potato 
inhibitor II on casein hydrolysis by crude gut extracts 
casein hydrolysis (% control) 
T2/T3, 
Insect NaPI C1 T4 
______________________________________ 
H. armigera 
33.2 32.7 30.3 
H. punctigera 26.6 29.3 28.5 
T. commodus 28.4 35.0 33.1 
A. infusa 37.5 40.2 43.3 
sugar cane 25.8 43.9 25.1 
grub 
W. cervinata 22.9 82.9 20.4 
E. postvitiana 39.7 45.4 41.2 
S. litura 28.1 33.6 24.8 
P. opercullela 95.8 100 98.5 
C. rufifacies 29.1 37.8 28.9 
L. serricata 59.2 100 63.0 
C. zealandica 31.7 54.7 32.0 
P. octo 57.1 67.2 57.4 
C. obliquana 51.1 49.1 45.5 
A. tsmaniae 28.3 34.2 39.5 
______________________________________ 
Legend to Table 3 
NaPI = N. alata proteinase inhibitors pooled 
C1 = N. alata chymotrypsin inhibitor (peak 1 from HPLC) 
T2/T3, T4 = N. alata trypsin inhibitors (peak 2 from HPLC) 
Heliothis armigera, Helicoverpa armigera, Tobacco budworm, Lepidoptera 
Heliothis punctigera, Helicoverpa punctigera Native budworm, Lepidoptera 
Teleogryllus commodus Black field cricket, Orthoptera 
Agrotis infusa Common cutworm, adults known as the Bogong moth, 
Lepidoptera 
Wiseana cervinata Porina, native to New Zealand, Lepidoptera 
Lucilla sericata Green blow fly, Diptera, assayed at pH 8 
Chrysomya rufifacies Hairy maggot blow fly, Diptera, assayed at pH 8 
Aphodius tasmaniae Tasmanian grass grub = Blackheaded pasture cockchafer, 
Coleoptera 
Costelytra zealandica New Zealand grass grub, Coleoptera 
Spodoptera litura Tropical armyworm, Lepidoptera 
Phthorimaea opercullela Potato tuber moth, Lepidoptera 
Epiphyas postvittana Lightbrown apple moth (leafroller), Lepidoptera 
Planototrix octo Greenheaded leafroller, Lepidoptera 
Ctenopseustis obliquana Brownheaded leafroller, Lepidoptera 
Sugar cane grub 
Those skilled in the art will appreciate that the invention described 
herein is susceptible to variations and modifications other than those 
specifically described. It is to be understood that the invention includes 
all such variations and modifications. The invention also includes all of 
the steps, features, compositions and compounds referred to or indicated 
in this specification, individually or collectively, and any and all 
combinations of any two or more of said steps or features. 
REFERENCES 
Anderson, M. A., McFadden, G. I., Bernatzky, R., Atkinson, A., Orpin, T., 
Dedman, H., Tregear, G., Fernley, R., Clarke, A. E. (1989) The Plant Cell 
1:483-491. 
Atkinson, A. H. (1992) PhD thesis, University of Melbourne, Victoria, 
Australia. 
Bernatzky, R., Tanksley, S. D. (1986) Theor. Appl. Genet. 72:314-321. 
Bradford, M. M. (1976) Anal. Biochem. 72:248-254 
Brown, W. E., Ryan, C. A. (1984) Biochemistry 23:3418-3422. 
Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. A. (1976) Biochemistry 
15:3418-3424. 
Cornish, E. C., Pettitt, J. M., Bonig, I., Clarke, A. E. (1987) Nature 326: 
99-102. 
Christeller, J. T., Shaw, B. D., Gardiner, S. E., Dymock, J. (1989) Insect 
Biochem. 19: 2217-231. 
Christeller, J. T., Laing, W. A., Markwick, N. P. and Burgess, E. P. J. 
(1992) Insect Biochem. Molec. Biol. 22:735-746 
Feinberg, A. P., Vogelstein, B. (1983) Anal. Biochem. 132: 6-13. 
Graham, J. S., Pearce, G., Merryweather, J., Titani, K., Ericsson, L. H., 
Ryan, C. A. (1985) J. Biol. Chem. 260: 6561-6564. 
Graham, J. S., Hall, G., Pearce, G., Ryan, C. A. (1986) Planta 169:399-405. 
