Subtilisin variants capable of cleaving substrates containing dibasic residues

The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing dibasic residues. A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates. Suitable substrates of the variant subtilisin were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues. This method identified a particularly good substrate, Asn-Leu-Met-Arg-Lys-, that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin variant. Accordingly, this variant subtilisin may be useful for cleaving fusion proteins with dibasic substrate linkers and processing hormones or other proteins (in vitro or in vivo) that contain dibasic cleavage sites.

FIELD OF THE INVENTION 
This invention relates to subtilisin variants having altered specificity 
from wild-type subtilisin useful for processing fusion proteins, 
especially those made in recombinant cell culture. Specifically, the 
subtilisin variants are modified so that they efficiently and selectively 
cleave substrates containing dibasic residues. 
BACKGROUND OF THE INVENTION 
Site-specific proteolysis is one of the most common forms of 
post-translational modifications of proteins (for review see Neurath, H. 
1989! Trends Biochem. Sci., 14:268). In addition, proteolysis of fusion 
proteins in vitro is an important research and commercial tool (for 
reviews see Uhlen, M. and Moks, T. 1990! Methods Enzymol., 185:129-143; 
Carter, P. 1990! in Protein Purification: From Molecular Mechanisms to 
Large-Scale Processes, M. R. Landisch, R. C. Wilson, C. D. Painton, S. E. 
Builder, Eds. ACS Symposium Series 427, American Chemical Society, 
Washington, D.C.!, Chap. 13, p.181-193; and Nilsson, B. et al. 1992! 
Current Opin. Struct. Biol., 2:569). Expressing a protein of interest as a 
fusion protein facilitates purification when the fusion contains an 
affinity domain such as glutathione-S-transferase, Protein A or a 
poly-histidine tail. The fusion domain can also facilitate high level 
expression and/or secretion. 
To liberate the protein product from the fusion domain requires selective 
and efficient cleavage of the fusion protein. Both chemical and enzymatic 
methods have been proposed (see references above). Enzymatic methods are 
generally preferred as they tend to be more specific and can be performed 
under mild conditions that avoid denaturation or unwanted chemical 
side-reactions. A number of natural and even designed enzymes have been 
applied for site-specific proteolysis. Although some are generally more 
useful than others (Forsberg, G., Baastrup, B., Rondahl, H., Holmgren, E., 
Pohl, G., Hartmanis, M. and Lake, M. 1992! J. Prot. Chem., 11:201-211), 
no one is applicable to every situation given the sequence requirements of 
the fusion protein junction and the possible existence of protease 
sequences within the desired protein product. Thus, an expanded array of 
sequence specific proteases, analogous to restriction endonucleases, would 
make site-specific proteolysis a more widely used method for processing 
fusion proteins or generating protein/peptide fragments either in vitro or 
in vivo. 
One of the most popular site-specific proteolysis events is the maturation 
of pro-hormones by the KEX2-family of enzymes that are present in 
eukaryotic cells (for reviews see Steiner, D. F., Smeekens, S. P., Ohagi, 
S. and Chan, S. J. 1992! J. Biol. Chem., 267:23435-23438 and Smeekens, S. 
P. 1993! Bio/Technology, 11:182-186). This family of proteases, that 
includes the yeast KEX2 and the mammalian PC2 and furin enzymes, are 
homologous to the bacterial serine protease subtilisin (Kraut, J. 1977! 
Annu. Rev. Biochem. . . , 46:331-358). Subtilisin has a broad substrate 
specificity that reflects its role as a scavenger protease. In contrast, 
these eukaryotic enzymes are very specific for cleaving substrates 
containing two basic residues and thus well-suited for site-specific 
proteolysis. However, the eukaryotic proteases are expressed in small 
amounts (Bravo, D. B., Gleason, J. B., Sanchez, R. I., Roth, R. A., and 
Fuller, R. S. 1994! J. Biol. Chem., 269:25830-25837 and Matthews, D. J., 
Goodman, L. J., Gorman, C. M., and Wells, J. A. 1994! Protein Science, 
3:1197-1205) making them impractical to apply presently to processing of 
fusion proteins in vitro. 
Despite the very narrow specificity of the pro-hormone processing enzymes, 
in some cases they are capable of rapid cleavage of target sequences. For 
example, the k.sub.cat /Km ratio for KEX2 to cleave a good substrate (e.g. 
Boc-RVRR-MCA) is 1.times.10.sup.6 M.sup.-1 s.sup.-1 (Brenner, C., and 
Fuller, R. S. 1992! Proc. Natl. Acad. Sci. USA, 89:922-926) compared to 
3.times.10.sup.5 for subtilisin cleaving a good substrate (e.g. 
suc-AAPF-pna) (Estell, D. A., Graycar, T. P., Miller, J. V., Powers, D. 
B., Burnier, J. P., Ng, P. G. and Wells, J. A. 1986! Science, 
233:659-663). Given the fact that subtilisin BPN' can be expressed in 
large amounts (Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A. and 
Chen, E. Y. 1983! Nucl. Acids Res., 11:7911-7929) we wondered if it would 
be possible to engineer the specificity of subtilisin to be like that of 
KEX2, to produce a useful subtilisin variant for processing fusion 
proteins or generating protein fragments by cleavage at designed dibasic 
sites. 
Previous attempts to introduce or reverse charge specificity in enzyme 
active sites have been met with considerable difficulty. This has 
generally been attributed to a lack of stabilization of the introduced 
charge or enzyme-substrate ion pair complex by the wild-type enzyme 
environment (Hwang, J. K. and Warshel, A. 1988! Nature, 334:270-272). For 
example, Stennicke et. al (Stennicke, H. R.; Ujje, H. M.; Christensen, U.; 
Remington, S. J.; and Breddam 1994! Prot. Eng. 7:911-916) made acidic 
(D/E) mutations at five residues in the P1' binding of carboxypeptidase Y 
in an attempt to change the P1' preference from Phe to Lys/Arg. Only the 
L272D and L272E mutations were found to alter the specificity in the 
desired direction, up to 1.5-fold preference in Lys/Arg over Phe, and the 
others simply resulted in less active enzymes having substrate preferences 
similar to wild-type. In the case of trypsin, a protease that is highly 
specific for basic P1 residues, recruitment of chymotrypsin-like 
(hydrophobic P1) specificity required not only mutations of the ion 
pair-forming Asp 189 to Ser, but also transplantation of two more distant 
surface loops from chymotrypsin (Graf, L., Jancso, A., Szilagyi, L., 
Hegyi, G., Pinter, K., Naray-Szabo, G., Hepp, J., Medzihradszky, K., and 
Rutter, W. J., Proc. Natl. Acad. Sci. USA 1988! 85:4961-4965 and 
Hedstrom, L., Szilagyi, L., and Rutter, W. J., Science 1992! 
255:1249-1253). 
In the present work, we have also verified that relatively low specificity 
is gained by introducing single ion-pairs between enzyme and substrate. 
However, when two choice ionic interactions were simultaneously engineered 
into subtflisin BPN', the resulting variant had higher specificity for 
basic residues in each of the subsites due to a non additive effect. 
Accordingly, it is an object to produce a subtilisin variant with dibasic 
specificity for use in processing pro-proteins made by recombinant 
techniques. 
SUMMARY OF THE INVENTION 
The invention includes subtilisin variants, having a substrate specificity 
which is substantially different from the substrate specificity of the 
precursor subtilisin from which the amino acid sequence of the mutant is 
derived. The substrate specificity of the preferred subtilisin variants is 
for substrates having dibasic amino acid residues. The preferred precursor 
subtilisin is subtilisin from Bacillus amyloliquefaciens, referred to as 
subtilisin BPN'. The amino acid sequence of the subtilisin variants are 
derived by the substitution of one or more amino acids of the precursor 
subtilisin amino acid sequence. The preferred subtilisin variants having 
substrate specificity for dibasic substrates have a different amino acid 
residue at residue position +62 than subtilisin naturally produced by 
Bacillus amyloliquefaciens. The naturally occurring Asn (N) at residue 
position +62 of subtilisin BPN' is preferably substituted with an acidic 
amino acid residue such as Glu (E) or Asp (D), most preferably D. The most 
preferred subtilisin variants, having substrate specificity for substrates 
having dibasic amino acid residues, additionally have an acidic residue, E 
or D, at residue position +62 of subtilisin BPN'. Thus the subtilisin BPN' 
variant N62D/G166D may be used to cleave fusion proteins with dibasic 
substrate linkers and processing hormones or other proteins (in vitro or 
in vivo) that contain dibasic cleavage sites. 
Preferred substrates for the subtilisin BPN' variant N62D/G166D contain 
either Lys (K) or Arg (R) at substrate positions P2 and P1, practically 
any residue at P3, a non-charged hydrophobic residue at P4, and again 
practically any residue at P5. Thus an exemplary good substrate would 
contain -Asn-Leu-Met-Arg-Lys- (SEQ ID NO: 35) at -P5-P4-P3-P2-P1- 
respectively. Additionally, good substrates would not have Pro at P1', 
P2', or P3' nor would Ile be present at P1'. Thus the invention includes a 
process comprising contacting the subtilisin variant having substrate 
specificity for dibasic amino acid residues with a substrate containing 
the above described amino acid residues under conditions. 
The invention also includes mutant DNA sequences encoding such subtilisin 
variants. These mutant DNA sequences are derived from a precursor DNA 
sequence which encodes a naturally occurring or recombinant precursor 
subtilisin. The mutant DNA sequence is derived by modifying the precursor 
DNA sequence to encode the substitution(s) of one or more amino acids 
encoded by the precursor DNA sequence. These recombinant DNA sequences 
encode mutants having an amino acid sequence which does not exist in 
nature and a substrate specificity which is substantially different from 
the substrate specificity of the precursor subtilisin encoded by the 
precursor DNA sequence. 
Further the invention includes expression vectors containing such mutant 
DNA sequences as well as host cells transformed with such vectors which 
are capable of expressing the subtilisin variants.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
Terms used in the claims and specification are defined as set forth below 
unless otherwise specified. 
The term amino acid or amino acid residue, as used herein, refers to 
naturally-occurring L .alpha.-arnino acids or residues, unless otherwise 
specifically indicated. The commonly used one- and three-letter 
abbreviations for amino acids are use herein (Lehninger, A. L., 
Biochemistry, 2d ed., pp. 71-92, Worth Publishers, N.Y. 1975!). 
