Bacterial surface protein expression

This invention provides a bacterium having an S-layer modified such that the bacterium S-layer protein gene contains one or more in-frame sequences coding for one or more heterologous polypeptides and, the S-layer is a fusion product of the S-layer protein and the heterologous polypeptide. The bacterium is preferably a Caulobacter which may be cultured as a film in a bioreactor or may be used to present an antigenic epitope to the environment of the bacterium. This invention also provides a method of expressing and presenting to the environment of a Caulobacter, a polypeptide that is heterologous to the S-layer of Caulobacter which comprises cloning a coding sequence for the polypeptide in-frame into an S-layer protein gene of Caulobacter whereby the polypeptide is expressed and presented on the surface of the Caulobacter as a fusion product of the S-layer protein and the polypeptide in the S-layer of the Caulobacter.

FIELD OF THE INVENTION 
This invention relates to the expression of heterologous proteins, or 
portions of such proteins, from genes cloned in a bacteria wherein the 
expressed protein is fused with a surface protein of the bacterium. 
BACKGROUND OF THE INVENTION 
Bacterial surface proteins have been used as carriers or vehicles of 
foreign epitopes expressed in the bacterium (particularly in Salmonella 
and E. coli) for various purposes, including the development of live 
vaccines. In some instances, the heterologous protein is expressed as a 
fusion product with a surface protein of the bacterium. Generally, the use 
of such surface proteins as a vehicle for expression and presentation of 
heterologous proteins has been limited by the characteristics of the 
particular surface protein involved. The bacterium's lipopolysaccharide 
layer, which tends to stimulate a strong immune response, also covers the 
integral outer membrane proteins of the organism and potentially affects 
efficient presentation of a cloned epitope. Also, where the surface 
protein is functional, for example, as part of a filamentous portion of 
the bacterial cell surface, there will be limited opportunities to express 
a fusion product and still retain the surface protein's function. 
Generally, the organisms that have been used for these purposes have been 
chosen because of the advantages presented in respect of the organism's 
relationship to its host. 
Many genera of bacteria assemble layers composed of repetitive, regularly 
aligned, proteinaceous sub-units on the outer surface of the cell. These 
layers are essentially two-dimensional paracrystalline arrays, and being 
the outer molecular layer of the organism, directly interface with the 
environment. Such layers are commonly known as S-layers and are found on 
members of every taxonomic group of walled bacteria including: 
Archaebacteria; Chlamydia; Cyanobacteria; Acinetobacter; Bacillus; 
Aquaspirrillum; Caulobacter; Clostridium; Chromatium. (see: Smit, J.; 
PROTEIN SURFACE LAYERS OF BACTERIA; in: "Offprints From Bacterial Outer 
Membranes As Model Systems" (1986) Dr. M. Inouye (Ed.); John Wylie and 
Sons, Inc.). 
Typically, an S-layer will be composed of an intricate, geometric array of 
at least one major protein having a repetitive regular structure. In many 
cases, such as in Caulobacter, the S-layer protein is synthesized by the 
cell in large quantities and the S-layer completely envelopes the cell and 
thus appears to be a protective layer. 
Caulobacter bacteria are natural inhabitants of most soil and freshwater 
environments and may persist in waste water treatment systems and 
effluents. The bacteria alternate between a stalked cell that is attached 
to a surface and a motile dispersal cell that has adhesive material 
already expressed and is searching to find a new surface upon which to 
stick and convert to a stalked cell. The bacteria attach tenaciously to 
nearly all surfaces and do so without producing the extracelluar enzymes 
or polysaccharide "slimes" that are characteristic of most other surface 
attached bacteria. They have simple requirements for growth. The organism 
is ubiquitous in the environment and has been isolated from oligotrophic 
to mesotrophic situations. Caulobacters are known for their ability to 
tolerate low nutrient level stresses, for example, low phosphate levels. 
This nutrient can be limiting in many leachate waste streams, especially 
those with high levels of iron or calcium. 
The S-layer of Caulobacter crescentus has been well characterised. Nearly 
all freshwater isolates of Caulobacter elaborate an S-layer visibly 
indistinguishable from the one produced by Caulobacter crescentus strains 
CB2 and CB15. The S-layer proteins from these strains have approximately 
100,000 m.w. The protein has been characterized both structurally and 
chemically. It is composed of ring-like structures spaced at 22 nm 
intervals arranged in a hexagonal manner on the outer membrane. The 
S-layer is bound to the bacterial surface by calcium ions and may be 
removed by low pH treatment or by treatment with a calcium chelator such 
as EGTA. 
The S-layer proteins of S-layer producing strains of Caulobacter have 
significant similarity. Thus a cloned S-layer protein gene of one 
Caulobacter strain will likely be useful to retrieve the corresponding 
genes in other Caulobacter strains (see: Walker, S. G., S. H. Smith, and 
J. Smit (1992) "Isolation and Comparison of the Paracrystalline Surface 
Layer Proteins of Freshwater Caulobacters". J. Bacteriol. 174: 1783-1792; 
and, MacRae, J. O. and, J. Smit (1991) "Characterization of Caulobacters 
Isolated from Wastewater Treatment Systems" Applied and Environmental 
Microbiology 57:751-758). 