Grego, B., van Driel, I. R., Stearne, P. A., Goding, J. W., Nice, E. G., 
Simpson, R. J. (1985) Eur. J. Biochem. 148:485-491. 
Green, T. R., Ryan, C. A. (1972) Science: 776-777. 
Hara-Nishimura, I., Inoue, K., Nishimura, M. (1991) FEBS Letters 294 89-93. 
Harlow, E., Lane, D. (1988) Antibodies. A Laboratory Manual. Cold Spring 
Harbour Laboratory, New York. 
Hass, G. M., Hermodson, M. A., Ryan, C. A., Gentry, L. (1982) Biochemistry 
21:752-756. 
Johnston, K. A., Gatehouse, J. A., Anstee, J. H. (1993) J. Insect Physiol. 
39, 657-664. 
King, L. A. Possee, R. D. (1992). The Baculovirus Expression system. A 
Laboratory guide. (Chapman & Hall: London, UK). 
Kuo, J., Pearce, G. Ryan C. A. (1984) Isolation and characterization of 
proteinase inhibitor I from etiolated tobacco leaves. Arch. Biochem. 
Biophys. 230: 504-510. 
Kyte, J., Doolittle, R. F. (1982) J. Mol. Biol. 157: 680-685. 
Laemmli, U. K. (1970) Nature 227:680-685. 
Lucknow, V. A. and Summers, M. D. (1989). Virology. 170:31-39. 
Mau, S-L., Williams, E. G., Atkinson, A., Anderson, M. A., Cornish, E. C., 
Grego, B., Simpson, R. J., Kheyr-Pour, A., Clarke, A. E. (1986) Planta 
169:184-191. 
Melville, J. C., Ryan, C. A. (1970) Archives of Biochemistry and Biophysics 
138: 700-702. 
Pearce, G., Ryan, C. A., Liljegren, D. (1988) Planta 175:527-531. 
Plunkett, G., Senear, D. F., Zuroske, G., Ryan, C. A. (1982) Arch. Biochem. 
Biophys. 213: 463-472. 
Richardson, M. (1977) Phytochemistry 16:159-169. 
Rickauer, M., Fournier, J., Esquerre-Tugaye, M. (1989) Plant Physiol. 
9:1065-1070. 
Ryan, C. A. (1984). Defense responses in plants. In: Plant Gene Research, 
Dennes, E. S., Hohn, B., Hohn, T., King, P., Schell, J., Verma, D. P. S., 
Eds. New York, Springer-Verlag, 375-386. 
Sanchez-Serrano, J. J., Schmidt, R., Schell, J., Willmitzer, L., (1986) 
Mol. Gen. Genet. 203:15-20. 
Scott, M. P., Jung, R., Muntz, K., Nielsen, N. C. (1992) Proc. Natl. Acad. 
Sci. USA 89 658-662. 
Thornburg, R. W. An, G., Cleveland, T. E., Johnson, R., Ryan, C. A. (1987). 