Substrates are described in triplet or single letter code as Pn . . . 
P2-P1-P1'-P2'. . . Pn'. The "P.sub.1 " residue refers to the position 
proceeding the scissile peptide bond (i.e. between the P1 and P1' 
residues) of the substrate as defined by Schechter and Berger (Schechter, 
I. and Berger, A., Biochem. Biophys. Res. Commun. 27: 157-162 1967!). 
"Subtilisins" are bacterial carbonyl hydrolases which generally act to 
cleave peptide bonds of proteins or peptides. As used herein, "subtilisin" 
means a naturally occurring subtilisin or a recombinant subtilisin. A 
series of naturally occurring subtilisins are known to be produced and 
often secreted by various bacterial species. Amino acid sequences of the 
members of this series are not entirely homologous. However, the 
subtilisins in this series exhibit the same or similar type of proteolytic 
activity. This class of serine proteases shares a common amino acid 
sequence defining a catalytic triad which distinguishes them from the 
chymotrypsin related class of serine proteases. The subtilisins and 
chymotrypsin related serine proteases both have a catalytic triad 
comprising aspartate, histidine and serine. In the subtilisin related 
proteases the relative order of these amino acids, reading from the amino 
to carboxy terminus is aspartate-histidine-serine. In the chymotrypsin 
related proteases the relative order, however is 
histidine-aspartate-serine. Thus, subtilisins as used herein refer to a 
serine protease having the catalytic triad of subtilisin related 
proteases. 
Generally, subtilisins are serine endoproteases' having molecular weights 
of about 27,500 which are secreted in large amounts from a wide variety of 
Bacillus species. The protein sequence of subtilisins have been determined 
from at least four different species of Bacillus. Markland, F. S., et al. 
(1971) in The Enzymes, ed. Boyer P. D., Acad Press, New York, Vol. III, 
pp. 561-608 and Nedkov, P. et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 
364:1537-1540. The three-dimensional crystallographic structure of 
subtilisin BPN' (from B. amyloliquefaciens) to 2.5A.sub.-- resolution has 
also been reported by Wright, C. S. et al. 1969! Nature 221:235-242 and 
Drenth, J. et al. 1972! Eur. J. Biochem. 26:177-181. These studies 
indicate that although subtilisin is genetically unrelated to the 
mammalian serine proteases, it has a similar active site structure. The 
x-ray crystal structures of subtilisin containing covalently bound peptide 
inhibitors (Robertus, J. D., et al. 1972! Biochemistry 11:2439-2449), 
product complexes (Robertus, J. D., et al. 1972! Biochemistry 
11:4293-4303), and transition state analogs (Matthews, D. A., et al. 
1975! J. Biol. Chem. 250:7120-7126 and Poulos, T. L., et al. 1976! J. 
Biol. Chem. 251:1097-1103), which have been reported have also provided 
information regarding the active site and putative substrate binding cleft 
of subtilisins. In addition, a large number of kinetic and chemical 
modification studies have been reported for subtilisins (Phillip, M., et 
al. 1983! Mol. Cell. Biochem. 51:5-32; Svendsen, I. B. 1976! Carlsberg 
Res. Comm. 41:237-291 and Markland, F. S. Id.) as well as at least one 
report wherein the side chain of methione at residue 222 of subtilisin was 
converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., 
et al. 1965! J. Biol. Chem. 244:5333-5338). 
"Recombinant subtilisin" refers to a subtilisin in which the DNA sequence 
encoding the subtilisin is modified to produce a mutant DNA sequence which 
encodes the substitution of one or more amino acids in the naturally 
occurring subtilisin amino acid sequence. Suitable methods to produce such 
modification include those disclosed in U. S. Pat. Nos. 4,760.025 and 
5,371,008 and in EPO Publication No. 0130756 and 0251446. 
When referring to mutants or variants, the wild type amino acid residue is 
30 followed by the residue number and the new or substituted amino acid 
residue. For example, substitution of D for wild type N in residue 
position 62 is denominated N62D. 
"Subtilisin variants or mutants" are designated in the same manner by using 
the single letter amino acid code for the wild-type residue followed by 
its position and the single letter amino acid code of the replacement 
residue. Multiple mutants are indicated by component single mutants 
separated by slashes. Thus the subtilisin BPN' variant N62D/G166D is a 
di-substituted variant in which Asp replaces Asn and Gly at residue 
positions 62 and 166 in wild-type subtilisin BPN' . 
Specific residues of B. amyloliquefaciens subtilisin are identified for 
substitution. These amino acid residue position numbers refer to those 
assigned to the B. amyloliquefaciens subtilisin sequence (SEQ ID NO: 74) 
see the mature sequence in FIG. 1. of U.S. Pat. No. 4,760,025). The 
invention, however, is not limited to the mutation of this particular 
subtilisin but extends to precursor carbonyl hydrolases containing amino 
acid residues which are "equivalent" to the particular identified residues 
in B. amyloliquefaciens subtilisin. An amino acid residue of a precursor 
carbonyl hydrolase is "equivalent" to a residue of B. amyloliquefaciens 
subtilisin if it is either homologous (i.e., corresponding in position in 
either primary or tertiary structure) or analogous to a specific residue 
or portion of that residue in B. amyloliquefaciens subtilisin (i.e., 
having the same or similar functional capacity to combine, react, or 
interact chemically). 
In order to establish homology to primary structure, the amino acid 
sequence of a precursor carbonyl hydrolase is directly compared to the B. 
amyloliquefaciens subtilisin primary sequence and particularly to a set of 
residues known to be invariant in all subtilisins for which the sequences 
are known (see e.g. FIG. 5-C in EPO 0251446). After aligning the conserved 
residues, allowing for necessary insertions and deletions in order to 
maintain alignment (i.e., avoiding the elimination of conserved residues 
through arbitrary deletion and insertion), the residues equivalent to 
particular amino acids in the primary sequence of B. amyloliquefaciens 
subtilisin are defined. Alignment of conserved residues should conserve 
100% of such residues. However, alignment of greater than 75% or as little 
as 50% of conserved residues is also adequate to define equivalent 
residues. Conservation of the catalytic triad, Asp32/His64/Ser221, is 
required. 
Equivalent residues homologous at the level of tertiary structure for a 
precursor carbonyl hydrolase whose tertiary structure has been determined 
by x-ray crystallography, are defined as those for which the atomic 
coordinates of 2 or more of the main chain atoms of a particular amino 
acid residue of the precursor carbonyl hydrolase and B. amyloliquefaciens 
subtilisin (N on N, CA on CA, C on C, and O on O) are within 0.13 nm and 
preferably 0.1 nm after alignment. Alignment is achieved after the best 
model has been oriented and positioned to give the maximum overlap of 
atomic coordinates of non-hydrogen protein atoms of the carbonyl hydrolase 
in question to the B. amyloliquefaciens subtilisin. The best model is the 
crystallographic model giving the lowest R factor for experimental 
diffraction data at the highest resolution available. 
##EQU1## 
Equivalent residues which are functionally analogous to a specific residue 
of B. amyloliquefaciens subtilisin are defined as those amino acids of the 
precursor carbonyl hydrolases which may adopt a conformation such that 
they either alter, modify or contribute to protein structure, substrate 
binding or catalysis in a manner defined and attributed to a specific 
residue of the B. amyloliquefaciens subtilisin as described herein. 
Further, they are those residues of the precursor carbonyl hydrolase (for 
which a tertiary structure has been obtained by x-ray crystallography), 
which occupy an analogous position to the extent that although the main 
chain atoms of the given residue may not satisfy the criteria of 
equivalence on the basis of occupying a homologous position, the atomic 
coordinates of at least two of the side chain atoms of the residue lie 
with 0.13 nm of the corresponding side chain atoms of B. amyloliquefaciens 
subtilisin. The three dimensional structures would be aligned as outlined 
above. 
Some of the residues. identified for substitution are conserved residues 
whereas others are not. In the case of residues which are not conserved, 
the replacement of one or more amino acids is limited to substitutions 
which produce a mutant which has an amino acid sequence that does not 
correspond to one found in nature. In the case of conserved residues, such 
replacements should not result in a naturally occurring sequence. The 
subtilisin mutants of the present invention include the mature forms of 
subtilisin mutants as well as the pro- and prepro-forms of such subtilisin 
mutants. The prepro-forms are the preferred construction since this 
facilitates the expression, secretion and maturation of the subtilisin 
mutants. 
"Prosequence" refers to a sequence of amino acids bound to the N-terminal 
portion of the mature form of a subtilisin which when removed results in 
the appearance of the "mature" form of the subtilisin. Many proteolytic 
enzymes are found in nature as translational proenzyme products and, in 
the absence of post-translational processing, are expressed in this 
fashion. The preferred prosequence for producing subtilisin mutants, 
specifically subtilisin BPN' mutants, is the putative prosequence of B. 
amyloliquefaciens subtilisin although other subtilisin prosequences may be 
used. 
A "signal sequence" or "presequence" refers to any sequence of amino acids 
bound to the N-terminal portion of a subtilisin or to the N-terminal 
portion of a prosubtilisin which may participate in the secretion of the 
mature or pro forms of the subtilisin. This definition of signal sequence 
is a functional one, meant to include all those amino acid sequences, 
encoded by the N-terminal portion of the subtilisin gene or other 
secretable carbonyl hydrolases, which participate in the effectuation of 
the secretion of subtilisin or other carbonyl hydrolases under native 
conditions. The present invention utilizes such sequences to effect the 
secretion of the subtilisin mutants as defined herein. 
A "prepro" form of a subtilisin mutant consists of the mature form of the 
subtilisin having a prosequence operably linked to the amino-terminus of 
the subtilisin and a "pre" or "signal" sequence operably linked to the 
amino terminus of the prosequence. 
"Expression vector" refers to a DNA construct containing a DNA sequence 
which is operably linked to a suitable control sequence capable of 
effecting the expression of the DNA in a suitable host. Such control 
sequences include a promoter to effect transcription, an optional operator 
sequence to control such transcription, a sequence encoding suitable niRNA 
ribosome binding sites, and sequences which control termination of 
transcription and translation. The vector may be a plasmid, a phage 
particle, or simply a potential genomic insert. Once transformed into a 
suitable host, the vector may replicate and function independently of the 
host genome, or may, in some instances, integrate into the genome itself. 
