Segregated folding determinants for small disulfide-rich peptides

The preparation of small peptides with multiple disulfide bonds is accomplished by forming a prepropeptide with an N-terminal excised region separated from the cysteine-rich peptide by one or more cleavable amino acid residues. The excised region preferably consists of an N-terminal end providing a hydrophobic signal sequence domain having up to approximately 25 amino acids, and an intermediate central propeptide domain having a variable length of between about 5-50 amino acids. The N-terminal excised region serves as a folding template to direct the formation of specific disulfide bonds in the cysteine-rich peptide. The cysteine-rich peptide is cleaved by enzymes releasing the biologically active peptide.

BACKGROUND OF THE INVENTION 
This invention relates to a method for forming biologically active peptides 
with specific disulfide linkages. This invention also relates to the 
synthesis of prepropeptides containing an N-terminal template region 
which, when combined with a mature peptide region containing multiple 
cysteine residues, results in specific disulfide bonds being formed in the 
mature peptide region. 
The obtaining of small biologically active cysteine-rich disulfide 
containing peptides, i.e. those containing 10 to 30, or even more, amino 
acids, which have the same folding pattern presents a problem in 
synthesis. The obtaining of such peptides is highly desirable for 
designing drug delivery systems which are targeted to specific protein 
receptors, or which interfere with the function of these receptor targets. 
These peptides have applications in biotechnology, particularly for the 
pharmaceutical industry and in agriculture. However, when such small 
peptides, such as found in nature in the venom of the Conus snail, are 
synthesized by conventional synthetic methods, the formation of the 
disulfide bonds is non-specific and, when oxidized, the peptides appear to 
fold in many different disulfide configurations rather than in a specific 
biologically active form found in the naturally occurring peptides. 
The conotoxins are a set of small peptide ligands found in the venom of 
predatory cone snails. These peptides, used by the cone snails to capture 
their prey, bind and interfere with the function of a diverse set of 
receptors and ion channel targets. Compared with most polypeptide toxins, 
the cone snails use much smaller molecules (typically 10-30 amino acids) 
wherein the biologically active conformation is shaped by the formation of 
disulfide bonds between specific cysteine residues Conotoxin peptides have 
some of the highest known densities of disulfide bonding in any biological 
system. Peptides from Conus venoms have been isolated that are only 12 
amino acids long with three disulfide bonds. 
Despite their relatively small size, these peptide ligands can bind with 
remarkably high affinities to the receptor targets in the prey. 
.omega.-Conotoxins have subpicomolar K.sub.D s for certain neuronal Ca 
channel targets. In general, only one disulfide bonded configuration (i.e. 
the conformation found in the natural peptide) exhibits high affinity for 
the specific receptor target. 
Although some conotoxins have only two disulfide bonds (notably the 
.alpha.-conotoxins which target to nicotinic acetylcholine receptors), 
more commonly the major paralytic conotoxins found in Conus venom have 
three disulfide bonds (in the fish-hunting cones, the .mu.-conotoxins 
which target to muscle voltage-sensitive Na channels and the 
.omega.-conotoxins which target to presynaptic voltage sensitive Ca 
channels). For the latter, there are 15 possible disulfide bonded 
configurations. Folding smaller peptides into one specific configuration 
is a biochemical problem which the cone snails had to solve before they 
could efficiently use such small peptides as high affinity ligands for 
paralysing their prey. When the peptides are chemically synthesized and 
cysteine residues oxidized after synthesis, they often appear to fold in a 
variety of different disulfide configurations, which are different from 
the biologically active form found in the venom. In the chemical synthesis 
of .mu.-conotoxin, even after optimizing for the increased yield, the 
overall yield of the correctly disulfide bonded configuration was still 
&lt;10%. 
Small, conformationally constrained peptides are ideal for a wide variety 
of biotechnology applications. Their small size facilitates access to 
specific target receptors. Specific cross-linking of disulfide bonds 
allows these small peptides to assume a relatively rigid structure that 
increases the probability of high affinity interaction with target 
molecules. Still, the variation of peptide structure afforded by variation 
in amino acid sequence in such peptides is enormous. Natural variants 
among peptides following this architectural design have been found to 
target a great diversity of target types. Molecules of this type have an 
expanding usefulness as agents capable of targeting a vast variety of 
receptors and ion channels on the surface of many different cell types. 
These molecules are useful in the design and testing of drugs targeting to 
variety of therapeutically important components, and in the design of 
agriculturally important agents. In addition, they have a more general 
potential as ligands used for interaction with broad classes of proteins 
and other biologically relevant macromolecules. 