Proc. Nat. Acad. Sci. USA 84:744-748. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 14 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1104 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - AAGGCTTGTA CCTTAAACTG TGATCCAAGA ATTGCCTATG GAGTTTGCCC GC - 
#GTTCAGAA 60 
- - GAAAAGAAGA ATGATCGGAT ATGCACCAAC TGTTGCGCAG GCACGAAGGG TT - 
#GTAAGTAC 120 
- - TTCAGTGATG ATGGAACTTT TGTTTGTGAA GGAGAGTCTG ATCCTAGAAA TC - 
#CAAAGGCT 180 
- - TGTACCTTAA ACTGTGATCC AAGAATTGCC TATGGAGTTT GCCCGCGTTC AG - 
#AAGAAAAG 240 
- - AAGAATGATC GGATATGCAC CAACTGTTGC GCAGGCACGA AGGGTTGTAA GT - 
#ACTTCAGT 300 
- - GATGATGGAA CTTTTGTTTG TGAAGGAGAG TCTGATCCTA GAAATCCAAA GG - 
#CTTGTCCT 360 
- - CGGAATTGCG ATCCAAGAAT TGCCTATGGG ATTTGCCCAC TTGCAGAAGA AA - 
#AGAAGAAT 420 
- - GATCGGATAT GCACCAACTG TTGCGCAGGC AAAAAGGGTT GTAAGTACTT TA - 
#GTGATGAT 480 
- - GGAACTTTTG TTTGTGAAGG AGAGTCTGAT CCTAAAAATC CAAAGGCCTG TC - 
#CTCGGAAT 540 
- - TGTGATGGAA GAATTGCCTA TGGGATTTGC CCACTTTCAG AAGAAAAGAA GA - 
#ATGATCGG 600 
- - ATATGCACCA ACTGCTGCGC AGGCAAAAAG GGTTGTAAGT ACTTTAGTGA TG - 
#ATGGAACT 660 
- - TTTGTTTGTG AAGGAGAGTC TGATCCTAAA AATCCAAAGG CTTGTCCTCG GA - 
#ATTGTGAT 720 
- - GGAAGAATTG CCTATGGGAT TTGCCCACTT TCAGAAGAAA AGAAGAATGA TC - 
#GGATATGC 780 
- - ACAAACTGTT GCGCAGGCAA AAAGGGCTGT AAGTACTTTA GTGATGATGG AA - 
#CTTTTGTT 840 
- - TGTGAAGGAG AGTCTGATCC TAGAAATCCA AAGGCCTGTC CTCGGAATTG TG - 
#ATGGAAGA 900 
- - ATTGCCTATG GAATTTGCCC ACTTTCAGAA GAAAAGAAGA ATGATCGGAT AT - 
#GCACCAAT 960 
- - TGTTGCGCAG GCAAGAAGGG CTGTAAGTAC TTTAGTGATG ATGGAACTTT TA - 
#TTTGTGAA 1020 
- - GGAGAATCTG AATATGCCAG CAAAGTGGAT GAATATGTTG GTGAAGTGGA GA - 
#ATGATCTC 1080 
- - CAGAAGTCTA AGGTTGCTGT TTCC - # - # 
1104 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1360 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 97..1200 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - CGAGTAAGTA TGGCTGTTCA CAGAGTTAGT TTCCTTGCTC TCCTCCTCTT AT - 
#TTGGAATG 60 
- - TCTCTGCTTG TAAGCAATGT GGAACATGCA GATGCC AAG GCT TGT - #ACC TTA AAC 
114 
- # - # Lys Ala Cys Thr Leu Asn 
- # - # 1 - # 5 
- - TGT GAT CCA AGA ATT GCC TAT GGA GTT TGC CC - #G CGT TCA GAA GAA AAG 
162 
Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pr - #o Arg Ser Glu Glu Lys 
10 - # 15 - # 20 
- - AAG AAT GAT CGG ATA TGC ACC AAC TGT TGC GC - #A GGC ACG AAG GGT TGT 
210 
Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Al - #a Gly Thr Lys Gly Cys 
25 - # 30 - # 35 
- - AAG TAC TTC AGT GAT GAT GGA ACT TTT GTT TG - #T GAA GGA GAG TCT GAT 
258 
Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cy - #s Glu Gly Glu Ser Asp 
40 - # 45 - # 50 
- - CCT AGA AAT CCA AAG GCT TGT ACC TTA AAC TG - #T GAT CCA AGA ATT GCC 
306 
Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cy - #s Asp Pro Arg Ile Ala 
55 - # 60 - # 65 - # 70 
- - TAT GGA GTT TGC CCG CGT TCA GAA GAA AAG AA - #G AAT GAT CGG ATA TGC 
354 
Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Ly - #s Asn Asp Arg Ile Cys 
75 - # 80 - # 85 
- - ACC AAC TGT TGC GCA GGC ACG AAG GGT TGT AA - #G TAC TTC AGT GAT GAT 
402 
Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Ly - #s Tyr Phe Ser Asp Asp 
90 - # 95 - # 100 
- - GGA ACT TTT GTT TGT GAA GGA GAG TCT GAT CC - #T AGA AAT CCA AAG GCT 
450 
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pr - #o Arg Asn Pro Lys Ala 
105 - # 110 - # 115 
- - TGT CCT CGG AAT TGC GAT CCA AGA ATT GCC TA - #T GGG ATT TGC CCA CTT 
498 
Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Ty - #r Gly Ile Cys Pro Leu 
120 - # 125 - # 130 
- - GCA GAA GAA AAG AAG AAT GAT CGG ATA TGC AC - #C AAC TGT TGC GCA GGC 
546 
Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Th - #r Asn Cys Cys Ala Gly 
135 1 - #40 1 - #45 1 - 
#50 