Expression and presentation of a heterologous polypeptide as a fusion 
product with an S-layer protein of a bacterium would provide advantages 
not previously seen in systems using organisms such as E. coli and 
Salmonella where fusion products of other kinds of surface proteins have 
been expressed. Firstly, many bacteria producing S-layer proteins 
(particulary Caulobacter) are relatively harmless and ubiquitous in the 
environment. In contrast, many Salmonella and E. coli strains are 
pathogens. Consequently, expression and presentation of a heterologous 
polypeptide using Caulobacter as a vehicle will have the advantage that 
the expression system will be stable in a variety of outdoor environments 
and may not present problems associated with the use of a pathogenic 
organism. Second, many such bacteria, including Caulobacter, are natural 
biofilm forming species and may be adapted for use in fixed biofilm 
bioreactors. Finally, the quantity of the S-layer protein that is 
synthesized by the bacterium and the unique characteristics of the 
repetitive, two-dimensional S-layer would make such bacteria ideal for use 
as an expression system and a "presentation surface" for heterologous 
polypeptides. Such a presentation surface is desirable in a live vaccine 
so that presentation of a foreign epitope is maximized. In addition, use 
of the presentation surface to achieve maximal exposure of a desired 
polypeptide to the environment results in such bacteria being particularly 
suited for use in bioreactors or as carriers for the polypeptide in 
aqueous or terrestrial outdoor environments. 
SUMMARY OF INVENTION 
This invention provides a method of expressing and presenting to the 
environment of a Caulobacter, a polypeptide that is heterologous to an 
S-layer of the Caulobacter, which method comprises cloning a coding 
sequence for the polypeptide in-frame into an S-layer protein gene of 
Caulobacter whereby the polypeptide is expressed and presented on the 
surface of the Caulobacter as a fusion product with the S-layer protein in 
an S-layer of the Caulobacter. 
This invention provides a bacterium having an S-layer wherein a gene of the 
bacterium encoding an S-layer protein contains one or more in-frame 
sequences encoding one or more heterologous polypeptides and, said S-layer 
is a fusion product of the S-layer protein and the heterologous 
polypeptide. 
This invention provides the means for producing a bioreactor comprising a 
suitable substrate (e.g. a rotating biological contactor) in which is 
adhered the above described bacterium. This invention also provides the 
means for expressing and presenting vaccine candidate epitopes to the 
environment of the bacterium described above.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A preferred organism for use in this invention is Caulobacter, particularly 
C. crescentus. Most preferred are C. crescentus strains such as CB15A 
containing the rsaA gene encoding the paracrystalline S-layer protein as 
described in: Gilchrist, A. J. A. Fisher and, J. Smit (1992) "Nucleotide 
Sequence Analysis Of The Gene Encoding the Caulobacter crescentus 
Paracrystalline Surface Layer Protein". Can. J. Microbiol. 38:193-208. 
A heterologous polypeptide referred to herein may be a peptide, 
polypeptide, protein or a part of a protein. The heterologous polypeptide 
may be an enzyme, a ligand, an antigen or another functional sequence of 
amino acids. 
Once a particular bacterium's S-layer protein gene is characterized, this 
invention may be practised by implementing one or more known methods to 
clone a selected heterologous coding sequence into the S-layer protein 
gene so that both the S-layer protein and the heterologous sequence are 
transcribed "in-frame". Knowledge of the S-layer protein gene sequence 
permits one to identify potential sites to install the heterologous 
genetic material. The repetitive nature of the S-layer protein results in 
multiple copies of the heterologous protein being expressed and presented 
on the surface of the cell. 
The following general procedure lays out courses of action and specifies 
particular plasmid vectors or constructions that may be used to accomplish 
fusion of an S-Layer protein with a polypeptide of interest. The following 
description uses the rsaA (S-layer) gene of C. crescentus as an example 
(see FIG. 6 and SEQ ID NO:6). The latter gene sequence is characterized in 
Gilchrist, A.; J. A. Fisher and, J. Smit (1992) "Nucleotide Sequence 
Analysis of the Gene Encoding the Caulobacter crescentus Paracrystalline 
Surface Layer Protein". Can. J. Microbiol 38: 193-202, in contrast to an 
earlier partial characterization now shown to contain some errors (Fisher, 
J. A.; J. Smit et al, N. Agabian (1988) J. Bacteriol 170:4706-4713). 
The general procedure provides detailed steps allowing for the following 
possibilities: 
1) use of an available collection of potentially permissive sites in the 
S-layer gene to install the genetic information for a polypeptide of 
interest; 
2) use of an available carrier cassette for delivering a gene of interest 
to sites within the S-layer gene (the cassette offers several advantages 
over direct modification of a gene of interest, in preparation for 
insertion); 
3) creation of a collection of random insertion sites based on a 
restriction enzyme of choice, if the available collection of potentially 
permissive sites is for some reason unsuitable; and, 
4) preparation of DNA coding for a polypeptide of interest for direct 
insertion into permissive sites (ie, not using the carrier cassette) by a 
method best suited for the particular case (several options are 
suggested). 
The general procedure involves the following steps and alternative courses 
of action. 
As a first step the practitioner will choose an appropriate region (or 
specific amino acid position) of the S-layer for insertion of a desired 
polypeptide. 
Second, the practitioner will create a unique restriction site in the 
(preferably hexameric) rsaA (S-layer) gene at position within the gene 
encoding that region (or corresponding to a specific amino acid) using 
either standard linker mutagenesis (regional) or site directed mutagenesis 
(specific amino acid). The unique restriction site is to later act as a 
site for accepting DNA encoding the polypeptide of interest. The 
plasmid-based promoter-less version of the rsaA gene (pTZ18U:rsaA.DELTA.P) 
shown in FIG. 2 is preferably used because it contains an appropriate 
combination of 5' and 3' restriction sites useful for subsequent steps. 