If these small peptides containing multiple disulfide bonds are synthesized 
by conventional synthetic methods, the formation of the disulfide bonds is 
non-specific. In nature this problem is solved by the formation of 
prepropeptides that fold in a conformation that allows the cysteine 
residues of the toxin portion of the peptide chain to form the disulfide 
bonds in the positions necessary for activity. It would be desirable to 
provide an atmosphere where the portions of the prepropeptides that 
contain the folding instructions could be synthesized and attached to 
either the natural or synthetic small peptides that contain multiple 
cysteine residues. Such synthesis would desirably be performed by cloning 
techniques or by conventional means such as solution phase synthesis, 
solid phase synthesis or a combination of these two techniques. Thus, the 
final prepropeptide resulting from such combination would have the 
disulfide bonds in the proper position for activity. Once synthesized, the 
pre- and pro- portions of the molecule could be cleaved without disturbing 
the disulfide bonds, thereby releasing a cystiene-rich bioactive peptide. 
OBJECTS AND SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a class of N-terminal 
polypeptides which function as template region in the synthesis of 
prepropeptides for the formation at the C-terminal end of cysteine-rich 
peptides having specific disulfide linkages and configuration. 
It is also an object of this invention to provide a method for forming 
biological active peptides with specific disulfide linkages through the 
formation of prepropeptides wherein the C-terminal end of the 
prepropeptide consists of the desired bioactive peptide and the remainder 
through the N-terminal end, referred to as the excised end, contains a 
region having folding template activity, both in vivo and in vitro, for 
directing the formation of specific disulfide bonds in the bioactive 
cysteine-rich peptide at the C-terminal end. 
It is also an object of this invention to provide a method for the 
preparation of relatively small cysteine-rich peptides having specific 
disulfide bond configurations prepared by directing specific disulfide 
bond formation using a region of the excised end of the prepropeptide as a 
folding template. 
These and other objects may be accomplished by formation of a class of 
prepropeptide molecules having a general architectural theme consisting of 
a C-terminal cysteine-rich mature peptide region and an excised region 
preferably having two domains, the first being the N-terminal end which 
consists of a hydrophobic signal sequence domain and a central propeptide 
domain as more specifically defined in the following formula wherein the 
excised region serves as a folding template to direct the formation of 
specific disulfide bonds in the mature peptide region. Between the mature 
peptide region and the excised region there is locate a cleavage site for 
the excision of the mature cysteine-rich peptide from the prepropeptide 
molecule. 
The prepropeptide is made up of an N-terminal template peptide preferably 
consisting of an N-terminal end providing a hydrophobic signal sequence 
domain and an intermediate central propeptide domain having a variable 
length and a C-terminal end providing a cleavage site attached to the 
active region containing the multiple disulfide bonds from which said 
mature peptide can be enzymatically cleaved. Although described as having 
two domains, the N-terminal peptide may have only the hydrophobic signal 
or propeptide domain as long as that domain or region serves as a folding 
template to direct the formation of specific disulfide bonds in the mature 
peptide region. 
The N-terminal peptide generally has the formula: 
EQU [hAA].sub.c [pAA].sub.b [cAA].sub.a (Formula 1) 
wherein [cAA] is a cleavage site made up of one or more amino acids from 
which the adjoining mature cysteine-rich peptide can be cleaved by 
enzymatic action, a is an integer of from about 1 to 10 with integers of 
about 1 to 5 being preferred; [pAA].sub.b represents a propeptide domain 
where b is an integer of between about 5 to 50 with integers of between 
about 20 to 30 being preferred where pAA represents suitable amino acids 
forming a peptide chain for proper template homology; [hAA].sub.c 
represents a hydrophobic signal sequence where c is an integer of between 
about 0 and 25 with integers of between about 15 and 25 being preferred 
wherein the excised region serves as a folding template to direct the 
formation of said specific disulfide bonds in said cysteine-rich peptide. 
The amino acids in the N-terminal peptide will generally form a peptide 
chain [a+b+c] having between about 10 and 85 amino acid residues with a 
range of between about 15 and 75 being preferred. It has been found that 
the excised region of the prepropeptides have very significant sequence 
homology when coupled to mature peptides with similar arrangement of 
Cysteine residues. These homologies in excised regions appear to extend 
across species lines, at least in the genus Conus, indicating that it is 
the excised regions of the molecule which direct folding of specific 
disulfide bonds in mature peptides. Therefore, it has been found possible 
to direct formation of specific disulfide bonds in small peptides by 
coupling the small mature peptide with the appropriate folding template 
region. 
Once the excised region peptide sequence is known, the portions of the 
prepropeptides that contain the folding instructions can be synthesized 
and attached to either natural or synthetic small peptides that contain 
multiple cysteine residues to yield only peptides having the desired 
specific disulfide bonds. This synthesis can be performed by cloning 
techniques or by conventional means such as solution phase synthesis, 
solid phase synthesis or a combination of these techniques. The final 
prepropeptide resulting from this combination has the disulfide bonds in 
the proper position for bioactivity. The prepropeptide portion can be 
enzymatically cleaved from the excised region at site of the basic amino 
acid moieties as above described without disturbing the disulfide bonds 
thereby releasing the active cysteine-rich peptide.