In the present specification, "plasmid" and "vector" are sometimes used 
interchangeably as the plasmid is the most commonly used form of vector at 
present. However, the invention is intended to include such other forms of 
expression vectors which serve equivalent functions and which are, or 
become, known in the art. 
The "host cells" used in the present invention generally are procaryotic or 
eucaryotic hosts which preferably have been manipulated by the methods 
disclosed in EPO Publication No. 0130756 or 0251446 or U.S. Pat. No. 
5,371,008 to render them incapable of secreting enzymatically active 
endoprotease. A preferred host cell for expressing subtilisin is the 
Bacillus strain BG2036 which is deficient in enzymatically active neutral 
protease and alkaline protease (subtilisin). The construction of strain 
BG2036 is described in detail in EPO Publication No. 0130756 and further 
described by Yang, M. Y., et al. (1984) J. Bacteriol. 160:15-21. Such host 
cells are distinguishable from those disclosed in PCT Publication No. 
03949 wherein enzymatically inactive mutants of intracellular proteases in 
E. coli are disclosed. Other host cells for expressing subtilisin include 
Bacillus subtilis var. I168 (EPO Publication No. 0130756). 
Host cells are transformed or transfected with vectors constructed using 
recombinant DNA techniques. Such transformed host cells are capable of 
either replicating vectors encoding the subtilisin mutants or expressing 
the desired subtilisin mutant. In the case of vectors which encode the pre 
or prepro form of the subtilisin mutant, such mutants, when expressed, are 
typically secreted from the host cell into the host cell medium. 
"Operably linked" when describing the relationship between two DNA regions 
simply means that they are functionally related to each other. For 
example, a presequence is operably linked to a peptide if it functions as 
a signal sequence, participating in the secretion of the mature form of 
the protein most probably involving cleavage of the signal sequence. A 
promoter is operably linked to a coding sequence if it controls the 
transcription of the sequence; a ribosome binding site is operably linked 
to a coding sequence if it is positioned so as to permit translation. 
The genes encoding the naturally-occurring precursor subtilisin may be 
obtained in accord with the general methods described in U.S. Pat. No. 
4,760,025 or EPO Publication No. 0130756. As can be seen from the examples 
disclosed therein, the methods generally comprise synthesizing labeled 
probes having putative sequences encoding regions of the hydrolase of 
interest, preparing genomic libraries from organisms expressing the 
hydrolase, and screening the libraries for the gene of interest by 
hybridization to the probes. Positively hybridizing clones are then mapped 
and sequenced. 
The cloned subtilisin is then used to transform a host cell in order to 
express the subtilisin. The subtilisin gene is then ligated into a high 
copy number plasmid. This plasmid replicates in hosts in the sense that it 
contains the well-known elements necessary for plasmid replication: a 
promoter operably linked to the gene in question (which may be supplied as 
the gene's own homologous promotor if it is recognized, i.e., transcribed, 
by the host), a transcription termination and polyadenylation region 
(necessary for stability of the mRNA transcribed by the host from the 
hydrolase gene in certain eucaryotic host cells) which is exogenous or is 
supplied by the endogenous terminator region of the subtilisin gene and, 
desirably, a selection gene such as an antibiotic resistance gene that 
enables continuous cultural maintenance of plasmid-infected host cells by 
growth in antibiotic-containing media. High copy number plasmids also 
contain an origin of replication for the host, thereby enabling large 
numbers of plasmids to be generated in the cytoplasm without chromosomal 
limitations. However, it is within the scope herein to integrate multiple 
copies of the subtilisin gene into host genome. This is facilitated by 
procaryotic and eucaryotic organisms which are particularly susceptible to 
homologous recombination. 
Once the subtilisin gene has been cloned, a number of modifications are 
undertaken to enhance the use of the gene beyond synthesis of the 
naturally-occurring precursor subtilisin. Such modifications include the 
production of recombinant subtilisin as disclosed in U.S. Pat. No. 
5,371,008 or EPO Publication No. 0130756 and the production of subtilisin 
mutants described herein. 
Mutant Design and Preparation. 
A number of structures have been solved of subtilisin with a variety of 
inhibitors and transition state analogs bound (Wright, C. S., Alden, R. A. 
and Kraut, J. 1969! Nature, 221:235-242; McPhalen, C.A. and James, N.G. 
1988! Biochemistry, 27:6582-6598; Bode, W., Papamokos, E., Musil, D., 
Seemueller, U. and Fritz, M. 1986! EMBO J., 5:813-818; and Bott, R., 
Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B. and Power, S. 1988! J. 
Biol. Chem., 263:7895-7906). One of these structures, FIG. 1 was used to 
locate residues that are in close proximity to side chains at the P1 and 
P2 positions from the substrate. Previous work had shown that replacement 
residues at positions 156 and 166 in the S1 binding site with various 
charged residues lead to improved specificity for complementary charged 
substrates (Wells, J. A., Powers, D. B., Bott, R. R., Graycar, T. P. and 
Estell, D. A. 1987a! Proc. Natl. Acad. Sci. USA, 84:1219-1223). Although 
longer range electrostatic effects of substrate specificity have been 
noted (Russell, A. J. and Fersht, A. R. 1987! Nature, 328:496-500) these 
were generally much smaller than local ones. Therefore it seemed 
reasonable that local differences in charge between subtilisin BPN' and 
the eukaryotic enzymes may account for the differences in specificity. 
A detailed sequence alignment of 35 different subtilisins (Siezen, R. J., 
de Vos, W. M., Leunissen, A. M., and Dijkstra, B. W. 1991! Prot. Eng., 
4:719-737) allowed us to identify differences between subtilisin BPN' and 
the eukaryotic processing enzymes, KEX2, furin and PC2. Within the S1 
binding pocket there are a number of charged residues that appear in the 
pro-hormone processing enzymes and not in subtilisin BPN' (Table 1A). 
TABLE 1A 
______________________________________ 
S1 subsite 
125-131.sup.a 151-157 163-168 
______________________________________ 
Subtilisin 
SLGGPSG AAAGNEG ST-VGYP 
BPN' (SEQ ID NO: 3) 
(SEQ ID NO: 4) 
(SEQ ID NO: 5) 
Kex2 SWGPADD FASGNGG CNYDGYT 
(SEQ ID NO: 6) 
(SEQ ID NO: 7) 
(SEQ ID NO: 8) 
Furin SWGPEDD WASGNGG CNCDGYT 
(SEQ ID NO: 9) 
(SEQ ID NO: 10) 
(SEQ ID NO: 11) 
PC2 SWGPADD WASGDGG CNCDGYA 
(SEQ ID NO: 6) 
(SEQ ID NO: 12) 
(SEQ ID NO: 13) 
______________________________________ 
.sup.a numbering according to subtilisin BPN.sup.1 sequence 
For example, the eukaryotic enzymes have two conserved Asp residues at 130 
and 131 as well as an Asp at 165 that is preceded by insertion of a Tyr or 
Cys. However, in the region from 151-157, subtilisin BPN' contains a Glu 
and the eukaryotes a conserved Gly. 
In the S2 binding site there were two notable differences in sequence 
(Table 1B). 
TABLE 1B 
______________________________________ 
S2 subsite 
30-35 60-64 
______________________________________ 
Subtilisin VIDSGI DNNSH 
BPN' (SEQ ID NO: 14) 
(SEQ ID NO: 15) 
KEX2 IVDDGL SDDYH 
(SEQ ID NO: 16) 
(SEQ ID NO: 17) 
Furin ILDDGI NDNRH 
(SEQ ID NO: 18) 
(SEQ ID NO: 19) 
PC2 IMDDGI WFNSH 
(SEQ ID NO: 20) 
(SEQ ID NO: 21) 
______________________________________ 
Subtilisin contains a Ser at position 33 whereas the pro-hormone processing 
enzymes contain Asp. There is not as clear a consensus in the region of 
60-64, but one notable difference is at position 62. This side chain 
points directly at the P2 side chain (FIG. 1) and is Asn in subtilisin 
BPN', furin and PC2 but Asp in KEX2. Thus, not all substitutions were 
clearly predictive of the specificity differences. 
A variety of mutants were produced to probe and engineer the specificity of 
subtilisin BPN' using oligonucleotides described in Table 2. 
TABLE 2 
__________________________________________________________________________ 
Oligonucleotides used for site-directed mutagenesis on subtilisin. 
Specificity 
Activity 
Mutant Oligonucleotide Pocket 
Expressed 
__________________________________________________________________________ 
S33D 5'-GCGGTTATCGACG*A*CGGTATCGATTCT -3' 
S2 + 
(SEQ ID NO: 22) 
S33K 5'-GCGGTTATCGACAA*A*G*GTATCGATTCT -3' 
S2 + 
(SEQ ID NO: 23) 
S33E 5'-GCGGTTATCGACG*A*A*GGTATCGATTCT -3' 
S2 + 
(SEQ ID NO: 24) 
N62D 5'-CCAAGACAACG*ACTCTCACGGAA -3' 
S2 + 
(SEQ ID NO: 25) 
N62S 5'-CCAAGACAACAG*CTCTCACGGAA -3' 
S2 + 
(SEQ ID NO: 26) 
N62K 5'-CCAAGACAACAAA*TCTCACGGAA -3' 
S2 + 
(SEQ ID NO: 27) 
G166D 5'- S1 + 
CACTTCCGGCAGCTCG*T*C*G*ACAGTGGA*C*T 
ACCCTGGC.AAATA-3' 
(SEQ ID NO: 28) (lnserts Sal I site) 
G166E 5'- S1 + 
CACTTCCGGCAGCTCG*T*C*G*ACAGTGGA*GT 
ACCCTGGCAAATA-3' 
(SEQ ID NO: 29) (Inserts Sal I site) 
G128P/P129A 
5'- S1 - 
TTAACATGAGCCTCGGCC*C*AG*CTA*G*C*GGT 
TCTGCTGCTTTA -3' 
(SEQ ID NO: 30) (Inserts Nhe I site) 
G128P/P129A/ 
5'- S1 - 
S130D/G131D 
TTAACATGAGCCTCGGCC*C*C*G*CGG*A*TGA* 
TTCTGCTGCTTTAAA -3' 
(SEQ ID NO: 31) (Inserts Sac II site) 
T164N/V165D 
5'- S1 - 
CGGCAGCTCAAGCA*A*C*G*A*T*GGCTAT*CCT 
GGCAAATACCCTTCTGTCA -3' 
(SEQ ID NO: 32) (Inserts BsaBI site) 
T164Y/V165D 
5'- S1 - 
CGGCAGCTCAAGCA*A*G*G*A*T*GGCTAT*CCT 
GGCAAATACCCTTCTGTCA -3' 
(SEQ ID NO: 32) (lnserts BsaBI site) 
T164N- 5'- S1 - 
Y(insert)- 
ACTTCCGGCAGCTCT*T*C*G*AA*C*T*A*C*G*A 
V165D *C*GGGTACCCTGGCAAATA-3' 
(SEQ ID NO: 33) (Inserts BstBI site) 
N62D/G166D 
See individual mutations 
S1/S2 
+ 
N62D/G166E 
See individual mutations 
S1/S2 
+ 
__________________________________________________________________________ 
*Asterisks indicate base changes from the pSSb (wildtype) template. 