The restriction site should not occur in rsaA, its carrier plasmid or the 
DNA sequence coding for the polypeptide of interest. 
If it is unclear which region of the S-layer would be suitable for 
insertion of a polypeptide of interest, a random linker mutagenesis 
approach is used to randomly insert a unique linker-encoded restriction 
site (preferably hexameric) at various positions in the rsaA gene. Sites 
for insertion of the linker are created using an endonuclease, either of a 
sequence specific nature (eg. tetrameric recognition site restriction 
enzyme) or sequence non-specific nature (eg. Deoxyribonuclease I [DNase 
I]). A particularly suitable method is the generalized selectable linker 
mutagenesis approach based on any desired restriction site of: Bingle, W. 
H., and J. Smit. (1991) "Linker Mutagenesis Using a Selectable Marker: A 
Method for Tagging Specific Purpose Linkers With an Antibiotic-Resistance 
Gene". Biotechniques 10: 150-152. Because endonuclease digestion is 
carried out under partial digestion conditions, a library of linker 
insertions at different positions in rsaA is created. 
If restriction endonucleases are used to create sites for subsequent 
insertion of a linker encoding a hexameric restriction site, mutagenesis 
is preferably done with a mixture of 3 different linkers incorporating 
appropriate spacer nucleotides in order to satisfy reading frame 
considerations at a particular restriction site (only 1 of the 3 linker 
insertions will be useful for subsequent acceptance of DNA encoding the 
polypeptide of interest). With DNase I, only one linker is needed, but 
again only 1 of 3 linker insertions may be useful for accepting DNA 
encoding the polypeptide of interest depending on the position of the 
DNase I cleavage with respect to the 3 bases of each amino acid codon. 
Next, a linker tagged with a marker is used to insert DNA of interest at a 
restriction site. For example, if BamHI sites are appropriate as sites for 
the introduction of DNA encoding a polypeptide of interest, BamHI linkers 
tagged with a kanamycin-resistance gene for selectable linker mutagenesis 
may be used. One such 12-bp linker carried in plasmid pUC1021K was 
described by Bingle and Smit (1991) [supra]. Two additional 15-bp linkers 
(pUC7165K and pTZ6571K) constructed for creating the other 2 possible 
translation frames within the linker insert itself are described in FIGS. 
3 and 4 (SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; and, SEQ ID NO:5). Any one 
of the above three kanamycin-resistance tagged BamHI linkers is suitable 
for mutagenesis at sites created in rsaA by DNase I. As outlined above, a 
mixture of all three linkers is preferably used for mutagenesis at sites 
created in rsaA by restriction enzyme digestion. 
Once a library composed of linker insertions encoding desired hexameric 
restriction site at different positions in rsaA has been created, the DNA 
encoding a polypeptide of interest is inserted into the sites en masse 
(the library of mutated rsaA genes may be manipulated as one unit). The 
library is digested with the restriction enzyme specific for the 
newly-introduced linker encoded restriction site and ligated to a DNA 
fragment encoding the polypeptide of interest and carrying the appropriate 
complementary cohesive termini. The DNA specifying the polypeptide of 
interest can be prepared by a number of standard methods, which may 
include oligonucleotide synthesis of 2 anti-complementary strands, 
polymerase chain reaction procedures, or addition of linkers whose termini 
are compatible with the introduced sites in rsaA to a suitably modified 
segment of DNA. 
In order to facilitate the rapid recovery of useful rsaA genes carrying 
newly inserted DNA at BamHI sites encoding the polypeptide of interest, 
the carrier oligonucleotide shown in FIG. 1 may be used. The DNA of 
interest is first directionally cloned, if possible, using the XhoI, StuI, 
or SalI sites or non-directionally cloned using any one of the sites in 
the same orientation as a promoterless chloramphenicol resistance (CmR) 
gene. To do this the DNA of interest must be provided with the appropriate 
termini for cloning and spacer nucleotides for maintaining correct reading 
frame within the cassette and should not contain a BglII site. Pot 
insertion into the BamHi linker library, the DNA of interest is recovered 
as a BamHI fragment tagged with a CmR gene. When ligated to the BamHI 
digested rsaA linker library, only those colonies of the bacterium (eg. E. 
coli) used for the gene modification steps that are recovered will be 
those carrying insertions of the desired DNA in the correct orientation, 
since the promoter on the plasmid is 5' to rsaA.DELTA.P and the CmR gene. 
This eliminates screening for DNA introduction and increases the recovery 
of useful clones by 100% (1 of 3 versus 1 of 6). While still manipulating 
the library as one unit, the CmR gene is removed using BglII. The carrier 
oligonucleotide also provides the opportunity to add DNA 5' or 3' to the 
DNA of interest at SalI, XhoI or StuI sites providing the DNA of interest 
does not contain any of these sites. This allows some control over spacing 
between rsaA sequences and the sequence of the DNA of interest. 
Next, the rsaA genes carrying the DNA of interest in the correct 
orientation is excised from the plasmid (eg. from the pTZ18U:rsaA.DELTA.P 
plasmid) and is transferred to a suitable vector providing a promoter 
recognized by Caulobacter. Preferably, such a vector is pWB9 or pWB10 
(Bingle, W. H., and J. Smit (1990) "High Level Plasmid Expression Vectors 
for Caulobacter crescentus Incorporating the Transcription and 
Transcription-Translation Initiation Regions of the Paracrystalline 
Surface Layer Protein Gene". Plasmid 24: 143-148) with EcoRI/SstI sites. 