DETAILED DESCRIPTION OF THE INVENTION 
For most polypeptides to assume a fixed conformation, a minimum size is 
necessary. Smaller peptides much below fifty amino acids in size are 
generally considered too small to assume a fixed conformation. In these 
small peptides the number of hydrogen bonds and other weak attractions 
between the amino acids of the chain is insufficient to form a rigid or a 
specific configuration. Therefore, a large number of different 
conformations can occur in these small peptides. Nature has solved this 
problem in certain cases by dispersing cysteine residues to form disulfide 
bonds thereby greatly constraining the small peptide chain. In the Conus 
venoms an unprecedented diversity of small (10-30 amino acids) 
disulfide-rich peptides is found. Since most of the small peptides in the 
Conus system have six cysteine residues, fifteen disulfide bonded 
configurations are theoretically possible However, in vivo, a single 
specific disulfide bonded configuration is generated for each peptide 
Cloning data from several different Conus species has revealed the 
existence of prepropeptide precursors to the conotoxins. Table 1 contains 
the amino acid sequence of 5 biologically active "mature" conotoxin 
molecules. These sequences have been determined by direct peptide 
sequencing of purified venom duct components. Also shown are amino acid 
sequences of prepropeptide precursors of each of these toxins as 
determined by cDNA sequencing of messages expressed in venom duct tissue. 
Each of these prepromolecules conforms to a general architectural theme. 
They contain an N terminal hydrophobic signal sequence domain of 
approximately 20 amino acids followed by a domain of variable length 
(25-30 amino acids) which is neither signal sequence nor the final 
biologically active toxin. These two N-terminal domains are excised from 
the precursor molecule during processing. The third region, at the 
C-terminal end contains toxin specific sequences and is referred to as the 
"mature peptide". In each case, one or more basic amino acid residues 
separate the second and third domains of the prepropeptide, affording a 
cleavage site for excision of the "mature peptide". Therefore, the present 
cloning data indicate that most if not all conotoxins are derived from 
precursor peptides which are each in the size range of molecules such as 
BPTI (bovine pancreatic trypsin inhibitor) which are capable of 
spontaneous precise folding into a specific conformation. 
Cloning of several "families" of conotoxins has revealed striking 
relationships among these prepropeptide precursor molecules. It has been 
observed that mature conotoxins which have very different amino acid 
sequences but share a common organization (framework) of cysteine residues 
are derived from precursor molecules which share significant sequence 
homology at their N-terminal domains. This data indicates that the two 
N-terminal domains which are excised during processing (hereinafter called 
the "template" region) are necessary for the formation of specific 
disulfide bonds in the mature peptide. The basis for this invention lies 
in the fact that the same template region can be used to specifically fold 
mature peptides of considerable sequence diversity, i.e., although the 
arrangement of cysteine residues is conserved, a wide variety of 
non-cysteine residue sequences are acceptable. This discovery provides 
applications for folding small disulfide-rich peptides in vitro and in 
vivo. 
The amino acid sequence of conotoxin precursor molecules has been 
determined by sequencing cDNA clones which encode these molecules. For 
these studies, two different types of plasmid based cDNA libraries have 
been employed. Data for the King Kong family of conotoxins (KK0, KK1, and 
KK2) has been derived from clones screened from a vector-primed library of 
Conus textile cDNA inserts. This library was constructed using polyA 
selected mRNA transcripts purified from Conus textile venom duct tissue 
and cloned into the Okyama-Berg oligo-dT primed plasmid pSV7186. cDNA 
clones were selected using standard colony hybridization procedures and 
oligonucleotide probes encoding the prepropeptide region of KK0 mRNA. 
Oligonucleotide probes for this experiment were synthesized based on 
partial sequencing of KK0 transcript directly from Conus textile polyA 
selected venom duct mRNA using reverse transcriptase. Data for the MVIIb, 
GVIA, Circler, and GI peptides were derived from clones screened from 
vector-primed cDNA libraries of Conus magus and Conus geographus venom 
duct transcripts. These libraries were created using oligo-dT primed pUC13 
vector. cDNA clones were identified using mixed oligonucleotide probes 
encoding all permutations of the universal codon sequence for 6 amino acid 
portions of each mature toxin. Colonies were plated onto nitrocellulose 
filters and grown on nutrient broth plates containing 50 .mu./ml kanamycin 
sulfate and 50 .mu.g/ml ampicillin. Replica filters were transferred to 
Whatman filter paper wet with lysis buffer containing 0.5M NaOH and 1.5M 
NaCl. Following serial neutralization in 0.5M tris pH 8.0, 1.5M tris pH 
8.0 and 1.5M NaCl, filters were dried at 80.degree. C. in a vacuum oven. 