After producing the mutant plasmids they were transformed into a protease 
deficient strain of B. subtilis (BG2036) that lacks an endogenous gene for 
secretion of subtilisin. These were then tested for protease activity on 
skim milk plates. 
The first set of mutants tested were ones where segments of the S1 binding 
site were replaced with sequences from KEX2. None of these segment 
replacements produced detectable activity on skim milk plates even though 
variants of subtilisin whose catalytic efficiencies are reduced by as much 
as 1000-fold do produce detectable halos (Wells, J. A., Cunningham, B. C., 
Graycar, T. P. and Estell, D. A. (1986) Philos. Trans. R. Soc. Lond. A. 
317.415423). We went on to produce single residue substitutions that 
should have less impact on the stability. These mutants at positions 166 
in the S1 site, and 33 and 62 in the S2 site, were chosen based on the 
modeling and sequence considerations described above. Fortunately all 
single mutants as well as combination mutants produced activity on skim 
milk plates and could be purified to homogeneity. 
Kinetic Analysis of Variant Subtilisins. 
To probe the effects of the G166E and G166D on specificity at the P1 
position we used substrates having the form suc-AAPX-pna (SEQ ID NO: 69) 
where X was either Lys (SEQ ID NO: 58), Arg (SEQ ID NO: 59), Phe (SEQ ID 
NO: 56), Met (SEQ ID NO: 60) or Gln (SEQ ID NO: 61). The k.sub.cat /Km 
values were determined from initial rate measurements and results reported 
in FIG. 2. Whereas the wild-type enzyme preferred Phe&gt;Met&gt;Lys&gt;Arg&gt;Gln, the 
G166E preferred Lys.about.Phe&gt;Arg.about.Met&gt;Gln, and G166D preferred 
Lys&gt;Phe.about.Arg.about.Met&gt;Gln. Thus, both the acidic substitutions at 
position 166 caused a shift in preference for basic residues as previously 
reported (Wells, J. A., Powers, D. B., Bott, R. R., Graycar, T. P.and 
Estell, D. A. (1987a), Proc. Natl. Acad. Sci. USA 84:1219-1223). 
The effects of single and double substitutions in the S2 binding site were 
analyzed with substrates having the form, suc-Ala-Ala-X-Phe-pna and are 
shown in FIG. 3. At the P2 position the wild-type enzyme preferred 
Ala&gt;Pro&gt;Lys&gt;Arg&gt;Asp. In contrast, the S33D preferred 
Ala&gt;Lys.about.Arg.about.Pro&gt;Asp and the N62D preferred 
Lys&gt;Ala&gt;Arg&gt;Pro&gt;Asp. Although the effects were most dramatic for the N62D 
mutant, the S33D variant also showed significant improvement toward basic 
P2 residues and corresponding reduction in hydrolysis of the Ala and Asp 
P2 substrates. We then analyzed the double mutant, but found it exhibited 
the catalytic efficiency of the worse of the two single mutants for each 
of the substrates tested. 
Despite the less than additive effects seen for the two charged 
substitutions in the S2 site, we decided to combine the best S2 site 
variant (N62D) with either of the acidic substitutions in the S1 site. The 
two double mutants, N62D/G166E and N62D/G166D, were analyzed with 
substrates having the form, suc-AAXX-pna (SEQ ID NO: 71) where XX was 
either KK (SEQ ID NO: 66), KR (SEQ ID NO: 67), KF (SEQ ID NO: 62), PK (SEQ 
ID NO: 58), PF (SEQ ID NO: 56) or AF (SEQ ID NO: 63)(FIG. 4). The 
wild-type preference was AF&gt;PF.about.KF&gt;KK.about.PK&gt;KR, whereas the double 
mutants had the preference KK&gt;KR&gt;KF&gt;PK.about.AF&gt;PF. Thus for the double 
mutants there was a dramatic improvement toward cleavage of dibasic 
substrates and away from cleaving the hydrophobic substrates. 
The greater than additive effect (or synergy) of these mutants can be seen 
from ratios of the catalytic efficiencies for the single and multiple 
mutants. For example, the G166E variant cannot distinguish Lys from Phe at 
the P1 position. Yet the N62D/G166E variant cleaves the Lys-Lys substrate 
about 8 times faster than the Lys-Phe substrate. Similarly the G166D 
cleaves the Lys P1 substrate about 3 times faster than the Phe P1 
substrate, but the N62D/G166D double mutant cleaves a Lys-Lys substrate 18 
times faster than a Lys-Phe substrate. Thus, as opposed to the reduction 
in specificity seen for the double mutant in the S2 site, the S1-S2 double 
mutants enhance specificity for basic residues. It is possible that these 
two sites bind the dibasic substrates in a cooperative manner analogous to 
a chelate effect. 
Substrate Phage Selection and Cleavage of a Fusion Protein 
Subtilisin has the capability to bind substrates from the P4 to P3' 
positions (McPhalen, C. A. and James, N. G. (1988) Biochemistry 
27:6582-6598 and Bode, W., Papamokos, E., Musil, D., Seemueller, U. and 
Fritz, M. (1986) EMBO J. 5:813-818). Given this extensive binding site and 
the apparent cooperative nature in the way the substrate can bind the 
enzyme we wished to explore more broadly the substrate preferences for the 
enzyme. To do this we utilized a method we call substrate phage selection 
(Matthews, D. J., Goodman, L. J., Gorman, C. M., and Wells, J. A. (1994) 
Protein Science 3:1197-1205 and Matthews, D. J. and Wells, J. A. (1993) 
Science 260:1113-1117). In this method a five-residue substrate linker 
that was flanked by diglycine residues is inserted between an affinity 
domain (in this case a high affinity variant of hGH) and the 
carboxy-termnal domain of gene III, a minor coat protein displayed on the 
surface of the filamentous phage, M13. The five residue substrate linker 
is fully randomized to generate a library of 20.sup.5 different protein 
sequence variants. These are displayed on the phage particles which are 
allowed to bind to the hGHbp. The protease of interest is added and if it 
cleaves the phage particle at the substrate linker it will release that 
particle. The particles released by protease treatment can be propagated 
and subjected to another round of selection to further enrich for good 
protease substrates. Sequences that are retained can also be propagated to 
enrich for poor protease substrates. By sequencing the isolated phage 
genes at the end of either selection one can identify good and poor 
substrates for further analysis. 
We chose to focus on the subtilisin BPN' variant N62D/G166D as it was 
slightly better at discriminating the synthetic dibasic substrates from 
the others. We subjected the substrate phage library to nine rounds of 
selection with the subtilisin variant and isolated clones that were either 
increasingly sensitive or resistant to cleavage. Of twenty-one clones 
sequenced from the sensitive pool eighteen contained dibasic residues, 
eleven of which had the substrate linker sequence Asn-Leu-Met-Arg-Lys (SEQ 
ID NO: 35)(Table 3). 
TABLE 3 
______________________________________ 
Substrate phage sequences sensitive or resistant to N62D/G166D 
subtilisin from a GG-xxxxx-GG library after 9 rounds of 
______________________________________ 
selection.sup.a. 
Protease Sensitive Pool 
No Basic Sites (0) 
Monobasic Sites (3) 
Dibasic Sites (18) 
______________________________________ 
N L T A R (3) 
N L M R K (11) 
(SEQ ID NO: 34) 
(SEQ ID NO: 35) 
T A S R R (4) (SEQ ID NO: 36) 
L T R R S (SEQ ID NO: 37) 
A L S R K (SEQ ID NO: 38) 
L M L R K (SEQ ID NO: 39) 
______________________________________ 
Protease Resistant Pool 
No Basic Sites (7) 
Monobasic Sites (2) 
Dibasic Sites (1) 
______________________________________ 
A S T H F Q K P N F R K P T H (SEQ ID NO: 42) 
(SEQ ID NO: 40) 
(SEQ ID NO: 41) 
I Q Q Q Y R P G A M 
(SEQ ID NO: 43) 
(SEQ ID NO: 44) 
Q G E L P 
(SEQ ID NO: 45) 
A P D P T 
(SEQ ID NO: 46) 
Q L L E H 
(SEQ ID NO: 47) 
V N N N H 
(SEQ ID NO: 48) 
A Q S N L 
(SEQ ID NO: 49) 
______________________________________ 
.sup.a Numbers in parentheses indicate the number of times a particular 
DNA sequence was isolated. 
Three (3) of the sensitive sequences were monobasic, Asn-Leu-Thr-Ala-Arg 
(SEQ ID NO: 34). It is known that subtilisin has a preference for 
hydrophobic residues at the P4 position. If these and the other selected 
substrates were indeed cleaved after the last basic residue they all would 
have a Leu, Met or Ala at the P4 position. Almost no basic residues were 
isolated in the protease resistant pool and those that were had a Pro 
following the mono- or dibasic residue. It is known that subtilisin does 
not cleave substrates containing Pro at the P1' position (Carter, P., 
Nilsson, B., Burnier, J., Burdick, D. and Wells, J. A. 1989! Proteins: 
Struct., Funct., Genet. 6:240-248). Thus, di-basic substrates where highly 
selected and these had the additional feature of Leu, Met or Ala at the P4 
position. 
We wished to analyze how efficiently the most frequently selected sequences 
were cleaved in the context of a fusion protein. For this we applied an 
alkaline phosphatase-fusion protein assay (Matthews, D. J., Goodman, L. 
