Title: Proteinase inhibitor, precursor thereof and genetic sequences encoding same
Abstract: 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.
Patent Number: 6,946,278 Issued on 09/20/2005 to Anderson, et al.
| Inventors:
|
Anderson; Marilyn Anne (Keilor, AU);
Atkinson; Angela Hilary (Montrose, AU);
Heath; Robyn Louise (Williamstown, AU);
Clarke; Adrienne Elizabeth (Parkville, AU)
|
| Assignee:
|
Hexima Limited (Melbourne, AU)
|
| Appl. No.:
|
157622 |
| Filed:
|
May 29, 2002 |
Foreign Application Priority Data
| Current U.S. Class: |
435/213; 435/19; 435/252.3; 435/320.1; 536/23.2 |
| Intern'l Class: |
C12N 009/76; C12N 009/50; C12N 015/00; C07H 021/04 |
| Field of Search: |
435/213,219,252.3,320.1,691
536/232,231,236
800/278,295
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Bryant, et al. (1976) "Proteinase Inhibitor II from Potatoes: Isolation and Characterization
of its Protomer Components": Biochemistry 15(16): 3418-3423.
Choi, et al (1990) "Primary structure of two Proteinase Inhibitor genes closely
linked in the Potato genome": Hanguk Saenghwahakho Chi 23: 214-220.
Suggs, et al. (1981) "Use of synthetic oligonucleotides as hybridization probes:
Isolation of cloned cDNA sequences for human β2-microglobulin":
Proc. Nat'l. Acad. Sci. USA 78(11): 6613-6617.
Richardson (1979), Sequence Search Comparison, Result 14, FEBS Lett. 104: 322-326.
|
Primary Examiner: Saidha; Tekchand
Attorney, Agent or Firm: Scully, Scott, Murphy & Presser
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of Ser. No. 09/431,499, filed Nov.
1, 1999, now U.S. Pat. No. 6,451,573, which is a divisional of Ser. No. 08/454,295,
filed Sep. 1, 1995, now U.S. Pat. No. 6,031,087, which corresponds to PCT/AU93/00659
having an international filing date of Dec. 16, 1993.
Claims
1. A recombinant type II serine protein inhibitor (PI) precursor from a plant
of the genus
Nicotiana, said precursor comprising at least three PI monomers
covalently linked to each other, wherein at least one of the monomers has a chymotrypsin
site and at least one other of the monomers has a trypsin specific site, wherein
said PI precursor comprises the amino acid sequence as set forth in SEQ ID NO:
3, and the monomers are set forth in SEQ ID NOS: 5-9.
2. A monomer of the recombinant PI precursor according to claim 1.
3. A recombinant type II serine protein inhibitor (PI) precursor from a plant,
said precursor comprising at least three PI monomers covalently linked to each
other, wherein at least one of the monomers has a chymotrypsin site and at least
one other of the monomers has a trypsin specific site, wherein said PI precursor
is encoded by a nucleotide sequence which hybridizes to the complement sequence
of SEQ ID NO: 1 under conditions comprising (1) hybridization at 68° C., and
washing at 68° C. in 2×SSC, 0.1% w/v SDS or in 0.2×SSC, 1% w/v SDS,
or (2) hybridization at 40° C. in 50% v/v formamide, and washing sequentially
in 4×SSC at room temperature, 2×SSC at room temperature and 1×SSC
at 40° C.
4. A recombinant PI precursor according to claim 3 wherein said PI precursor
comprises at least four monomers.
5. A recombinant PI precursor according to claim 3 wherein said PI precursor
comprises at least five monomers.
6. A recombinant PI precursor according to claim 3 wherein said PI precursor
comprises at least six monomers.