Filters were pre-washed in a solution containing 3M tetramethylammonium 
chloride (TMAC), 50 mM tris and 0.2% SDS. Filters were pre-hydribized at 
48.degree. C. for 4 hours and hybridized for 40-60 hours in a solution 
containing 3M TMAC, 0.1M NaPhosphate, 1 mM EDTA, 5.times.Denhardt's 
solution, 0.6% SDS and 100 .mu.g/ml salmon sperm DNA. In addition, the 
hybridization solution contained mixed oligonucleotide probes end-labelled 
using .tau.P.sup.32 ATP and T4 DNA kinase. Following hybridization, 
filters were washed at room temperature in a solution containing 3M 
TMAC,50mM tris pH 8.0 and 0.2% SDS. Final washing was done at 50.degree. 
C. in a solution containing 2XSSC and 0.1% SDS. Following drying and 
autoradiography on photographic film, putative conotoxin cDNA clones were 
picked from replica filters kept on agar plates. In all cases, nucleic 
acid sequence was determined by direct double strand sequencing of cDNA 
clones using standard procedures. 
EXAMPLE 1 
Calcium Channel 4 loop Toxins 
Several different conotoxins which target presynaptic calcium channels have 
previously been characterized. These toxins have largely overlapping 
specificities for different calcium targets in neuronal tissue 
preparations. Structurally, these toxins are found to conform to a 
conserved number and arrangement (framework) of cysteine residues. Each 
member of this class forms a 4 loop folded toxin molecule with a specific 
arrangement of cysteines referred to as the omega (.omega.) pattern. cDNA 
clones for several members of the .omega.-conotoxin family were obtained 
and their nucleic acid sequence determined by the general cloning and 
sequencing methods above. 
Sequences for two members of this family, MVIIB from Conus magus and GVIA 
from Conus geographus are illustrated in Table 1. MVIIB and GVIA are 4 
loop .omega.-toxins which contain 25 and 27 amino acids respectively. 
Their cysteine frameworks vary only in the exact amino acid spacing of the 
two carboxy terminal inter-cys domains. Beyond the similarity of 
framework, the two mature toxins are remarkably divergent. Only nine of 
the 21 non-cysteine amino acids of the .omega.-GVIA are conserved in 
.omega.-MVIIA. sequence for each of these toxins. MVIIB and GVIA template 
domains are each 45 amino acids in length. In addition, they show a &gt;90% 
conservation of amino acid sequence with only 4 positions of amino acid 
non-identity. These two sequences therefore illustrate the existence of 
two highly conserved template domains associated with two structurally 
dissimilar toxins. These features are illustrated in the following 
sequence representation wherein the respective domains or regions are 
identified as in Formula 1 where ".rarw..fwdarw." represents an interface 
or melding of the regions: 
##STR1## 
TABLE 1 
__________________________________________________________________________ 
Mature Toxin 
Type/ 
determined by amino Prepropeptide 
# Name 
acid sequencing** determined from cDNA clones* 
__________________________________________________________________________ 
I 4 loop 
[seq. ID NO: 2] [seq. ID NO: 1] 
a. 
M-7B 
CKGKGASCHRTSYDCCTGSCNRGKC 
MKLTCVVIVAVLLLTACQLITADDSRGTQ 
[seq. ID NO: 4] [seq. ID NO: 3] 
b. 
G-6A 
CKS -PGSSCS -PTSYNCCRSCN -PYTKRCY 
MKLTCVVIVAVLLLTACQLITADDSRGTQ 
##STR2## 
II 
4 loop 
[seq. ID NO: 6] [seq. ID NO: 5] 
a. 
KK0 WCKQSGEMCNLLDQNCCDGYCIVLVCT 
MKLTCMMIVAVLFLTAWTFATADDPRNGL 
##STR3## 
[seq. ID NO: 8] [seq. ID NO: 7] 
b. 
KK1 CIEQFDPCEMIRHTCCVGVLFLMACI 
MKLTCMMIVAVLFLTAWTFATADDSGNGL 
##STR4## 
[seq. ID NO: 10] [seq. ID NO: 9] 
c. 
KK2 CAPFLHPCTFFFPTCCNSYCVQFICL 
MKLTCMMIVAVLFLTAWTFVTADDSGNGL 
##STR5## 
III 
3 loop 
[seq. ID NO: 12] [seq. ID NO: 11] 
CIRC 
CCPPVACNMGCKPCC MSKLGALLTICLLLFSLTAVPLDGDQHAD 
##STR6## 
IV 
2 loop 
[seq. ID NO: 14] [seq. ID NO: 13] 
GI ECCNGRHYSC MGMRMMFTVFLLVVLATTVVSFFSERASD 
##STR7## 
__________________________________________________________________________ 
*Sequences with G or GR at the Cterminus are posttranslationally processe 
to give a Cterminal amide in the mature toxin or peptide. 
**-P in a sequence stands for hydroxyproline. 
Example 2 
Another family of conotoxin prepropeptides has been derived from sequencing 
of cDNA done from the mollusk hunting species Conus textile. The king Kong 
peptide is a 27 amino acid toxin which is phenotypically inactive when 
injected either intraperitoneally or intracerebrally in mice. However, it 
has been shown to elicit dominant posturing when injected into lobsters 
and to cause a rhythmic undulation in mollusks. 