J., Gorman, C. M., and Wells, J. A. 1994!Protein Science 3:1197-1205 and 
Matthews, D. J. and Wells, J. A. 1993!Science 260:1113-1117). The hGH 
substrate linker domains were excised from the phage vector by PCR and 
fused in front of the gene for E. coli AP. The fusion protein was 
expressed and purified on an hGH receptor affinity column. The fusion 
protein was bound to the hGH receptor on a plate and treated with the 
subtilisin variant. The rate of cleavage of the fusion protein from the 
plate was monitored by collecting soluble fractions as a function of time 
and assaying for AP activity (FIG. 5). The most frequently isolated 
substrate sequence, Asn-Leu-Met-Arg-Lys (SEQ ID NO: 35) was cleaved about 
ten times faster than the next most frequently isolated clones 
(Thr-Ala-Ser-Arg-Arg (SEQ ID NO: 50) and Asn-Leu-Thr-Ala-Arg (SEQ ID NO: 
34)). We also tested the dibasic sequence isolated from the resistant 
pool, namely Arg-Lys-Pro-Thr-His (SEQ ID NO: 42). We observed no 
detectable cleavage above background for this substrate during the assay. 
Cleavage of a Fusion Proteins With Subtilisin Variants 
A fusion protein is any polypeptide that contains within it an affinity 
domain (AD) that usually aids in protein purification, a protease cleavage 
sequence or substrate linker (SL), which is cleaved by a protease and a 
protein product of interest (PP). Such fusion proteins are generally 
expressed by recombinant DNA technology. The genes for fusion proteins are 
designed so that the SL is between the AD and PP. These usually take the 
form AD-SL-PP such that the domain closest to the N-terminus is AD and PP 
is closest to the C-terminus. 
Examples of AD would include, glutathione-Stransferase which binds to 
glutathione, protein A (or derivatives or fragments thereof) which binds 
IgG molecules, poly-histidine sequences, particularly (His).sub.6 (SEQ ID 
NO: 51) that bind metal affinity columns, maltose binding protein that 
binds maltose, human growth hormone that binds the human growth hormone 
receptor or any of a variety of other proteins or protein domains that can 
bind to an immobilized affinity support with an association constant (Ka) 
of &gt;10.sup.5 M.sup.-1. 
The SL can be any sequence which is cleaved by the N62D/G166D subtilisin 
variant but preferably ones with di-basic residues. The SL should be at 
least four residues and preferably contain a large hydrophobic residue at 
P4 (such as Leu or Met) and dibasic residues at P2 and P1 (such as Arg and 
Lys). A particularly good substrate is Leu-Met-Arg-Lys- (SEQ ID NO: 52), 
but a variety of other sequences may work including Ala-Ser-Arg-Arg (SEQ 
ID NO: 50) and even Leu-Thr-Ala-Arg (SEQ ID NO: 53). It is often useful 
that the SL contain a flexible segment on its N-terminus to better 
separate it from the AD and PP. Such sequences include Gly-Pro-Gly-Gly 
(SEQ ID NO: 54) but can be as simple as Gly-Gly or Pro-Gly. Thus, an 
example of a particularly good SL would have the sequence 
Gly-Pro-Gly-Gly-Leu-Met-Arg-Lys (SEQ ID NO: 55). This sequence would be 
inserted between the AD and PP domains. 
The PP can be virtually any protein or peptide of interest but preferably 
should not have a Pro, Ile, Thr, Val, Asp or Glu as its first residue 
(P1'), or Pro or Gly at the second residue (P2') or Pro at the third 
residue (P3'). Such residues are poor substrates for the enzyme and may 
impair the ability of the N62D/G166D subtilisin variant to cleave the SL 
sequence. 
The conditions for cleaving the fusion protein are best done in aqueous 
solution, although it should be possible to immobilize the enzyme and 
cleave the soluble fusion protein. It may also be possible to cleave the 
fusion protein as it remains immobilized on a solid support (e.g. bound to 
the solid support through AD) with the soluble N62D/G166D subtilisin 
variant. It is preferable to add the enzyme to the fusion protein so that 
the enzyme is less than one part in 100 (1:100) by weight. A good buffer 
is 10-50 mM Tris (pH 8.2) in 10 mM NaCl. A preferable temperature is about 
25.degree. C. although the enzyme is active up to 65.degree. C. The extent 
of cleavage can be assayed by applying samples to SDS-PAGE. Generally 
suitable conditions for using the subtilisin variants of this invention do 
not depart substantially from those known in the art for the use of other 
subtilisins. 
EXAMPLES 
In the examples below and elsewhere, the following abbreviations are 
employed: subtilisin BPN', subtilisin from Bacillus amyloliquefaciens; 
Boc-RVRR-MCA (SEQ ID NO: 73), N-t-butoxy 
carbonyl-arginine-valine-arginine-arginine-7-amido4-methyl coumarin (SEQ 
ID NO: 73); suc-Ala-Ala-Pro-Phe-pna (SEQ ID NO: 56), 
N-succinyl-alanine-alanine-proline-phenylalanyl-p-nitroanalide (SEQ ID NO: 
56); hGH, human growth hormone; hGHbp, extracellular domain of the hGH 
receptor; PBS, phosphate buffered saline; AP, alkaline phosphatase; 
Example 1 
Construction and Purification of Subtilisin Mutants. 
Site-directed mutations were introduced into the subtilisin BPN' gene 
cloned into the phagemid pSS5 (Wells, J. A., Ferrari, E., Henner, D. J., 
Estell, D. A. and Chen, E. Y. 1983! Nucl. Acids Res. 11:7911-7929). 
Single-stranded uracil-containing pSS5 template was prepared and 
mutagenesis performed using the method of Kunkel (Kunkel, T. A., Bebenek, 
K and McClary, J. 1991! Methods Enzymol. 204:125-139). For example, the 
synthetic oligonucleotide N62D, 
EQU (5'-CCAAGACAACG*ACTCTCACGGAA-3') (SEQ ID NO: 25) 
in which the asterisk denotes a mismatch to the wild-type sequence, was 
used to construct the N62D mutant. The oligonucleotide was first 
phosphorylated at the 5' end using T4 polynucleotide kinase according to a 
described procedure (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) 
in "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring 
Harbor, N.Y.). The phosphorylated oligonucleotide was annealed to 
single-stranded uracil-containing pSS5 template, the complementary DNA 
strand was filled in with deoxynucleotides using T7 polynucleotide kinase, 
and the resulting nicks ligated using T4 DNA ligase according to a 
previously described procedure (Sambrook, J., Fritsch, E. F., and 
Maniatis, T. (1989) in "Molecular Cloning: A Laboratory Manual," Second 
Edition, Cold Spring Harbor, N.Y.). Heteroduplex DNA was transformed into 
the E. coli host JM101(Yanish-Perron, C., Viera, J., and Messing, J. 
(1985) Gene 33: 103-199), and putative mutants were confirmed by 
preparation and dideoxy nucleotide sequencing of single stranded DNA 
(Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. 
USA 74:5463-5467) according to the Sequenase.RTM. protocol (USB 
Biochemicals). Mutant single-stranded DNA was then retransformed into 
JM101 cells and double stranded DNA prepared according to a previously 
described procedure (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) 
in "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring 
Harbor, N.Y.). For other mutations also requiring the use of one primer, 
the oligonucleotides used are listed in Table 2. For several of these 
oligonucleotides, additional silent mutations emplacing new restriction 
sites were simultaneously introduced to provide an alternative 
verification of mutagenesis. 
To construct the double mutants N62D/G166D, and N62D/G166E, pSS5 DNA 
containing the N62D mutation was produced in single-stranded 
uracil-containing form using the Kunkel procedure (Kunkel, T. A. , 
Bebenek, K and McClary, J. (1991) Methods Enzymol. 204, 125-139). This 
mutant DNA was used as template for the further introduction of the G166D 
or G166E mutations, using the appropriate oligonucleotide primers (see 
sequences in Table 2), following the procedures described above. 
For expression of the subtilisin BPN' mutants, double stranded mutant DNA 
was transformed into a protease-deficient strain (BG2036) of Bacillus 
Subtilis (Yang, M. Y., Ferrari, E. and Henner, D. J. (1984) Journal of 
Bacteriology 160:15-21) according to a previous method (Anagnostopolouus, 
C. and Spizizen, J. (1961) Journal of Bacteriology 81:741-746) in which 
transformation mixtures were plated out on LB plus skim milk plates 
containing 12.5 .mu.g/mL chloramphenicol. The clear halos indicative of 
skim milk digestion surrounding transformed colonies were noted to roughly 
estimate secreted protease activity. 
The transformed BG2036 strains were cultured by inoculating 5 mL of 
2.times.YT media (Miller, J. H., (1972) in "Experiments in Molecular 
Genetics," Cold Spring Harbor, N.Y.) containing 12.5 .mu.g/mL 
chloramphenicol and 2 mM CaCl.sub.2 at 37.degree. C. for 18-20 h, followed 
by 1:100 dilution in the same medium and growth in shake flasks at 
37.degree. C. for 18-22 h with vigorous aeration. The cells were harvested 
by centrifugation (6000 g, 15 min, 4.degree. C.), and to the supernatant 
20 mM (final) CaCl.sub.2 and one volume of ethanol (-20.degree. C.) were 
added. After 30 min at 4.degree. C., the solution was centrifuged (12,000 
g, 15 min, 4.degree. C.), and one volume of ethanol (-20.degree. C.) added 
to the supernatant. After 2 h at -20.degree. C., the solution was 
centrifuged (12,000 g, 15 min., 4.degree. C.) and the pellet resuspended 
in and dialyzed against MC (25 mM 2-N-Morpholino!ethanesulfonic acid 
(MES), 5 mM CaCl.sub.2 at pH 5.5) overnight at 4.degree. C. The dialysate 
was passed through a 0.22 .mu.m syringe filter and loaded onto a mono-S 
cation exchange column run by an FPLC system (Pharmacia Biotechnology). 
The column was washed with 20 volumes of MC and mutant subtilisin eluted 
over a linear gradient of zero to 0.15M NaCl in MC, all at a flow rate of 
1 mL/min. Peak fractions were recovered and the subtilisin mutant 
quantitated by measuring the absorbance at 280 nm (E.sub.280 0.1% =1.17) 
(Matsubara, H.; Kasper, C. B.; Brown, D. M.; and Smith, E. L. (1965) J. 
Biol. Chem., 240:1125-1130.). 
Example 2 
Kinetic Characterizations 
Subtilisins were assayed by measuring the initial rates of hydrolysis of 
p-nitroanilide tetrapeptide substrates in 0.4 mL 20 mM Tris-Cl pH 8.2, 4% 
(v/v) dimethyl sulfoxide at (25.+-.0.2).degree. C. as described previously 
(Estell, D. A., Graycar, T. P., Miller, J. V., Powers, D. B., Burnier, J. 