7. A monomer of the recombinant PI precursor according to claim 3.
Description
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 usefu 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 |
| Gys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pro Arg Ser Glu Clu 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 TTG 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 CAA 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 CGG |
| 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 CGC 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 CAG 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 Cly Ile Gys 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 preterred 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
1S
3, S
3S
3
or S
6S
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
1-X
1-X
2-Asn-Asp-R
2
where R
1, R
2, X
1 and X
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:
wherein X
1 and X
2 are any amino acid but are preferably
both Lys residues. The protease sensitive peptide may also be represented as:
wherein X
1 and X
2 are preferably the same and are preferably
both Lys residues and wherein R
1 and R
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, substituted alkyl, alkenyl and substituted
alkenyl and the like is meant to encompass straight and branched molecules, lower
(C
1-C
6) and higher (more than C
6) derivatives.
The term "substituted" includes all the substituents set forth above.
In its most preferred embodiment, the protease sensitive peptide is:
wherein R
1 and R
2 are the same or different and are
peptides or polypeptides and wherein X
1 and X
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:
wherein X
1 and X
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 PIs 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 polyclonal 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.
The present invention is further described by reference to the following non-limiting
Figures and Examples.
IN THE FIGURES
FIG. 1 shows the nucleic acid sequence (SEQ ID NO. 2) of the pNA-PI-2 insert
and the corresponding amino acid sequence (SEQ ID NO. 3) of the
N. alata PI
protein. The amino acid sequence is numbered beginning with 1 for the first amino
acid of the mature protein. The signal sequence is encoded by nucleotides 1 to
97 and the amino acid residues have been assigned negative numbers. The reactive
site residues of the inhibitor are boxed. The
N. alata PI sequence contains
six similar domains (domain 1, residues 1 to 58, domain 2, residues 59-116, domain
3, residues 117-174, domain 4, residues 175-232, domain 5, residues 233-290 and
domain 6, residues 291-343).
FIG. 2 is a photographic representation showing a gel blot analysis of RNA from
various organs of
N. alata. Gel Blot of RNA isolated from organs of
N.
alata and from stigmas and styles of
N. tabacum and
N. sylvestris,
hybridised with the cDNA clone NA-PI-2. St, stigma and style; Ov, ovaries; Po,
pollen; Pe, petals; Se, sepals; L, non-wounded leaves; L4, leaves 4 h after wounding;
L24, leaves 24 h after wounding; Nt,
N. tabacum stigma and style; Ns,
N.
sylvestris stigma and style; Na HindIII restriction fragments of Lambda-DNA.
The NA-PI-2 clone hybridised to 2 mRNA species (1.0 and 1.4 kb). The larger mRNA
was predominant in stigma and styles, whereas the smaller mRNA species was more
dominant in other tissues. After high stringency washes, the 1.0 kb mRNA from stigma
and style no longer hybridises to the NA-PI-2 probe.
FIG. 3 is a photographic presentation depicting in situ localisation of RNA
homologous to NA-PI-2 in stigma and style.
- (a) Autoradiograph of a longitudinal cryosection through the stigma
and style of a 1 cm long bud after hybridisation with the 32P-labelled
NA-PI-2 cDNA probe.
- (b) The same section as (a), stained with toluidine blue. c, cortex;
v, vascular bundles; tt, transmitting tract; s, stigmatic tissue.
The cDNA probe labelled the cells of the stigma heavily and some hybridisation
to the vascular bundles can be seen. There was no hybridisation to the epidermis,
cortical tissue or transmitting tissue. Scale bars=200 μm.
FIG. 4 is a photographic representation of a gel blot analysis of genomic DNA
of
N. alata. Gel blot analysis of
N. alata genomic DNA digested with
the restriction enzymes EcoRI or HindIII, and probed with radiolabelled NA-PI-2.
Size markers (kb) are HindIII restriction fragments of Lambda-DNA.
EcoRI produced two hybridising fragments (11 kb and 7.8 kb), while HindIII
gave three large hybridising fragments (16.6, 13.5 and 10.5 kb). The NA-PI-2 clone
appears to belong to a small multigene family consisting of at least two members.
FIG. 5 is a graphic representation of PI activity in various organs of
N.
alata. Buffer soluble extracts from various organs were tested for their ability
to inhibit trypsin and chymotrypsin. Stigma and sepal extracts were the most effective
inhibitors of both trypsin (A) and chymotrypsin (B).