The primary sequence of the King Kong peptide [Seq. ID No: 6] (shown below) 
was determined by amino acid sequencing as described by Hillyard et al., 
Biochemistry, 28, 358-361(1988). 
EQU W--C--K--Q--S--G--E--M--C--N--L--L--D--Q--N--C--C--D--G--Y--C--I--V--L--V-- 
C--T 
The horizontal bars show the amino acids encoded by the 14 and 20 
nucleotide mixed probes that were used as primers in the RNA sequencing 
reaction. The probe to the more N-terminal region was a mixture of 16 
different oligonucleotides, and the more C-terminal probe had a degeneracy 
of 64. The mixed oligonucleotides were then used for sequencing 
poly(a)+RNA from Conus textile venom ducts. The sequence obtained was used 
for probing a Conus textile venom duct cDNA library. Detailed methods 
follow: 
SYNTHESIS OF OLIGONUCLEOTIDES 
Mixed oligonucleotides corresponding to two regions of the King-Kong 
peptide were synthesized using an Applied Biosystems 380B synthesizer, a 
20 base oligonucleotide 5' CCRTCRCARCARTTYTGRTC 3' [Seq. ID No: 15] and a 
14 base oligonucleotide 5' TTRCACATYTCNCC 3' [Seq. ID No: 16](R=A or G, 
Y=T or C and N=ATG or C). These correspond to the amino acid sequences 
DQNCCDG [Seq. ID No: 17] and GEMCN [Seq. ID No: 18] of the King-Kong 
peptide. 
SEQUENCING OF RNA 
RNA was isolated from .about.20 C.textiIe venom ducts that had been 
previously dissected and frozen at -80.degree. C. The tissue was 
pulverized using a mortar ad pestle in liquid nitrogen and lysed in the 
presence of guanidinium thiocyanate according to published procedures 
(Chirgwin et al., Biochemistry, 18, 5294-5299,(1979)). Poly(A).sup.+ RNA 
was prepared by two passes of the total RNA over an oligo(dT) column 
(Maniatis et al., Molecular Cloning: Laboratory Manual, Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, NY, (1982)). 
Approximately 1 .mu.g of poly(A) RNA in distilled H.sub.2 O was denatured 
by heating to 95.degree. C. for 10 min, then quenched on ice for 5 min. 
Sixteen picomols of either of the two oligomers above that had been 
labeled using T4 DNA kinase and [.gamma.-.sup.32 P] ATP were added and 
annealed in the presence of 5 mM Tris-HCI, pH 8.3, 6 mM NaCl, 1 mM DTT at 
37.degree. C. for 10 min. Mg(OAc).sub.2 was added to 6 mM and the mixture 
divided into four tubes corresponding to the following mixes of 
nucleotides: 0.36 mM each of dATP, dGTP, dCTPPP and dTTP, 0.2 mM of either 
ddATP, ddCTP, ddGTP or ddTTP and 0.5 U of reverse transcriptase 
(Seikagaku) in a total volume of 5.0 ml, incubated for a total of 30 min 
at 52.degree. C. The reactions were stopped with 10 ml of loading dye 
consisting of 0.04% xylene cyanol, 0.04% bromophenol blue, 1.times.TBE 
buffer (0.089M Tris-borate, 0.089M boric acid, 2 mM EDTA) and formamide, 
and heated to 95.degree. C. for 2 min before loading on a 7.5% 
acrylamide/7M urea sequencing gel. The sequence obtained spanned 178 
nucleotides including five N-terminal amino acids of the King-Kong 
peptides. 
cDNA LIBRARY CONSTRUCTION 
A library was constructed in the Okayama-Berg vector pSV7186 (Okayama and 
Berg, 1982) using .about.1.5 mg of oligo(dT) selected poly(A) .sup.+ RNA 
from venom duct isolated as above. Approximately 3.2 .times.10 independent 
colonies were obtained after transformation into Escherichia coli DH5 
competent cells. DNA from 24 clones was prepared from minipreps of 5 ml 
overnight cultures. Upon restriction digestion and analysis, 92% of these 
clones appeared to have inserts ranging in size from 150 to 2700 bases. 
The library was amplified and stored in 15% DMSO AT -80.degree. C. 
SCREENING OF cDNA LIBRARIES 
To identify individual clones containing the King-Kong peptide sequence, 
two 16 base oligonucleotides were synthesized. Probe C corresponds to the 
mRNA sequence, as determined above, of the five most N-terminal acids, 
WCKSQ, and probe D corresponds to the complementary sequence. 
Approximately 16 pmol of probes C and D Were kinased and hybridized to 
duplicate blots for 10 h at 42.degree. C. in 6.times.NET, 
5.times.Denhardt's, 0.1% SDS and 100 mg/ml sonicated salmon testis DNA. 