P., Ng, P. G. and Wells, J. A. 1986! Science 233:659-663). Enzyme 
concentrations E!.sub.0 were determined spectrophotometrically using 
E.sub.280 nm 0.1% =1.17 (Matsubara, H.; Kasper, C. B.; Brown, D. M.; and 
Smith, E. L. (1965) J. Biol. Chem., 240:1125-1130.), and were typically 
5-50 nM in reactions. Initial rates were determined for nine to twelve 
different substrate concentrations over the range of 0.001-2.0 mM. Plots 
of initial rates (v) versus substrate concentration S! were fitted to the 
Michaelis-Menton equation, 
##EQU2## 
to determine the kinetic constants k.sub.cat and K.sub.m (Fersht, A. in 
"Enzyme Structure and Mechanism", Second edition, Freeman and Co., N.Y.) 
using the program Kaleidagraph (Synergy Software, Reading, Pa.). 
Example 3 
Substrate Phage 
Substrate phage selections were performed as described by Matthews and 
Wells (Matthews, D. J. and Wells, J. A. (1993) Science 260:1113-1117), 
with minor modifications. Phage sorting was carried out using a library in 
which the linker sequence between the gene III coat protein and a 
tight-binding variant of hGH was GPGGX.sub.5 GGPG (SEQ ID NO: 57). The 
library contained 2.times.10.sup.6 independent transformants. Phage 
particles were prepared by infecting 1 mL of log phase 27C7 (F'/tet.sup.R 
/Ompt.sup.- /degP.sup.-) Escherichia coli with .about.10.sup.8 library 
phage for 1 h at 37.degree. C., followed by 18-24 h of growth in 25 mL 2YT 
medium containing 10.sup.10 M13K07 helper phage and 50 .mu.g/mL 
carbenicillin at 37.degree. C. Wells of a 96-well Nunc Maxisorb microtiter 
plate were coated with 2 .mu.g/mL of hGHbp in 50 mM NaHCO.sub.3 at pH 9.6 
overnight at 4.degree. C. and blocked with PBS (10 mM sodium phosphate at 
pH 7.4 nd 150 mM NaCl) containing 2.5% (w/v) skim milk for 1 h at room 
temperature. Between 10.sup.11 and 10.sup.12 phage in 0.1 mL 10 mM tris-Cl 
(pH 7.6), 1 mM EDTA, and 100 mM NaCl were incubated in the wells at room 
temperature for 2 h with gentle agitation. The plate was washed first with 
20 rinses of PBS plus 0.05% Tween 20 and then twice with 20 mM tris-Cl at 
pH 8.2. The N62D/G166D subtilisin was added in 0.1 mL of 20 mM tris-Cl at 
pH 8.2 and protease sensitive phage were eluted after a variable reaction 
time. The concentration of protease and incubation times for elution of 
sensitive phage were decreased gradually over the course of sorting 
procedure to increase selectivity, with protease concentrations of 0.2 nM 
(rounds 1-3) and 0.1 nM (rounds 4-9), and reaction times of 5 min (rounds 
1-6), 2.5 min (round 7), 40 s (round 8) and 20 s (round 9). Control wells 
in which no protease was added were also included in each round. For the 
resistant phage pool, the incubation time with protease remained constant 
at 5 min. The wells were then washed ten times with PBS plus 0.05% Tween 
20 and resistant phage eluted by treatment with 0.1 mL of 0.2M glycine at 
pH 2.0 in PBS plus 0.05% Tween 20 for 1 min at room temperature. Protease 
sensitive and resistant phage pools were titered and used to infect log 
phase 27C7 cells for 1 h at 37.degree. C., followed by centrifugation at 
4000 rpm, removal of supernatant, and resuspension in 1 mL 2YT medium. The 
infected cells were then grown 18-24 h in the presence of helper phage as 
described above and the process repeated 9 times. Selected substrates were 
introduced into AP fusion proteins and assayed for relative rates of 
cleavage as described by Matthews and Wells (Matthews, D. J., Goodman, L. 
J., Gorman, C. M., and Wells, J. A. (1994) Protein Science 3:1197-1205 and 
Matthews, D. J. and Wells, J. A. (1993)Science 260:1113-1117), except that 
the cleavage reactions were performed in 20 mM Tris-Cl at pH 8.2. 
The present invention has of necessity been discussed herein by reference 
to certain specific methods and materials. It is to be understood that the 
discussion of these specific methods and materials in no way constitutes 
any limitation on the scope of the present invention, which extends to any 
and all alternative materials and methods suitable for accomplishing the 
ends of the present invention. 
All references cited herein are expressly incorporated by reference. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 74 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8119 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GAATTCNGGTCTACTAAAATATTATTCCATACTATACAATTAATACACAG50 
AATAATCTGTCTATTGGTTATTCTGCAAATGAAAAAAAGGAGAGGATAAA100 
GAGTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTT138 
ValArgGlyLysLysValTrpIleSerLeuLeuPhe 
107-105-100 
GCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACA177 
AlaLeuAlaLeuIlePheThrMetAlaPheGlySerThr 
95-90-85 
TCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAG216 
SerSerAlaGlnAlaAlaGlyLysSerAsnGlyGluLys 
80-75-70 
AAATATATTGTCGGGTTTAAACAGACAATGAGCACGATG255 
LysTyrIleValGlyPheLysGlnThrMetSerThrMet 
65-60 
AGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGC294 
SerAlaAlaLysLysLysAspValIleSerGluLysGly 
55- 50-45 
GGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCT333 
GlyLysValGlnLysGlnPheLysTyrValAspAlaAla 
40-35 
TCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAA372 
SerAlaThrLeuAsnGluLysAlaValLysGluLeuLys 
30-25-20 
AAAGACCCGAGCGTCGCTTACGTTGAAGAAGATCACGTA411 
LysAspProSerValAlaTyrValGluGluAspHisVal 
15-10-5 
GCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCA450 
AlaHisAlaTyrAlaGlnSerValProTyrGlyValSer 
15 
CAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACT489 
GlnIleLysAlaProAlaLeuHisSerGlnGlyTyrThr 
101520 
GGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATC528 
GlySerAsnValLysValAlaValIleAspSerGlyIle 
253035 
GATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCC567 
AspSerSerHisProAspLeuLysValAlaGlyGlyAla 
4045 
AGCATGGTTCCTTCTGAAACAAATCCTTTCCAAGACAAC606 
SerMetValProSerGluThrAsnProPheGlnAspAsn 
505560 
GACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCT645 
AspSerHisGlyThrHisValAlaGlyThrValAlaAla 
6570 
CTTAATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGC684 
LeuAsnAsnSerIleGlyValLeuGlyValAlaProSer 
758085 
GCATCACTTTACGCTGTAAAAGTTCTCGGTGCTGACGGT723 
AlaSerLeuTyrAlaValLysValLeuGlyAlaAspGly 
9095100 
TCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGG762 
SerGlyGlnTyrSerTrpIleIleAsnGlyIleGluTrp 
105110 
GCGATCGCAAACAATATGGACGTTATTAACATGAGCCTC801 
AlaIleAlaAsnAsnMetAspValIleAsnMetSerLeu 
115120125 
GGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCGGCAGTT840 
GlyGlyProSerGlySerAlaAlaLeuLysAlaAlaVal 
130135 
GATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCA879 
AspLysAlaValAlaSerGlyValValValValAlaAla 
140145150 
GCCGGTAACGAAGGCACTTCCGGCAGCTCGTCGACAGTG918 
AlaGlyAsnGluGlyThrSerGlySerSerSerThrVal 
155160165 
GACTACCCTGGCAAATACCCTTCTGTCATTGCAGTAGGC957 
AspTyrProGlyLysTyrProSerValIleAlaValGly 
170175 
GCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGC996 
AlaValAspSerSerAsnGlnArgAlaSerPheSerSer 
180185190 
GTAGGACCTGAGCTTGATGTCATGGCACCTGGCGTATCT1035 
ValGlyProGluLeuAspValMetAlaProGlyValSer 
195200 
ATCCAAAGCACGCTTCCTGGAAACAAATACGGGGCGTAC1074 
IleGlnSerThrLeuProGlyAsnLysTyrGlyAlaTyr 
205210215 
AACGGTACCTCAATGGCATCTCCGCACGTTGCCGGAGCG1113 
AsnGlyThrSerMetAlaSerProHisValAlaGlyAla 
220225230 
GCTGCTTTGATTCTTTCTAAGCACCCGAACTGGACAAAC1152 
AlaAlaLeuIleLeuSerLysHisProAsnTrpThrAsn 
235240 
ACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAA1191 
ThrGlnValArgSerSerLeuGluAsnThrThrThrLys 
245250255 
CTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAAC1230 
LeuGlyAspSerPheTyrTyrGlyLysGlyLeuIleAsn 
260265 
GTACAGGCGGCAGCTCAGTAAAACATAAAAAACCGGCCTT1270 
ValGlnAlaAlaAlaGln 
270275 
GGCCCCGCCGGTTTTTTATTATTTTTCTTCCTCCGCATGTTCAATCCGCT1320 
CCATAATCGACGGATGGCTCCCTCTGAAAATTTTAACGAGAAACGGCGGG1370 
TTGACCCGGCTCAGTCCCGTAACGGCCAAGTCCTGAAACGTCTCAATCGC1420 
CGCTTCCCGGTTTCCGGTCAGCTCAATGCCGTAACGGTCGGCGGCGTTTT1470 
CCTGATACCGGGAGACGGCATTCGTAATCGGATCCGGAAATTGTAAACGT1520 
TAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTT1570 
TTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAG1620 
ACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATT1670 
AAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCT1720 
ATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGG1770 
TGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGC1820 
TTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGA1870 
AAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTA1920 
ACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCGGATC1970 
NGATCCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTT2020 
CCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTA2070 
GATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAG2120 
CCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCGCCT2170 
CGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATAC2220 
CTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTC2270 
GACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAG2320 
AATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACC2370 
CTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGG2420 
CGCATCTCGGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG2470 
ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA2520 
GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC2570 
TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTT2620 
CGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCG2670 
GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA2720 
GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG2770 
TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC2820 
AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA2870 
CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGC2920 
CAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC2970 
ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG3020 
AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG3070 
CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA3120 
AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC3170 
AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA3220 
TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT3270 
GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC3320 
TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG3370 
ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT3420 
CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC3470 
TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG3520 
CTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGC3570 
TCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA3620 
AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG3670 
CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT3720 
GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA3770 
GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGT3820 
CAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATC3870 
ATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT3920 
GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCAT3970 
CTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT4020 
GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT4070 
CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA4120 
GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCG4170 
CGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTAT4220 
CATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTC4270 
AAGAATTAATTCCTTAAGGAACGTACAGACGGCTTAAAAGCCTTTAAAAA4320 
CGTTTTTAAGGGGTTTGTAGACAAGGTAAAGGATAAAACAGCACAATTCC4370 
AAGAAAAACACGATTTAGAACCTAAAAAGAACGAATTTGAACTAACTCAT4420 
AACCGAGAGGTAAAAAAAGAACGAAGTCGAGATCAGGGAATGAGTTTATA4470 
AAATAAAAAAAGCACCTGAAAAGGTGTCTTTTTTTGATGGTTTTGAACTT4520 
GTTCTTTCTTATCTTGATACATATAGAAATAACGTCATTTTTATTTTAGT4570 
TGCTGAAAGGTGCGTTGAAGTGTTGGTATGTATGTGTTTTAAAGTATTGA4620 
AAACCCTTAAAATTGGTTGCACAGAAAAACCCCATCTGTTAAAGTTATAA4670 
GTGACTAAACAAATAACTAAATAGATGGGGGTTTCTTTTAATATTATGTG4720 
TCCTAATAGTAGCATTTATTCAGATGAAAAATCAAGGGTTTTAGTGGACA4770 
AGACAAAAAGTGGAAAAGTGAGACCATGGAGAGAAAAGAAAATCGCTAAT4820 
GTTGATTACTTTGAACTTCTGCATATTCTTGAATTTAAAAAGGCTGAAAG4870 
AGTAAAAGATTGTGCTGAAATATTAGAGTATAAACAAAATCGTGAAACAG4920 
GCGAAAGAAAGTTGTATCGAGTGTGGTTTTGTAAATCCAGGCTTTGTCCA4970 
ATGTGCAACTGGAGGAGAGCAATGAAACATGGCATTCAGTCACAAAAGGT5020 
TGTTGCTGAAGTTATTAAACAAAAGCCAACAGTTCGTTGGTTGTTTCTCA5070 
CATTAACAGTTAAAAATGTTTATGATGGCGAAGAATTAAATAAGAGTTTG5120 
TCAGATATGGCTCAAGGATTTCGCCGAATGATGCAATATAAAAAAATTAA5170 
TAAAAATCTTGTTGGTTTTATGCGTGCAACGGAAGTGACAATAAATAATA5220 
AAGATAATTCTTATAATCAGCACATGCATGTATTGGTATGTGTGGAACCA5270 
ACTTATTTTAAGAATACAGAAAACTACGTGAATCAAAAACAATGGATTCA5320 
ATTTTGGAAAAAGGCAATGAAATTAGACTATGATCCAAATGTAAAAGTTC5370 
AAATGATTCGACCGAAAAATAAATATAAATCGGATATACAATCGGCAATT5420 
GACGAAACTGCAAAATATCCTGTAAAGGATACGGATTTTATGACCGATGA5470 
TGAAGAAAAGAATTTGAAACGTTTGTCTGATTTGGAGGAAGGTTTACACC5520 
GTAAAAGGTTAATCTCCTATGGTGGTTTGTTAAAAGAAATACATAAAAAA5570 
TTAAACCTTGATGACACAGAAGAAGGCGATTTGATTCATACAGATGATGA5620 
CGAAAAAGCCGATGAAGATGGATTTTCTATTATTGCAATGTGGAATTGGG5670 
AACGGAAAAATTATTTTATTAAAGAGTAGTTCAACAAACGGGCCAGTTTG5720 
TTGAAGATTAGATGCTATAATTGTTATTAAAAGGATTGAAGGATGCTTAG5770 
GAAGACGAGTTATTAATAGCTGAATAAGAACGGTGCTCTCCAAATATTCT5820 
TATTTAGAAAAGCAAATCTAAAATTATCTGAAAAGGGAATGAGAATAGTG5870 
AATGGACCAATAATAATGACTAGAGAAGAAAGAATGAAGATTGTTCATGA5920 
AATTAAGGAACGAATATTGGATAAATATGGGGATGATGTTAAGGCTATTG5970 
GTGTTTATGGCTCTCTTGGTCGTCAGACTGATGGGCCCTATTCGGATATT6020 
GAGATGATGTGTGTCATGTCAACAGAGGAAGCAGAGTTCAGCCATGAATG6070 
GACAACCGGTGAGTGGAAGGTGGAAGTGAATTTTGATAGCGAAGAGATTC6120 
TACTAGATTATGCATCTCAGGTGGAATCAGATTGGCCGCTTACACATGGT6170 
CAATTTTTCTCTATTTTGCCGATTTATGATTCAGGTGGATACTTAGAGAA6220 
AGTGTATCAAACTGCTAAATCGGTAGAAGCCCAAACGTTCCACGATGCGA6270 
TTTGTGCCCTTATCGTAGAAGAGCTGTTTGAATATGCAGGCAAATGGCGT6320 
AATATTCGTGTGCAAGGACCGACAACATTTCTACCATCCTTGACTGTACA6370 
GGTAGCAATGGCAGGTGCCATGTTGATTGGTCTGCATCATCGCATCTGTT6420 
ATACGACGAGCGCTTCGGTCTTAACTGAAGCAGTTAAGCAATCAGATCTT6470 
CCTTCAGGTTATGACCATCTGTGCCAGTTCGTAATGTCTGGTCAACTTTC6520 
CGACTCTGAGAAACTTCTGGAATCGCTAGAGAATTTCTGGAATGGGATTC6570 
AGGAGTGGACAGAACGACACGGATATATAGTGGATGTGTCAAAACGCATA6620 
CCATTTTGAACGATGACCTCTAATAATTGTTAATCATGTTGGTTACGTAT6670 
TTATTAACTTCTCCTAGTATTAGTAATTATCATGGCTGTCATGGCGCATT6720 
AACGGAATAAAGGGTGTGCTTAAATCGGGCCATTTTGCGTAATAAGAAAA6770 
AGGATTAATTATGAGCGAATTGAATTAATAATAAGGTAATAGATTTACAT6820 
TAGAAAATGAAAGGGGATTTTATGCGTGAGAATGTTACAGTCTATCCCGG6870 
CAATAGTTACCCTTATTATCAAGATAAGAAAGAAAAGGATTTTTCGCTAC6920 
GCTCAAATCCTTTAAAAAAACACAAAAGACCACATTTTTTAATGTGGTCT6970 
TTATTCTTCAACTAAAGCACCCATTAGTTCAACAAACGAAAATTGGATAA7020 
AGTGGGATATTTTTAAAATATATATTTATGTTACAGTAATATTGACTTTT7070 
AAAAAAGGATTGATTCTAATGAAGAAAGCAGACAAGTAAGCCTCCTAAAT7120 
TCACTTTAGATAAAAATTTAGGAGGCATATCAAATGAACTTTAATAAAAT7170 
TGATTTAGACAATTGGAAGAGAAAAGAGATATTTAATCATTATTTGAACC7220 
AACAAACGACTTTTAGTATAACCACAGAAATTGATATTAGTGTTTTATAC7270 
CGAAACATAAAACAAGAAGGATATAAATTTTACCCTGCATTTATTTTCTT7320 
AGTGACAAGGGTGATAAACTCAAATACAGCTTTTAGAACTGGTTACAATA7370 
GCGACGGAGAGTTAGGTTATTGGGATAAGTTAGAGCCACTTTATACAATT7420 
TTTGATGGTGTATCTAAAACATTCTCTGGTATTTGGACTCCTGTAAAGAA7470 
TGACTTCAAAGAGTTTTATGATTTATACCTTTCTGATGTAGAGAAATATA7520 
ATGGTTCGGGGAAATTGTTTCCCAAAACACCTATACCTGAAAATGCTTTT7570 
TCTCTTTCTATTATTCCATGGACTTCATTTACTGGGTTTAACTTAAATAT7620 
CAATAATAATAGTAATTACCTTCTACCCATTATTACAGCAGGAAAATTCA7670 
TTAATAAAGGTAATTCAATATATTTACCGCTATCTTTACAGGTACATCAT7720 
TCTGTTTGTGATGGTTATCATGCAGGATTGTTTATGAACTCTATTCAGGA7770 
ATTGTCAGATAGGCCTAATGACTGGCTTTTATAATATGAGATAATGCCGA7820 
CTGTACTTTTTACAGTCGGTTTTCTAATGTCACTAACCTGCCCCGTTAGT7870 
TGAAGAAGGTTTTTATATTACAGCTCCAGATCCATATCCTTCTTTTTCTG7920 
AACCGACTTCTCCTTTTTCGCTTCTTTATTCCAATTGCTTTATTGACGTT7970 
GAGCCTCGGAACCCNTATAGTGTGTTATACTTTACTTGGAAGTGGTTGCC8020 
GGAAAGAGCGAAAATGCCTCACATTTGTGCCACCTAAAAAGGAGCGATTT8070 
ACATATGAGTTATGCAGTTTGTAGAATGCAAAAAGTGAAATCAGGATCN8119 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 382 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ValArgGlyLysLysValTrpIleSerLeuLeuPheAlaLeuAla 
107-105-100-95 
LeuIlePheThrMetAlaPheGlySerThrSerSerAlaGlnAla 
90-85-80 
AlaGlyLysSerAsnGlyGluLysLysTyrIleValGlyPheLys 
75-70-65 
GlnThrMetSerThrMetSerAlaAlaLysLysLysAspValIle 
60-55-50 
SerGluLysGlyGlyLysValGlnLysGlnPheLysTyrValAsp 
45-40-35 
AlaAlaSerAlaThrLeuAsnGluLysAlaValLysGluLeuLys 
30-25-20 
LysAspProSerValAlaTyrValGluGluAspHisValAlaHis 
15-10-5 
AlaTyrAlaGlnSerValProTyrGlyValSerGlnIleLysAla 
1510 
ProAlaLeuHisSerGlnGlyTyrThrGlySerAsnValLysVal 
152025 
AlaValIleAspSerGlyIleAspSerSerHisProAspLeuLys 
303540 
ValAlaGlyGlyAlaSerMetValProSerGluThrAsnProPhe 
455055 
GlnAspAsnAspSerHisGlyThrHisValAlaGlyThrValAla 
606570 
AlaLeuAsnAsnSerIleGlyValLeuGlyValAlaProSerAla 
758085 
SerLeuTyrAlaValLysValLeuGlyAlaAspGlySerGlyGln 
9095100 
TyrSerTrpIleIleAsnGlyIleGluTrpAlaIleAlaAsnAsn 
105110115 
MetAspValIleAsnMetSerLeuGlyGlyProSerGlySerAla 
120125130 
AlaLeuLysAlaAlaValAspLysAlaValAlaSerGlyValVal 
135140145 
ValValAlaAlaAlaGlyAsnGluGlyThrSerGlySerSerSer 
150155160 
ThrValAspTyrProGlyLysTyrProSerValIleAlaValGly 
165170175 
AlaValAspSerSerAsnGlnArgAlaSerPheSerSerValGly 
180185190 
ProGluLeuAspValMetAlaProGlyValSerIleGlnSerThr 
195200205 
LeuProGlyAsnLysTyrGlyAlaTyrAsnGlyThrSerMetAla 
210215220 
SerProHisValAlaGlyAlaAlaAlaLeuIleLeuSerLysHis 
225230235 
ProAsnTrpThrAsnThrGlnValArgSerSerLeuGluAsnThr 
240245250 
ThrThrLysLeuGlyAspSerPheTyrTyrGlyLysGlyLeuIle 
255260265 
AsnValGlnAlaAlaAlaGln 
270275 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
SerLeuGlyGlyProSerGly 