Therefore, the DNA of interest should not contain the latter sites. These 
vectors allow expression of rsaA hybrids in S-layer negative mutants of C. 
crescentus such as CB15KSac (Edwards, P. and, J. Smit (1991) "A 
Transducing Bacteriophage for Caulobacter crescentus Uses The 
Paracrystalline Surface Layer Protein As a Receptor" J. Bacteriol. 
173:5568-5572); or, CB2A described in: Smit, J. and, N. Agabian (1984) J. 
Bacteriol. 160:1137-1145. 
Those Caulobacter surviving transfer are examined for S-layer assembly and 
presentation of the new polypeptide activity, antigenicity, etc. by 
methods specific to the needs of the investigator or the capabilities of 
the inserted sequence. Many of the sites created are "benign" as they have 
no effect on the functional regions of the protein involved with export, 
self assembly, etc. However, not every site that results in an absence of 
functional disruption of the S-layer is best for insertion of new 
activities. Some sites may not be well exposed on the surface of the 
organism and other sites may not tolerate insertion of much more DNA than 
the linker sequence. 
Use of the S-layer protein as a vehicle for production and presentation of 
a heterologous polypeptide has several advantages. Firstly, the S-layer 
protein is synthesized in large quantities and has a generally repetitive 
sequence. This permits the development of systems for synthesis of a 
relatively large amount of heterologous material as a fusion product with 
an S-layer protein. It may be desirable to retain the fusion product as 
part of the bacterial cell envelope or, the fusion product may be 
separated from the organism, such as by the method described in: Walker, 
S. G.; S. H. Smith; and J. Smit (1992) "Isolation and Comparison of the 
Paracrystalline Surface Layer Proteins of Freshwater Caulobacters". J. 
Bacteriol. 174:1783-1792. Alternatively, the Caulobacter strain that is 
used to express the fusion product may be derived from a strain such as 
CB15Ca5 that sheds its S-layer (Edwards and J. Smit (1991) [Supra]. 
Second, this invention is particularly suitable for use in a bioreactor 
systems. An example would be the use of a modified Caulobacter expressing 
a polypeptide having the activity of a metallothionein in a bioreactor, to 
bind toxic metals in sewage, waste water etc. Caulobacters are ideal 
candidates for fixed-cell bioreactors, the construction of which is well 
known. An example of such a bioreactor is a rotating biological contactor. 
Although other bacteria are found in the environment that are capable of 
binding metals, they often do so by producing copious polysaccharide 
slimes that quickly plug filtration systems. In some cases, the bacteria 
are not surface-adherent or the bacteria do not show selectivity towards 
key toxic metals. By taking advantage of the natural biofilm forming 
characteristics of Caulobacter, bioreactors may be formed comprising a 
substrate and a single layer of cells adhered thereon, with the cells 
distributed at high density. A variety of substrates may be used such as a 
column of chemically derivatized glass beads or a porous ceramic material 
such as ceramic foam. 
Metallothioneins are small cysteine-rich proteins induced by many organisms 
in response to exposure to heavy metals. They are generally expressed 
internally and are designed to limit exposure of other aspects of cell 
physiology to the toxic metals. Typically, metallothioneins are composed 
of about 60 amino acids and the genes from a variety of mammalian 
organisms have been cloned and sequenced. Metallothioneins bind metals 
such as cadmium, zinc, cobalt, copper and mercury in significant amounts 
(such as from 4-12 moles of metal per mole of protein). Modification of a 
Caulobacter such that its S-layer protein is a fusion product with a 
metallothionein or a polypeptide with a similar activity would provide a 
useful component for a bioreactor. 
Another advantageous application for this invention is in the production of 
batch cultures of modified Caulobacter wherein the S-layer protein is a 
fusion product with an enzyme. For example, such Caulobacter could be 
grown in wood pulp suspensions at an appropriate juncture of the pulping 
process in order to provide for enzymatic decomposition of the wood-pulp 
structure (e.g. with an enzyme having an activity like xylanase or 
cellulase). Such an application may permit more effective penetration of 
bleaching agents in the wood-pulp bleaching process thereby reducing the 
use of chlorine-based bleaching agents. 
Another advantageous application of this invention is the production of 
organisms that present vaccine-candidate epitopes at the organism's cell 
surface. For example, modified Caulobacter may be readily cultured in 
outdoor freshwater environments and would be particularly useful in fish 
vaccines. The two-dimensional crystalline array of the S-protein layer of 
Caulobacter, which has a geometrically regular, repetitive structure, 
provides an ideal means for dense packing and presentation of a foreign 
epitope to an immune system. 
EXAMPLE 1 
Production of Permissive Insertion Sites in C. crescentus 
Using the restriction enzyme TaqI, a partial digestion of the rsaA gene in 
pTZ18U:rsaA.DELTA.P produced a group of linearized segments with random 
TaqI sites cleaved. The linearized segments were modified by use of the 
tagged linker mutagenesis procedure of Bingle and Smit (1991) [supra], 
using the 12-bp BamHI linker carried in plasmid pUC102K discussed in the 
general procedure above. Those products that produced a full-length 
protein in E. coli were ultimately transferred to pWBI (a minor variation 
of pWB9 that is replicated by Caulobacter), as described in the general 
procedure. The resulting construction was introduced into a C. crescentus 
strain. Distinguishable events were retrieved and analyzed for the ability 
to produce a full-length protein in C. crescentus and to produce the 
crystalline S-layer on their surface and the approximate location of the 
insertion. The results of this screening together with the approximate 
positions of six successful events (and subsequently determined exact or 
specific insertion positions) are illustrated in FIG. 5. 