FIG. 6 depicts the steps of the purification of PI from
N. alata stigmas.
- (a) Sephadex G-50 gel filtration chromatography of ammonium sulphate
precipitated proteins from stigma extracts. The PI activity eluted late in the profile.
- (b) 20% w/v SDS-polyacrylamide gel (Laemmli, 1970) of fractions across
the gel filtration column. The gel was silver stained and molecular weight markers
(Pharmacia peptide markers) are in kilodaltons. A protein of about 6 kD (arrowed)
coelutes with the proteinase inhibitor activity.
- (c) Analysis of PI-containing fractions at different stages of the purification
procedure, by SDS-PAGE. Lane 1, crude stigma extract (5 μg); Lane 2, stigma
proteins precipitated by 80% w/v ammonium sulphate (5 μg); Lane 3, PI protein
eluted from the chymotrypsin affinity column (1 μg).
The PI is a 6 kD protein and is a major component in unfractionated buffer soluble
extracts from stigmas.
FIG. 7 is a graphical representation showing hydropathy plots of the PI proteins
encoded by the NA-PI-2 clone from
N. alata and the potato and tomato PI
II cDNAs. Values above the line denote hydrophobic regions and values below the
line denote hydrophilic regions. The putative signal peptides are shaded. The hydrophobicity
profile was generated using the predictive rules of Kyte and Doolittle (1982) and
a span of 9 consecutive amino acids.
- (a) Hydropathy profile of the N. alata PI protein. The six repeated
domains in the predicted precursor protein are labelled I-VI. The hydrophilic regions
containing the putative cleavage sites for production of the 6 kD PI species are
arrowed. The regions corresponding to the peptides that would be produced by cleavage
at these sites are marked C for chymotrypsin inhibitor, T for typsin inhibitor
and x for the two flanking peptides.
- (b) Hydropathy profile of the potato PI II protein. (Sanchez-Serrano
et al., 1986). The two repeated domains in the PI II protein are labelled I and
II. The putative cleavage sites for production of PCI-1 are arrowed (Hass et al.,
1982) and the region spanned by PCI-1 is marked.
- (c) Hydropathy profile of the polypeptide encoded by the tomato PI II
cDNA (Graham et al., 1985). The two domains are labelled, I and II and the residues
which would be potential processing sites are arrowed. These sites are not present
in regions predicted to be hydrophilic and consequently a cleavage product is not marked.
FIG. 8 shows an immunoblot analysis of the PI protein in stigmas of developing flowers.
- (a) Developing flowers of N. alata.
- (b) SDS-PAGE of stigma proteins at the stages of development shown in
(a) 5 μg of each exact was loaded. The peptide gel was silver stained and
molecular weight markers (LKB Low Molecular weight and Pharmacia peptide markers)
are in kilodaltons.
- (c) Immunoblot of a gel identical to (b), probed with anti-PI antiserum.
Stigmas from developing flowers contain four proteins of approximately 42
kD, 32 kD, 18 kD and 6 kD that bind to the anti-PI antibody. The 42 kD and the
18 kD components decrease in concentration as the flowers mature, while the 6 kD
PI protein reaches a maximum concentration just before anthesis. The level of the
32 kD component, which runs as a doublet, does not alter significantly during flower development.
FIG. 9 shows the separation and identification of the 6 kD proteinase inhibitor
species from
N.alata stigmas
A. Separation of the 6 kD PIs by Reversed Phase HPLC Chromatography
Four major peaks were obtained with retention times of about 15.5 min(peak1),
20.5 min(peak2), 22.5 min(peak3), 24 min(peak4). The peptides in each peak have
been identified by a combination of N-terminal analysis and mass spectrometry.
See B for description of C1 and T1-T4.
B. The five homologous peptides produced from the PI precursor protein: C1, chymotrypsin
inhibitor, T1-T4 trypsin inhibitors. The solid bars represent the reactive sites
of the inhibitors. The precursor protein is drawn minus the signal sequence.
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region of the
six repeated domains (amino acids 1-343, FIG. 1).
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non-repeated sequence (amino acids 344-368,
FIG. 1). The arrows point to the processing sites in the precursor protein.