Blots were washed to a final stringency of 2 SSC, 0.1% SDS at 42.degree. 
C. To identify clones containing the 5' flanking sequences two overlapping 
90 base oligonucleotides (probes A and B), corresponding to the region 5' 
of the King-Kong peptide, who sequence was determined by the RNA 
sequencing, were synthesized as above. Approximately 16 pmol each of 
probes A and B were kinased and hybridized to duplicate blots at 
52.degree. C. in 6.times.NET, 5.times.Denhardt's, 0.1% SDS and 100 mg/ml 
sonicated salmon testes DNA. Blots were washed at a final stringency of 
0.1.times.SSC, 0.1% SDS at 65.degree. C. for 15 min. 
A total of 20 000 colonies were plated out and duplicate membrane filter 
lifts prepared One set of filters was hybridized to probes A and B and the 
other set to probes C or D using the same conditions as above. The filters 
using intensifying screens at -80.degree. C. for 24 h. Colonies that were 
positive for both oligomer probes A and B or probes C. and D were picked, 
single colonies isolated and 5 ml mini-plasmid preparations prepared. 
Putative plasmid clones were sequenced using a double stranded sequencing 
protocol with Sequenase (Kraft et al., Biotechniques, 6, 544-546, (1988)) 
and sequencing primers corresponding to both ends of the vector pSV7186 
(Okayama, H. and Berg, P. Mol. Cell. Biol., 2, 161-170 (1982)). 
The sequences obtained encoded the prepropeptide sequences shown in Table I 
and FIGS. 2A and 2B below. 
##STR8## 
The above tables show the propeptides encoded by the KK0, KK1 and KK2 
sequences. It is notable that in the 51 amino acid excised N-terminal 
flanking region there is only one position which is different In all three 
sequences [Seq. ID No: 21, Seq. ID No: 22, and Seq. ID No: 23]; in 
contrast, except for cysteine residues, no residues are entirely conserved 
and 16 out of 20 positions have different residues in all three sequences 
in the peptide toxin region. The putative final processed peptides 
predicted form potential protease cleavage sites and stop signals are 
shown in Part B. 
OTHER EXAMPLES 
The cDNA sequencing of toxins representative of two other cysteine 
framework types have been determined. The circler toxin is found in the 
venom of Conus textile and has a 3 loop cysteine framework as shown in Row 
III in Table I. This framework is also seen in toxins which target 
skeletal muscle Na+ channels. When the sequence of the circler toxin 
"excised region" [Seq. ID No: 24] is compared to any of the 4 loop 
prepropeptides cited above, no significant sequence homology is noted. 
This is true even for toxins from the King Kong family which are derived 
from the same species as the circler toxin. Sequence comparisons for the 
acetylcholine receptor targeting toxin GI from Conus geographus have also 
been made. This toxin has 4 cysteine residues and forms a two loop 
cysteine framework as shown in Row IV of Table I. The "excised region" 
[Seq. ID No: 25] of the GI prepropeptide shows no significant sequence 
homology to any of the 3 or 4 loop conotoxin precursors. 
In summary, it can be seen that the methods and compositions of the above 
invention provide an advancement in the art of synthesizing small 
disulfide-rich peptides. In particular, the invention demonstrates that a 
segregated template sequence directs specific disulfide bond formation in 
mature peptides that have considerable diversity in amino acid sequence. 
The segregation of template and mature peptide sequences, and the ability 
of essentially conserved templates to direct specific disulfide bonding in 
diverse peptides, gives the invention more general applicability to 
synthesizing disulfide-rich peptides, particularly by cloning methods. 
Specific disulfide bonding can be directed even under conditions where 
sequences in the mature peptide are being varied. 
While certain representative embodiments of the invention have been 
described herein for purposes of illustration, the invention may be 
embodied in other specific forms without departing from its spirit or 
essential characteristics, and the described examples should not be 
considered restrictive, but only illustrative. The scope of the invention 
is therefore defined by the appended claims rather than by the foregoing 
examples. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 25 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 71 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for four-loop 
MVIIB Omega conotoxin from Conus magus. 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo-dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetLysLeuThrCysValValIleValAlaValLeuLeuLeuThrAla 
45-40-35-30 
Cys GlnLeuIleThrAlaAspAspSerArgGlyThrGlnLysHisArg 
25-20-15 
AlaLeuArgSerAspThrLysLeuSerMetSerThrArgCysLysGly 
10-51 
LysGlyAlaSerCysHisArgThrSerTyrAspCysCysThrGlySer 
51015 
CysAsnArgGlyLysCysGly 
20 25 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 amino acids 
(B) TYPE: amino acids 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: MVIIB Omega conotoxin. 