The above-described six positive events represent cases where the 4-amino 
acid insertion is tolerated with no effect on the S-layer function. The 
S-layers of the modified Caulobacter were indistinguishable from a 
wild-type S-layer. Thus, they have a higher potential for tolerating the 
addition of more foreign peptide material than less characterized sites. 
By producing 3 versions of the gene of interest, representing each 
possible reading frame (using standard linker addition technology), one 
may test each of these sites for suitability in expressing the desired 
activity. Also, by using restriction enzymes other than TaqI (such as 
AciI, HinPI or MspI) a larger library of BamHI insertions may be created. 
EXAMPLE 2 
Insertion of Metallothionein Into a Specific Site 
An insertion of the above described 12 bp linker was made at the TaqI site 
that corresponds to amino acid #188, frame #3 (see FIG. 6; SEQ ID NO:6; 
and, SEQ ID NO:7). This created a unique BamHI site at that position. 
Because the precise position of the TaqI site could be assessed from the 
DNA sequence information available for the rsaA gene, the necessary 
translation frame was known and thus a single construction of the 
metallothionein gene was made. This was done by excision of the coding 
sequence of monkey metallothionein II peptide (which is 60 amino acids 
with a molecular weight of about 5000) at known restriction sites and 
adapting the gene ends with BamHI linkers with appropriate base pair 
spacers for the needed translation frame. 
After insertion into the BamHI site created at position 188, frame 3, 
several clones were examined by determining whether they could bind 
elevated levels of cadmium. This is a functional assay for the 
metallothionein, explained in more detail below. The assay was necessary 
because the segment had equal probability of being inserted backwards. One 
clone that gave positive results was examined by electron microscopy and 
the presence of a normal S-layer was confirmed. The plasmid in the clone 
that gave positive results was also examined by DNA sequencing analysis, 
sequencing across the junction between the position 188 site and the 5' 
side of the metallothionein gene. The sequence data confirmed correct 
orientation. 
The plasmid-containing clone and relevant control strains were examined for 
the ability to bind several metals known to be bound by native 
metallothionein. This was done by growing the strains of bacteria in the 
presence of the metals at a concentration of 5 .mu.g/ml. After extensive 
washing of the cells to remove unbound metal, the cells were ashed by 
treatment at 500.degree. C. and the residue was dissolved in dilute nitric 
acid and examined for metal content by atomic absorption spectroscopy. The 
results from one round of data collection is shown in Table 1. In the case 
of cadmium and copper, an elevated level of bound metal is noted in the 
metallothionein-expressing strains. 
TABLE 1 
______________________________________ 
Metal Ion Tested (.mu.g/metal/ 
OD unit of cells 
Copper 
Trial 
Caulohacter 1 2 Cadmium Zinc 
______________________________________ 
CB15 1.79 1.0 0.71 4.15 
(wild-type,S-layer[+]) 
CB15KSAC 2.18 1.33 1.07 4.08 
(S-layer negative strain) 
CB15KSAC/p188.3 2.01 1.30 11.1 3.66 
(containes S-layer 
with linker insert only) 
CB15KSAC/p188.3MT 2.79 3.09 19.1 3.00 
(S-layer with Metallothionein 
inserted) 
______________________________________ 
EXAMPLE 3 
Investigation of Other Permissive Sites in rsaA Gene 
A library of 240 BamHI linker insertions was created using the procedures 
of Example 1. Of the 240 insertions, 45 target sites in the rsaA gene were 
made with TaqI. 34 of the latter insertions were discarded because the 
clones contained deletions of rsaA DNA as well as the linker insertions. 
The remaining 11 resulted in 5 non-permissive and the 6 permissive sites 
described in Example 1. The remaining 195 insertions in the library were 
made using the enzymes HinPI, AciI, and MspI to create target sites as 
outlined in Example 1. Of the latter 195 insertions, 49 permissive sites 
were located for a total of 55. Of those sites scored as non-permissive, 
some may have had deletions of rsaA DNA at the linker insertion site. 
The results of the screening for permissive sites using the above-mentioned 
restriction enzymes is shown in FIG. 7 which illustrates the approximate 
location of the 55 permissive sites. While most of the TaqI sites may be 
specifically identified from the rsaA gene sequence, many of the sites for 
the other restriction enzymes are only known by their approximate location 
in the gene since closely spaced alternate sites are known from the rsaA 
sequence. The results show that sites that will accept 2-4 amino acids 
while still allowing the protein to be made and assembled into an S-layer 
are scattered up and down the protein. Furthermore, there is an 
unexpectedly high proportion of sites at which such insertions do not 
prevent expression and assembly of the S-layer. The results indicate that 
approximately 25-50% of in-frame linker insertions will be tolerated by 
the S-layer protein and the Caulobacter and that diverse regions of the 
protein will tolerate insertions. Thus, Caulobacter is an ideal candidate 
for expression of polypeptides fused with the S-layer and the presence of 
multiple permissive sites extending along the rsaA gene will permit the 
insertion of a plurality of the same or different peptides into the same 
rsaA protein molecule and expressed on the surface of a single 
Caulobacter. 