157 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AlaAlaAlaGlyAsnGluGly 
157 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
SerThrValGlyTyrPro 
156 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
SerTrpGlyProAlaAspAsp 
157 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
PheAlaSerGlyAsnGlyGly 
157 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CysAsnTyrAspGlyTyrThr 
157 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
SerTrpGlyProGluAspAsp 
157 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TrpAlaSerGlyAsnGlyGly 
157 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CysAsnCysAspGlyTyrThr 
157 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
TrpAlaSerGlyAspGlyGly 
157 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CysAsnCysAspGlyTyrAla 
157 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ValIleAspSerGlyIle 
156 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AspAsnAsnSerHis 
15 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
IleValAspAspGlyLeu 
156 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
SerAspAspTyrHis 
15 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
IleLeuAspAspGlyIle 
156 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AsnAspAsnArgHis 
15 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
IleMetAspAspGlyIle 
156 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
TrpPheAsnSerHis 
15 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
GCGGTTATCGACGACGGTATCGATTCT27 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GCGGTTATCGACAAAGGTATCGATTCT27 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GCGGTTATCGACGAAGGTATCGATTCT27 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CCAAGACAACGACTCTCACGGAA23 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CCAAGACAACAGCTCTCACGGAA23 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CCAAGACAACAAATCTCACGGAA23 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CACTTCCGGCAGCTCGTCGACAGTGGACTACCCTGGCAAATA42 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
CACTTCCGGCAGCTCGTCGACAGTGGAGTACCCTGGCAAATA42 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
TTAACATGAGCCTCGGCCCAGCTAGCGGTTCTGCTGCTTTA41 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 43 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
TTAACATGAGCCTCGGCCCCGCGGATGATTCTGCTGCTTTAAA43 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 47 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CGGCAGCTCAAGCAACGATGGCTATCCTGGCAAATACCCTTCTGTCA47 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ACTTCCGGCAGCTCTTCGAACTACGACGGGTACCCTGGCAAATA44 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
AsnLeuThrAlaArg 
15 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
AsnLeuMetArgLys 
15 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
ThrAlaSerArgArg 
15 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
LeuThrArgArgSer 
15 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
AlaLeuSerArgLys 
15 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
LeuMetLeuArgLys 
15 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
AlaSerThrHisPhe 
15 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
GlnLysProAsnPhe 
15 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
ArgLysProThrHis 
15 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
IleGlnGlnGlnTyr 
15 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
ArgProGlyAlaMet 
15 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
GlnGlyGluLeuPro 
15 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
AlaProAspProThr 
15 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
GlnLeuLeuGluHis 
15 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
ValAsnAsnAsnHis 
15 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
AlaGlnSerAsnLeu 
15 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
ThrAlaSerArgArg 
15 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
HisHisHisHisHisHis 
156 
(2) INFORMATION FOR SEQ ID NO:52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
LeuMetArgLys 
14 
(2) INFORMATION FOR SEQ ID NO:53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
LeuThrAlaArg 
14 
(2) INFORMATION FOR SEQ ID NO:54: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
GlyProGlyGly 
14 
(2) INFORMATION FOR SEQ ID NO:55: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
GlyLeuMetArgLys 
15 
(2) INFORMATION FOR SEQ ID NO:56: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
AlaAlaProPhe 
14 
(2) INFORMATION FOR SEQ ID NO:57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
GlyProGlyGlyXaaXaaXaaXaaXaaGlyGlyProGly 
151013 
(2) INFORMATION FOR SEQ ID NO:58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
AlaAlaProLys 
14 
(2) INFORMATION FOR SEQ ID NO:59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
AlaAlaProArg 
14 
(2) INFORMATION FOR SEQ ID NO:60: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
AlaAlaProMet 
14 
(2) INFORMATION FOR SEQ ID NO:61: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
AlaAlaProGln 
14 
(2) INFORMATION FOR SEQ ID NO:62: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
AlaAlaLysPhe 
14 
(2) INFORMATION FOR SEQ ID NO:63: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
AlaAlaAlaPhe 
14 
(2) INFORMATION FOR SEQ ID NO:64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
AlaAlaArgPhe 
14 
(2) INFORMATION FOR SEQ ID NO:65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
AlaAlaAspPhe 
14 
(2) INFORMATION FOR SEQ ID NO:66: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
AlaAlaLysLys 
14 
(2) INFORMATION FOR SEQ ID NO:67: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
AlaAlaLysArg 
14 
(2) INFORMATION FOR SEQ ID NO:68: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
AlaAlaLysPhe 
14 
(2) INFORMATION FOR SEQ ID NO:69: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
AlaAlaProXaa 
14 
(2) INFORMATION FOR SEQ ID NO:70: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
AlaAlaXaaPhe 
14 
(2) INFORMATION FOR SEQ ID NO:71: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
AlaAlaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:72: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 275 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
AlaGlnSerValProTyrGlyValSerGlnIleLysAlaProAla 
151015 
LeuHisSerGlnGlyTyrThrGlySerAsnValLysValAlaVal 
202530 
IleAspSerGlyIleAspSerSerHisProAspLeuLysValAla 
354045 
GlyGlyAlaSerMetValProSerGluThrAsnProPheGlnAsp 
505560 
AsnAspSerHisGlyThrHisValAlaGlyThrValAlaAlaLeu 
657075 
AsnAsnSerIleGlyValLeuGlyValAlaProSerAlaSerLeu 
808590 
TyrAlaValLysValLeuGlyAlaAspGlySerGlyGlnTyrSer 
95100105 
TrpIleIleAsnGlyIleGluTrpAlaIleAlaAsnAsnMetAsp 
110115120 
ValIleAsnMetSerLeuGlyGlyProSerGlySerAlaAlaLeu 
125130135 
LysAlaAlaValAspLysAlaValAlaSerGlyValValValVal 
140145150 
AlaAlaAlaGlyAsnGluGlyThrSerGlySerSerSerThrVal 
155160165 
AspTyrProGlyLysTyrProSerValIleAlaValGlyAlaVal 
170175180 
AspSerSerAsnGlnArgAlaSerPheSerSerValGlyProGlu 
185190195 
LeuAspValMetAlaProGlyValSerIleGlnSerThrLeuPro 
200205210 
GlyAsnLysTyrGlyAlaTyrAsnGlyThrSerMetAlaSerPro 
215220225 
HisValAlaGlyAlaAlaAlaLeuIleLeuSerLysHisProAsn 
230235240 
TrpThrAsnThrGlnValArgSerSerLeuGluAsnThrThrThr 
245250255 
LysLeuGlyAspSerPheTyrTyrGlyLysGlyLeuIleAsnVal 
260265270 
GlnAlaAlaAlaGln 
275 
(2) INFORMATION FOR SEQ ID NO:73: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
ArgValArgArg 
14 
(2) INFORMATION FOR SEQ ID NO:74: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 275 amino acids 
(B) TYPE: Amino Acid 
(D) TOPOLOGY: Linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
AlaGlnSerValProTyrGlyValSerGlnIleLysAlaProAla 
151015 
LeuHisSerGlnGlyTyrThrGlySerAsnValLysValAlaVal 
202530 
IleAspSerGlyIleAspSerSerHisProAspLeuLysValAla 
354045 
GlyGlyAlaSerMetValProSerGluThrAsnProPheGlnAsp 
505560 
AsnAsnSerHisGlyThrHisValAlaGlyThrValAlaAlaLeu 
657075 
AsnAsnSerIleGlyValLeuGlyValAlaProSerAlaSerLeu 
808590 
TyrAlaValLysValLeuGlyAlaAspGlySerGlyGlnTyrSer 
95100105 
TrpIleIleAsnGlyIleGluTrpAlaIleAlaAsnAsnMetAsp 
110115120 
ValIleAsnMetSerLeuGlyGlyProSerGlySerAlaAlaLeu 
125130135 
LysAlaAlaValAspLysAlaValAlaSerGlyValValValVal 
140145150 
AlaAlaAlaGlyAsnGluGlyThrSerGlySerSerSerThrVal 
155160165 
GlyTyrProGlyLysTyrProSerValIleAlaValGlyAlaVal 
170175180 
AspSerSerAsnGlnArgAlaSerPheSerSerValGlyProGlu 
185190195 
LeuAspValMetAlaProGlyValSerIleGlnSerThrLeuPro 
200205210 
GlyAsnLysTyrGlyAlaTyrAsnGlyThrSerMetAlaSerPro 
215220225 
HisValAlaGlyAlaAlaAlaLeuIleLeuSerLysHisProAsn 
230235240 
TrpThrAsnThrGlnValArgSerSerLeuGluAsnThrThrThr 
245250255 
LysLeuGlyAspSerPheTyrTyrGlyLysGlyLeuIleAsnVal 
260265270 
GlnAlaAlaAlaGln 
275 
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