(C) IDENTIFICATION METHOD: Direct peptide 
sequencing of purified Conus magus venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CysLysGlyLysGlyAlaSerCysHisArgThrSerTyrAspCysCys 
51015 
ThrGlySerCysAsnArgGlyLysCys 
20 25 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 73 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for four-loop 
GVIA Omega conotoxin from Conus geographus. 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetLysLeuThrCysValValIleValAlaValLeuLeuLeuThrAla 
45-40-35-30 
CysGlnLeuIleThrAlaAspAspSerArgGly ThrGlnLysHisArg 
25-20-15 
AlaLeuGlySerThrThrGluLeuSerLeuSerThrArgCysLysSer 
10-5 1 
ProGlySerSerCysSerProThrSerTyrAsnCysCysArgSerCys 
51015 
AsnProTyrThrLysArgCysTyrGly 
2025 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: GVIA Omega conotoxin. 
(C) IDENTIFICATION METHOD: Direct peptide 
sequencing of purified Conus geographus 
venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CysLysSerProGlySerSerCysSerProThrSerTyrAsnCysCys 
151015 
ArgSerCysAsnProTyrThrLysArgCysTyr 
20 25 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 78 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for four loop 
King Kong (kk0) conotoxin from Conus textile. 
(C) IDENTIFICATION METHOD: Library was constructed 
using polyA selected mRNA transcripts purified 
from Conus textile venom duct tissue and cloned 
into the Okyama-Berg oligo-dT primed plasmid 
pSV7186. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla 
50-45 -40 
TrpThrPheAlaThrAlaAspAspProArgAsnGlyLeuGlyAsnSer 
35-30-25-20 
PheSerAsnAlaHisHisGluMetLysAsnProGluAlaSerLysLe u 
15-10-5 
AsnLysArgTrpCysLysGlnSerGlyGluMetCysAsnLeuLeuAsp 
1510 
GlnAsnCysCysA spGlyTyrCysIleValLeuValCysThr 
152025 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: King Kong (kk0) conotoxin 
(C) IDENTIFICATION METHOD: Direct peptide sequencing 
of purified Conus textile venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TrpCysLysGlnSerGlyGluMetCysAsnLeuLeuAspGlnAsnCys 
151015 
CysAspGlyTyrCysIleValLeuValCysThr 
2025 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 77 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for four loop 
King Kong (kk1) conotoxin from Conus textile. 
(C) IDENTIFICATION METHOD: Library was constructed 
using polyA selected mRNA transcripts purified 
from Conus textile venom duct tissue and cloned 
into the Okyama-Berg oligo-dT primed plasmid 
pSV7186. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetLysLeuThrCysMetMetIleValA laValLeuPheLeuThrAla 
50-45- 40 
TrpThrPheAlaThrAlaAspAspSerGlyAsnGlyLeuGluAsnSer 
35-30-25 -20 
PheSerLysAlaHisHisGluMetLysAsnProGluAlaSerLysLeu 
15-10-5 
AsnLysArgCysIleGluGlnPheAspProCysGluMetIleArgHis 
1510 
ThrCysCysValGlyValCysPheLeuMetAlaCysIle 
152025 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: King Kong (kk1) conotoxin. 
(B) IDENTIFICATION METHOD: Direct peptide sequencing 
of purified Conus textile venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CysIleGluGlnPheAspProCysGluMetIleArgHisThrCy sCys 
151015 
ValGlyValCysPheLeuMetAlaCysIle 
2025 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 77 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for four loop 
King Kong (kk2) conotoxin from Conus textile. 
(C) IDENTIFICATION METHOD: Library was constructed 
using polyA selected mRNA transcripts purified 
from Conus textile venom duct tissue and cloned 
into the Okyama-Berg oligo-dT primed plasmid 
pSV7186. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla 
50-45- 40 
TrpThrPheValThrAlaAspAspSerGlyAsnGlyLeuGluAsn Ser 
35-30-25-20 
PheSerLysAlaHisHisGluMetLysAsnProGluAlaSerAsnLeu 
15-10-5 
As nLysArgCysAlaProPheLeuHisProCysThrPhePhePhePro 
1510 
ThrCysCysAsnSerTyrCysValGlnPheIleCysLeu 
1520 25 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: King Kong (kk2) conotoxin. 
(C) IDENTIFICATION METHOD: Direct peptide sequencing 
of purified Conus textile venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CysAlaProPheLeuHisProCysThrPhePhePheProThrCysCys 
151015 
AsnSerTyrCysValGlnPheIleCysLeu 
20 25 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 68 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for three loop 
conotoxin from Circler conotoxin from Conus 
textile 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetSerLysLeuGlyAlaLeuLeuThrIleCysLeuLeuLeuPheSer 
50-45-40 
LeuThrAlaValProLeuAspGlyAspGlnHis AlaAspGlnProAla 
35-30- 25 
GlnArgLeuGlnAspArgIleProThrGluAspHisProLeuPheAsp 
20-15-10-5 
ProAsnLysArgCysCysProProValAlaCysAsnMetGlyCysLys 
1510 
ProCysCysGly 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Circler conotoxin. 