- - AAA AAG GGT TGT AAG TAC TTT AGT GAT GAT GG - #A ACT TTT GTT TGT 
GAA 594 
Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gl - #y Thr Phe Val Cys Glu 
155 - # 160 - # 165 
- - GGA GAG TCT GAT CCT AAA AAT CCA AAG GCC TG - #T CCT CGG AAT TGT GAT 
642 
Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cy - #s Pro Arg Asn Cys Asp 
170 - # 175 - # 180 
- - GGA AGA ATT GCC TAT GGG ATT TGC CCA CTT TC - #A GAA GAA AAG AAG AAT 
690 
Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Se - #r Glu Glu Lys Lys Asn 
185 - # 190 - # 195 
- - GAT CGG ATA TGC ACC AAC TGC TGC GCA GGC AA - #A AAG GGT TGT AAG TAC 
738 
Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr 
200 - # 205 - # 210 
- - TTT AGT GAT GAT GGA ACT TTT GTT TGT GAA GG - #A GAG TCT GAT CCT AAA 
786 
Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Lys 
215 2 - #20 2 - #25 2 - 
#30 
- - AAT CCA AAG GCT TGT CCT CGG AAT TGT GAT GG - #A AGA ATT GCC TAT 
GGG 834 
Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly 
235 - # 240 - # 245 
- - ATT TGC CCA CTT TCA GAA GAA AAG AAG AAT GA - #T CGG ATA TGC ACA AAC 
882 
Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn As - #p Arg Ile Cys Thr Asn 
250 - # 255 - # 260 
- - TGT TGC GCA GGC AAA AAG GGC TGT AAG TAC TT - #T AGT GAT GAT GGA ACT 
930 
Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Ph - #e Ser Asp Asp Gly Thr 
265 - # 270 - # 275 
- - TTT GTT TGT GAA GGA GAG TCT GAT CCT AGA AA - #T CCA AAG GCC TGT CCT 
978 
Phe Val Cys Glu Gly Glu Ser Asp Pro Arg As - #n Pro Lys Ala Cys Pro 
280 - # 285 - # 290 
- - CGG AAT TGT GAT GGA AGA ATT GCC TAT GGA AT - #T TGC CCA CTT TCA GAA 
1026 
Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Il - #e Cys Pro Leu Ser Glu 
295 3 - #00 3 - #05 3 - 
#10 
- - GAA AAG AAG AAT GAT CGG ATA TGC ACC AAT TG - #T TGC GCA GGC AAG 
AAG 1074 
Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cy - #s Cys Ala Gly Lys Lys 
315 - # 320 - # 325 
- - GGC TGT AAG TAC TTT AGT GAT GAT GGA ACT TT - #T ATT TGT GAA GGA GAA 
1122 
Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Ph - #e Ile Cys Glu Gly Glu 
330 - # 335 - # 340 
- - TCT GAA TAT GCC AGC AAA GTG GAT GAA TAT GT - #T GGT GAA GTG GAG AAT 
1170 
Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Va - #l Gly Glu Val Glu Asn 
345 - # 350 - # 355 
- - GAT CTC CAG AAG TCT AAG GTT GCT GTT TCC TA - #AGTCCTAA CTAATAATAT 
1220 
Asp Leu Gln Lys Ser Lys Val Ala Val Ser 
360 - # 365 
- - GTAGTCTATG TATGAAACAA AGGCATGCCA ATATGCTCTG TCTTGCCTGT AA - 
#TCTGTAAT 1280 
- - ATGGTAGTGG AGCTTTTCCA CTGCCTGTTT AATAAGAAAT GGAGCACTAG TT - 
#TGTTTTAG 1340 
- - TTAAAAAAAA AAAAAAAAAA - # - # 
136 - #0 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 368 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Il - #e Ala Tyr Gly Val Cys 
1 5 - # 10 - # 15 
- - Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Il - #e Cys Thr Asn Cys Cys 
20 - # 25 - # 30 
- - Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser As - #p Asp Gly Thr Phe Val 
35 - # 40 - # 45 
- - Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Ly - #s Ala Cys Thr Leu Asn 
50 - # 55 - # 60 
- - Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pr - #o Arg Ser Glu Glu Lys 
65 - # 70 - # 75 - # 80 
- - Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Al - #a Gly Thr Lys Gly Cys 
85 - # 90 - # 95 
- - Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cy - #s Glu Gly Glu Ser Asp 
100 - # 105 - # 110 
- - Pro Arg Asn Pro Lys Ala Cys Pro Arg Asn Cy - #s Asp Pro Arg Ile Ala 
115 - # 120 - # 125 
- - Tyr Gly Ile Cys Pro Leu Ala Glu Glu Lys Ly - #s Asn Asp Arg Ile Cys 
130 - # 135 - # 140 
- - Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Ly - #s Tyr Phe Ser Asp Asp 
145 1 - #50 1 - #55 1 - 