EXAMPLE 4 
Further Studies with Metallothionein 
The results described for Example 3 indicate that it may be possible to 
insert metallothionein at multiple places in the rsaA protein and thereby 
enhance the metal binding capacity of such a transformed Caulobacter. 
However, when the procedures of Example 2 were repeated to insert the 
metallothionein coding sequence into the 55 permissive sites identified in 
the preceding Example, the transformed Caulobacter did not synthesize an 
S-layer. Furthermore, the transformed Caulobacter of Example 2 is stable 
as long as the transformants are frozen immediately after isolation. When 
continuously cultured for approximately one week, the tranformants delete 
the metallothionein portion of the S-layer and the S-layer protein returns 
to its normal size. Consideration of the predicted amino acid sequence of 
the rsaA protein shows that the latter protein lacks cysteine residues 
whereas metallothionein has a high cysteine content. It appears that for 
long term expression of a fusion product with rsaA protein, the 
heterologous polypeptide should not have a high cysteine content and 
preferably, not be capable of producing an internal disulphide bond in an 
aerobic environment. 
EXAMPLE 5 
Expression and Presentation of Antigenic Epitomes on Caulobacter Cell 
Surface 
Using the library of the 49 permissive sites other than those made with 
TaqI described in Example 3, the coding sequence for a 12-amino acid 
epitope lacking cysteine residues from Pseudomonas aeruginosa PAK pilin 
was inserted at the sites using the procedures described above and 
employing the carrier cassette shown in FIG. 1. DNA coding for the pilus 
epitope shown in FIG. 8 (SEQ ID NO:8) consisting of the amino acids 
numbered 1-12 in superscript was prepared by oligonucleotide synthesis of 
two anti-complementary strands. The transformed bacteria were screened for 
both production and presentation of the epitopes by the transformed 
Caulobacter by using standard Western immunoblot analysis (see: Burnette, 
W. N. 1981 "Western Blotting; Electrophoretic Transfer of Protein from 
Sodium Dodecyl-Polyacrylamide Gels to Unmodified Nitrocellulose and 
Radiographic Detection Antibody and Radioiodinated Protein A"; Analytical 
Biochemistry 112:195-203) and by colony immunoblot tests in which the 
cells were not disrupted (see: Engleberg, N. C., et al 1984 "Cloning an 
Expression of Legionella pneumophilia Antigens in Escherichia coli"; 
Infection and Immunity 44:222-227). Anti-pilus monoclonal antibody 
obtained from Dr. William Paranchych, Dept. of Microbiology, University of 
Alberta (Canada) was used in the immunoblot analyses to detect the 
presence of the pilus epitope insert. The antibody (called PK99H) was 
prepared using purified Pseudomonas aeruginosa PAK pilin as the antigen 
and the monoclonal antibody against the 12 amino acid epitope was isolated 
by standard techniques using BALB/C mice as a source of ascites fluid. 
Reaction with the antibody in the whole cell colony immunoblot assay shows 
that the epitope is not only expressed in the transformed Caulobacter but 
is exposed on the S-layer surface overlying the cell in such a way that 
the epitope is available to the antibody. Of the organisms screened, 
insertions of the pilus epitope at the following sites in the rsaA gene 
(identified by restriction enzyme and approximate amino acid position) 
resulted in a positive reaction with the antibody in the whole cell Colony 
immunoblot analysis: HinPI 288, 538, and 671; AciI at 438; and, MspI at 
95, 521, 638, 705, 588, and 905. The results show that the permissive 
sites that will accept polypeptides of the size of the pilus epitope are 
numerous and scattered across the rsaA gene. Furthermore, analysis of the 
transformants indicated that multiple copies of the pilus epitope were 
successfully inserted at the sites made with MspI at approximate amino 
acid position 638. 
EXAMPLE 6 
Insertion of Large Polypeptides 
Bacterial surface proteins from organisms other than Caulobacter described 
in the prior art are not known to accept polypeptides larger than about 60 
amino acids within the structure of the surface protein. The procedures of 
the preceding Example were carried out in order to insert the coding 
sequence of a 110 amino acid epitope from IHNV virus coat glycoprotein at 
the approximate insertion sites identified in the preceding Examples as 
521; 588; 638; and 705. The IHNV epitope coding sequence was prepared by 
PCR and had the sequence shown in FIG. 9 (SEQ ID NO:10) which corresponds 
to amino acid residues 335-444 of the IHNV sequence described in: Koener, 
J. F. et al 1987 "Nucleotide Sequence of a cDNA Clone Carrying the 
Glycoprotein Gene of Infectious Hematopoietic Necrosis Virus, a Fish 
Rhabdovirus"; Journal of Virology 61:1342-1349. Anti-IHNV polyclonal 
antibody against whole IHNV obtained from Dr. Joann Leong, Dept. of 
Microbiology, Oregon State University, U.S.A. (see: Xu, L. et al 1991 
"Epitope Mapping and Characterization of the Infectious Hematopoietic 
Necrosis Virus Glycoprotein, Using Fusion Proteins Synthesized in 
Escherichia coli"; Journal of Virology 65:1611-1615) was used in the 
immunoblot assays described in the preceding Example to screen for 
Caulobacter that express and present the IHNV sequence on the surface of 
the S-layer of the Caulobacter. Reaction in the whole cell colony 
immunoblot assay was positive in respect of insertions at approximate site 
locations 521 and 705, and negative at approximate site locations 588 and 
638. The IHNV insert contains a single cysteine residue and is an 
extremely large insert for successful expression as a fusion product with 
a bacterial surface protein. Yet, 50% of the sites tested resulted in 
successful production and presentation of the epitope on the cell surface. 