C. The amino acid sequence of C1 and T1-T4 predicted from the cDNA clone and confirmed
by N-terminal sequencing of the purified peptides. The amino acid at the carboxy-terminus
of each peptide was obtained by accurate mass determination using an electro-spray
mass spectrometer. The C1 and T1 inhibitors differ by five amino acids (bold).
Two of these amino acids are located at the reactive site (underlined) and the
other two to three reside at the carboxy-terminus. Peptides T2-T4 have changes
in three amino acids (boxed) that are conserved between C1 and T1. Peptides T2
and T3 are identical to each other. Mass spectrometry was used to demonstrate that
other forms of C1 and T1-T4 occur due to non-precise trimming at the N- and C-termini.
That is, some forms are missing residue 1 or residue 53 and others are missing
both residue 1 and 53 (see Table 2).
FIG. 10 shows the amino-acid sequence around the processing sites in the precursor
PI protein.
The sequence in bold is the amino-terminal sequence obtained from the purified
PI protein. The sequence labelled with negative numbers is the flanking sequence
predicted from the cDNA clone. The predicted precursor protein contains six repeats
of this sequence.
FIG. 11 shows the PI precursor produced in a baculovirus expression system and
the products obtained after digestion of the affinity purified PI precursor by
the endoproteinase Asp-N.
A. The PI Precursor Produced by the Recombinant Baculovirus.
Immunoblot containing affinity and HPLC purified PI precursor from
N.alata
stigmas at the green bud stage of development (lane 1) and affinity purified
PI precursor produced by the recombinant baculovirus (lane 2). Proteins were fractionated
by electrophoresis on a 15% w/v SDS-polyacrylamide gel prior to electrophoretic
transfer to nitrocellulose. The blot was incubated with the antibody raised in
rabbits to the 6 kD PI species from stigmas. The recombinant virus produced an
immunorective protein of 42 kD that is the same size as the PI precursor protein
produced by stigmas (arrowed).
B. Cleavage of the PI Precursor by Endoproteinase Asp-N.
15% SDS-polyacrylamide gel stained with silver containing: 1, PI precursor, produced
by baculovirus, incubated without enzyme. 2, enzyme incubated without precursor.
6 kD, PI peptides of about 6 kD purified from
N.alata stigmas. 1 m, 5 m,
30 m, reaction products produced after 1, 5 and 30 minutes of incubation. 2 h and
24 h, reaction products after 2 and 24 h of incubation. Peptides of about 6-7 kD
were detected within one minute of incubation of the precursor with the enzyme.
After 24 h only peptides of 6-7 kD were detected. The bands smaller than 42 kD
in track 1 are due to truncated forms of the precursor produced by premature termination
of translation in the baculovirus expression system.
FIG. 12 Preparative chromatography by reversed phase HPLC of the peptides produced
from the precursor by Asp-N digestion
HPLC profile of peptides produced by Asp-N digestion of the PI precursor. The
major peaks had a retention time of 19 min (termed Asp-N1) and 21 min (termed Asp-N2).
The peptides in these peak fractions (1 & 2) had a slightly slower mobility on
SDS-PAGE than the 6 kD peptides from stigmas (C, inset). The proteinase inhibitory
activity of Asp-N1 and Asp-N2 was tested against trypsin and chymotrypsin.
FIG. 13 shows a comparison of the trypsin and chymotrypsin inhibition activity
of the PI precursor, PI peptides from stigmas and in vitro produced PI peptides
from the PI precursor.
PI precursor or PI peptides (0-1.0 μg) were tested for their ability to
inhibit 1.0 μg of trypsin or chymotrypsin as described in the materials and
methods. Inhibitory activity is expressed as the percentage of proteinase activity
remaining after the proteinase had been preincubated with the PI with 100% remaining
activity taken as the activity of the proteinase preincubated with no PI. Experiments
were performed in duplicate and mean values were plotted. Deviation from the mean
was 8% or less.