(C) IDENTIFICATION METHOD: Direct peptide 
sequencing of purified Conus textile venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CysCysProProValAlaCysAsnMetGlyCysLysProCysCys 
1 51015 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 64 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Prepropeptide sequence for two loop GI 
conooxin from Conus geographus. 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
MetGlyMetArgMetMetPheThrValPheLeuLeuValValLeuAla 
45-40-35 
ThrTh rValValSerPheProSerGluArgAlaSerAspGlyArgAsp 
30-25-20 
AspThrAlaLysAspGluGlySerAspMetAspLysLeuValGluLys 
15 -10-5 
LysGluCysCysAsnProAlaCysGlyArgHisTyrSerCysGlyArg 
151015 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: GI Conotoxin 
(C) IDENTIFICATION METHOD: Direct peptide 
sequencing of purified Conus geographus venom 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GluCysCysAsnProAlaCysGlyArgHisTyrSerCys 
510 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(D) OTHER INFORMATION: In sequence R =A or G and 
Y =T or C 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CCRTCRCARCARTTYTGRTC20 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(D) OTHER INFORMATION: In sequence R =A or G, 
Y =T or C and N =ATG or C 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
TTRCACATYTCNCC14 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: amino acid sequence from King Kong KKO 
conotoxin 
(B) IDENTIFICATION METHOD: sequencer 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
AspGlnAsnCysCysAspGly 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: amino acid sequence from King Kong KKO 
conotoxin 
(B) IDENTIFICATION METHOD: sequencer 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GlyGluMetCysAsn 
5 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
four-loop MVIIB Omega conotoxin 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
MetLysLeuThrCysValValIleValAlaValLeuLeuLeuThrAla 
151015 
CysGlnLeuIleThrAlaAspAspSerArgGlyThrGlnLysHisArg 
202530 
AlaLeuArgSerAspThrLysLeuSerMetSerThrArg 
354045 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
four-loop GVIA Omega conotoxin 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
MetLysLeuThrCysValValI leValAlaValLeuLeuLeuThrAla 
151015 
CysGlnLeuIleThrAlaAspAspSerArgGlyThrGlnLysHisArg 
2025 30 
AlaLeuGlySerThrThrGluLeuSerLeuSerThrArg 
354045 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
four loop King Kong (kk0) conotoxin 
(C) IDENTIFICATION METHOD: Library was constructed 
using polyA selected mRNA transcripts purified 
from Conus textile venom duct tissue and cloned 
into the Okyama-Berg oligo-dT primed plasmid 
pSV7186. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla 
151015 
TrpThrPheAlaThrAlaAspAspProArgAsnGlyLeuGlyAsnSer 
202530 
PheSerAsnAlaHisHisGluMetLysAsnProGluAlaSerLysLeu 
354045 
AsnLysArg 
50 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
four loop King Kong (kk1) conotoxin 
(C) IDENTIFICATION METHOD: Library was constructed 
using polyA selected mRNA transcripts purified 
from Conus textile venom duct tissue and cloned 
into the Okyama-Berg oligo-dT primed plasmid 
pSV7186. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla 
151015 
TrpThr PheAlaThrAlaAspAspSerGlyAsnGlyLeuGluAsnSer 
202530 
PheSerLysAlaHisHisGluMetLysAsnProGluAlaSerLysLeu 
35 4045 
AsnLysArg 
50 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
four loop King Kong (kk2) conotoxin 
(C) IDENTIFICATION METHOD: Library was constructed 
using polyA selected mRNA transcripts purified 
from Conus textile venom duct tissue and cloned 
into the Okyama-Berg oligo-dT primed plasmid 
pSV7186. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla 
1 51015 
TrpThrPheValThrAlaAspAspSerGlyAsnGlyLeuGluAsnSer 
202530 
PheSerLysAlaHis HisGluMetLysAsnProGluAlaSerAsnLeu 
354045 
AsnLysArg 
50 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 52 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
three loop conotoxin from Circler conotoxin 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
MetSerLysLeuGlyAlaLeuLeuThrIleCysLeuLeuLeuPheSer 
1 51015 
LeuThrAlaValProLeuAspGlyAspGlnHisAlaAspGlnProAla 
202530 
GlnArgLeuGlnAspA rgIleProThrGluAspHisProLeuPheAsp 
354045 
ProAsnLysArg 
50 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Signal/Pro sequence for synthesis of 
two loop GI conooxin 
(C) IDENTIFICATION METHOD: Libraries were created 
using oligo- dT primed pUC13 vector 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
MetGlyMetArgMetMetPheThrValPheLeuLeuValValLeuAla 
1 51015 
ThrThrValValSerPheProSerGluArgAlaSerAspGlyArgAsp 
202530 
AspThrAlaLysAsp GluGlySerAspMetAspLysLeuValGluLys 
354055 
Lys