This invention now being described, it will be apparent to one of ordinary 
skill in the art that changes and modifications can be made thereto 
without departing from the spirit or scope of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 10 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ACGTCCTAGGC GAGCTCCAGCTGGCTCCGGAGGTCTAGACCTAG44 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GTCGACGGGATCC 13 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGATCCGCGTCGAC 14 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GTCGACGCGGATCC 14 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GGATCCCGTCGAC 13 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3300 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Caulobacter crescentus 
(B) STRAIN: CB 15 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GCTATTGTCGACGTATGACGTTTGCTCTATAGCCATCGCTGCTCCCATGCGCGCCACTCG60 
GTCGCAGGGGGTGTGGGATTTTTTTTGGGAGACAATCCTCATGGCCTATACGACGGCCCA120 
GTTGGTGACTGCGTAC ACCAACGCCAACCTCGGCAAGGCGCCTGACGCCGCCACCACGCT180 
GACGCTCGACGCGTACGCGACTCAAACCCAGACGGGCGGCCTCTCGGACGCCGCTGCGCT240 
GACCAACACCCTGAAGCTGGTCAACAGCACGACGGCTGTTGCCATCCAGACCTACCAGTT 300 
CTTCACCGGCGTTGCCCCGTCGGCCGCTGGTCTGGACTTCCTGGTCGACTCGACCACCAA360 
CACCAACGACCTGAACGACGCGTACTACTCGAAGTTCGCTCAGGAAAACCGCTTCATCAA420 
CTTCTCGATCAACCTGGCCACGGGCGCCGGCGCC GGCGCGACGGCTTTCGCCGCCGCCTA480 
CACGGGCGTTTCGTACGCCCAGACGGTCGCCACCGCCTATGACAAGATCATCGGCAACGC540 
CGTCGCGACCGCCGCTGGCGTCGACGTCGCGGCCGCCGTGGCTTTCCTGAGCCGCCAGGC600 
CAACATCGA CTACCTGACCGCCTTCGTGCGCGCCAACACGCCGTTCACGGCCGCTGCCGA660 
CATCGATCTGGCCGTCAAGGCCGCCCTGATCGGCACCATCCTGAACGCCGCCACGGTGTC720 
GGGCATCGGTGGTTACGCGACCGCCACGGCCGCGATGATCAACGACCTGTCG GACGGCGC780 
CCTGTCGACCGACAACGCGGCTGGCGTGAACCTGTTCACCGCCTATCCGTCGTCGGGCGT840 
GTCGGGTTCGACCCTCTCGCTGACCACCGGCACCGACACCCTGACGGGCACCGCCAACAA900 
CGACACGTTCGTTGCGGGTGAAGTCGC CGGCGCTGCGACCCTGACCGTTGGCGACACCCT960 
GAGCGGCGGTGCTGGCACCGACGTCCTGAACTGGGTGCAAGCTGCTGCGGTTACGGCTCT1020 
GCCGACCGGCGTGACGATCTCGGGCATCGAAACGATGAACGTGACGTCGGGCGCTGCGAT1080 
C ACCCTGAACACGTCTTCGGGCGTGACGGGTCTGACCGCCCTGAACACCAACACCAGCGG1140 
CGCGGCTCAAACCGTCACCGCCGGCGCTGGCCAGAACCTGACCGCCACGACCGCCGCTCA1200 
AGCCGCGAACAACGTCGCCGTCGACGGGCGCGCCAACGTCACCGT CGCCTCGACGGGCGT1260 
GACCTCGGGCACGACCACGGTCGGCGCCAACTCGGCCGCTTCGGGCACCGTGTCGGTGAG1320 
CGTCGCGAACTCGAGCACGACCACCACGGGCGCTATCGCCGTGACCGGTGGTACGGCCGT1380 
GACCGTGGCTCAAACGGCCG GCAACGCCGTGAACACCACGTTGACGCAAGCCGACGTGAC1440 
CGTGACCGGTAACTCCAGCACCACGGCCGTGACGGTCACCCAAACCGCCGCCGCCACCGC1500 
CGGCGCTACGGTCGCCGGTCGCGTCAACGGCGCTGTGACGATCACCGACTCTGCCGCCGC1 560 
CTCGGCCACGACCGCCGGCAAGATCGCCACGGTCACCCTGGGCAGCTTCGGCGCCGCCAC1620 
GATCGACTCGAGCGCTCTGACGACCGTCAACCTGTCGGGCACGGGCACCTCGCTCGGCAT1680 
CGGCCGCGGCGCTCTGACCGCCACGCCGACCGCCAACA CCCTGACCCTGAACGTCAATGG1740 
TCTGACGACGACCGGCGCGATCACGGACTCGGAAGCGGCTGCTGACGATGGTTTCACCAC1800 
CATCAACATCGCTGGTTCGACCGCCTCTTCGACGATCGCCAGCCTGGTGGCCGCCGACGC1860 