#60 
- - Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pr - #o Lys Asn Pro Lys 
Ala 
165 - # 170 - # 175 
- - Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Ty - #r Gly Ile Cys Pro Leu 
180 - # 185 - # 190 
- - Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Th - #r Asn Cys Cys Ala Gly 
195 - # 200 - # 205 
- - Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gl - #y Thr Phe Val Cys Glu 
210 - # 215 - # 220 
- - Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cy - #s Pro Arg Asn Cys Asp 
225 2 - #30 2 - #35 2 - 
#40 
- - Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Se - #r Glu Glu Lys Lys 
Asn 
245 - # 250 - # 255 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr 
260 - # 265 - # 270 
- - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg 
275 - # 280 - # 285 
- - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly 
290 - # 295 - # 300 
- - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn As - #p Arg Ile Cys Thr Asn 
305 3 - #10 3 - #15 3 - 
#20 
- - Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Ph - #e Ser Asp Asp Gly 
Thr 
325 - # 330 - # 335 
- - Phe Ile Cys Glu Gly Glu Ser Glu Tyr Ala Se - #r Lys Val Asp Glu Tyr 
340 - # 345 - # 350 
- - Val Gly Glu Val Glu Asn Asp Leu Gln Lys Se - #r Lys Val Ala Val Ser 
355 - # 360 - # 365 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Il - #e Ala Tyr Gly Val Cys 
1 5 - # 10 - # 15 
- - Pro Arg Ser Glu Glu Lys Lys Asn 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Th - #r Lys Gly Cys Lys Tyr 
1 5 - # 10 - # 15 
- - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg 
20 - # 25 - # 30 
- - Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pr - #o Arg Ile Ala Tyr Gly 
35 - # 40 - # 45 
- - Val Cys Pro Arg Ser Glu Glu Lys Lys Asn 
50 - # 55 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Th - #r Lys Gly Cys Lys Tyr 
1 5 - # 10 - # 15 
- - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg 
20 - # 25 - # 30 
- - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Pr - #o Arg Ile Ala Tyr Gly 
35 - # 40 - # 45 
- - Ile Cys Pro Leu Ala Glu Glu Lys Lys Asn 
50 - # 55 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr 
1 5 - # 10 - # 15 
- - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Lys 
20 - # 25 - # 30 
- - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly 
35 - # 40 - # 45 
- - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 
50 - # 55 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr 
1 5 - # 10 - # 15 
- - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Lys 
20 - # 25 - # 30 
- - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly 
35 - # 40 - # 45 
- - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 
50 - # 55 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr 
1 5 - # 10 - # 15 
- - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg 
20 - # 25 - # 30 
- - Asn Pro Lys Ala Cys Pro Arg Asn Cys Pro Gl - #y Arg Ile Ala Tyr Gly 
35 - # 40 - # 45 
- - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 
50 - # 55 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr 
1 5 - # 10 - # 15 
- - Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gl - #y Glu Ser Glu Thr Ala 
20 - # 25 - # 30 
- - Ser Lys Val Asp Glu Tyr Val Gly Glu Val Gl - #u Asn Asp Leu Gln Lys 
35 - # 40 - # 45 
- - Ser Lys Val Ala Val Ser 
50 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Th - #r Lys Gly 
1 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly 
1 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - Lys Ala Cys Thr Leu Asn 
1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - Glu Glu Lys Lys Asn 
1 5 
__________________________________________________________________________