FIG. 14 is a graphical representation showing a growth curve for
T. commodus
nymphs reared on control artificial diet, soybean Bowman-Birk inhibitor and
N. alata PI. The vertical axis represents the mean weight of the crickets
in each treatment (+/- standard error) in mg. The horizontal axis represents the
week number. The crickets reared on the
N. alata PI showed a lower mean
weight than those reared on both the control diet and the diet containing the soybean
inhibitor, throughout the experiment.
| SEQ ID NO. 1 |
Nucleotide coding region of N. alata PI precursor |
| SEQ ID NO. 2 |
Full length nucleotide sequence of N. alata PI |
| |
precursor |
| SEQ ID NO. 3 |
Amino acid sequence corresponding to SEQ ID NO. 1 |
| SEQ ID NO. 4 |
Residues 1-24 of SEQ ID NO. 2 (peptide 1) |
| SEQ ID NO. 5 |
Residues 25-82 of SEQ ID NO. 2 (peptide 2) |
| SEQ ID NO. 6 |
Residues 83-140 of SEQ ID NO. 2 (peptide 3) |
| SEQ ID NO. 7 |
Residues 141-198 of SEQ ID NO. 2 (peptide 4) |
| SEQ ID NO. 8 |
Residues 199-256 of SEQ ID NO. 2 (peptide 5) |
| SEQ ID NO. 9 |
Residues 257-314 of SEQ ID NO. 2 (peptide 6) |
| SEQ ID NO. 10 |
Residues 315-368 of SEQ ID NO. 2 (peptide 7) |
| SEQ ID NO. 11 |
N-terminal amino acid sequence of 6kD PI protein |
| SEQ ID NO. 12 |
N-terminal amino acid sequence of 6kD PI protein |
EXAMPLE 1
1. Materials and Methods
Plant Material
Nicotiana alata (Link et Otto) plants of self-incompatibility genotype
S
1S
3, S
3S
3 and S
6S
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° 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 from stigmas and styles, isolated
from mature flowers of
N. alata (genotype S
3S
3), and
used to construct a cDNA library in Lambda gt10 (Anderson et al., 1989). Single
stranded
32P-labelled cDNA was prepared from mRNA from stigmas and styles
of
N. alata (genotype S
3S
3 and S
6S
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™
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
32P
using random hexanucleotides (1×10
8 cpm μg
-1;
1×10
7 cpm ml
-1)(Feinberg and Vogelstein, 1983). Prehybridisation
and hybridisation, at 68° C., were as described by Anderson et al. (1989).
The filters were washed in 2×SSC, 0.1% w/v SDS or 0.2×SSC, 1% w/v SDS
at 68° 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
8 cpm μg
-1 by random hexanucleotide priming
(Feinberg and Vogelstein, 1983). The labelled probe was precipitated, and resuspended
in hybridisation buffer (50 μl), and 5 μl was applied to the sections.
The sections were covered with coverslips, and incubated overnight at 40°
C. in a closed box containing 50% v/v formamide. After incubation, sections were
washed sequentially in 4×SSC at room temperature, 2×SSC at room temperature,
and 1×SSC at 40° 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
2O,
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 μg) 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×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
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
2, 14 μM β-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 chymotrypsin as described by Rickauer et al. (1989). Inhibitory
activity was measured against 1 μg of trypsin (TPCK-treated; Sigma) or 3
μg of chymotrypsin (TLCK-treated; Sigma). The rate of hydrolysis of synthetic
substrates N-α-P-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-tyrosine
ethyl ester (BTEE) by trypsin and chymotrypsin, respectively, were taken as the
unhibited 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
2,
and extracted in buffer (100 mM Tris-HCl, pH8.5, 10 mM EDTA, 2 mM CaCl
2,
14 μM β-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×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 α-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 μl 1M Tris-HCl pH 8, and dialyzed extensively against
deionised H
2O.
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. 6
c, 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
2O, 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 mn 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 μg 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®Semi-dry electrophoretic transfer
cell (12V, 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 μg/ml in 1% w/v BSA, Tris Buffered Saline) overnight at room temperature.
Bound antibody was 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°
C. with endoproteinase Asp-N (2 μg) in 100 mM NH
4HCO
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
μm, 4.6×100 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