GACGACCCTGAA CATCTCGGGCGACGCTCGCGTCACGATCACCTCGCACACCGCTGCCGC1920 
CCTGACGGGCATCACGGTGACCAACAGCGTTGGTGCGACCCTCGGCGCCGAACTGGCGAC1980 
CGGTCTGGTCTTCACGGGCGGCGCTGGCCGTGACTCGATCCTGCTGGGCGCCACGA CCAA2040 
GGCGATCGTCATGGGCGCCGGCGACGACACCGTCACCGTCAGCTCGGCGACCCTGGGCGC2100 
TGGTGGTTCGGTCAACGGCGGCGACGGCACCGACGTTCTGGTGGCCAACGTCAACGGTTC2160 
GTCGTTCAGCGCTGACCCGGCCTTCGGCGG CTTCGAAACCCTCCGCGTCGCTGGCGCGGC2220 
GGCTCAAGGCTCGCACAACGCCAACGGCTTCACGGCTCTGCAACTGGGCGCGACGGCGGG2280 
TGCGACGACCTTCACCAACGTTGCGGTGAATGTCGGCCTGACCGTTCTGGCGGCTCCGAC2340 
CGGTA CGACGACCGTGACCCTGGCCAACGCCACGGGCACCTCGGACGTGTTCAACCTGAC2400 
CCTGTCGTCCTCGGCCGCTCTGGCCGCTGGTACGGTTGCGCTGGCTGGCGTCGAGACGGT2460 
GAACATCGCCGCCACCGACACCAACACGACCGCTCACGTCGACACGCTG ACGCTGCAAGC2520 
CACCTCGGCCAAGTCGATCGTGGTGACGGGCAACGCCGGTCTGAACCTGACCAACACCGG2580 
CAACACGGCTGTCACCAGCTTCGACGCCAGCGCCGTCACCGGCACGGCTCCGGCTGTGAC2640 
CTTCGTGTCGGCCAACACCACGG TGGGTGAAGTCGTCACGATCCGCGGCGGCGCTGGCGC2700 
CGACTCGCTGACCGGTTCGGCCACCGCCAATGACACCATCATCGGTGGCGCTGGCGCTGA2760 
CACCCTGGTCTACACCGGCGGTACGGACACCTTCACGGGTGGCACGGGCGCGGATATCTT2820 
CGATATCAACGCTATCGGCACCTCGACCGCTTTCGTGACGATCACCGACGCCGCTGTCGG2880 
CGACAAGCTCGACCTCGTCGGCATCTCGACGAACGGCGCTATCGCTGACGGCGCCTTCGG2940 
CGCTGCGGTCACCCTGGGCGCTGCTGCGACCCTGGCTCAGT ACCTGGACGCTGCTGCTGC3000 
CGGCGACGGCAGCGGCACCTCGGTTGCCAAGTGGTTCCAGTTCGGCGGCGACACCTATGT3060 
CGTCGTTGACAGCTCGGCTGGCGCGACCTTCGTCAGCGGCGCTGACGCGGTGATCAAGCT3120 
GACCGGTCTGGTCACG CTGACCACCTCGGCCTTCGCCACCGAAGTCCTGACGCTCGCCTA3180 
AGCGAACGTCTGATCCTCGCCTAGGCGAGGATCGCTAGACTAAGAGACCCCGTCTTCCGA3240 
AAGGGAGGCGGGGTCTTTCTTATGGGCGCTACGCGCTGGCCGGCCTTGCCTAGTTCCGGT 3300 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1026 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetAlaTyrThrThrAlaGlnLeuValThrAlaTyrThrAsnAlaAsn 
151015 
LeuGlyLysAlaProAspAlaAlaThrThrLeuThrLeuAspAlaTyr 
202530 
AlaThrGlnThrGlnThrGlyGlyLeuSerAspAlaAlaAlaLeuThr 
354045 
AsnThrLeuLysLeuValAsnSerThrThrAlaValAlaIleGlnThr 
505560 
TyrGlnPhePheThrGlyValAlaProSerAlaAlaGlyLeuAspPhe 
65707580 
LeuValAspSerThrThrAsnThrAsnAspLeuAsnAspAlaTyrTyr 
859095 
SerLysPheAlaGlnGluAsnArgPheIleAsnPheSerIleAsn Leu 
100105110 
AlaThrGlyAlaGlyAlaGlyAlaThrAlaPheAlaAlaAlaTyrThr 
115120125 
GlyValSerTyrAlaGlnThrValAlaThrAlaTyrAspLysIleIle 
130135140 
GlyAsnAlaValAlaThrAlaAlaGlyValAspValAlaAlaAlaVal 
145150155160 
AlaPheLeuSerArgGlnAlaAsnIleAspTyrLeuThrAlaPheVal 
165170 175 
ArgAlaAsnThrProPheThrAlaAlaAlaAspIleAspLeuAlaVal 
180185190 
LysAlaAlaLeuIleGlyThrIleLeuAsnAlaAlaTh rValSerGly 
195200205 
IleGlyGlyTyrAlaThrAlaThrAlaAlaMetIleAsnAspLeuSer 
210215220 
AspGlyAlaLeuSerThrAspAsnAlaAlaGlyValAsnLeuPheThr 
225230235240 
AlaTyrProSerSerGlyValSerGlySerThrLeuS erLeuThrThr 
245250255 
GlyThrAspThrLeuThrGlyThrAlaAsnAsnAspThrPheValAla 
260265 270 
GlyGluValAlaGlyAlaAlaThrLeuThrValGlyAspThrLeuSer 
275280285 
GlyGlyAlaGlyThrAspValLeuAsnTrpVal -lnAlaAlaAlaVal 
290295300 
ThrAlaLeuProThrGlyValThrIleSerGlyIleGluThrMetAsn 
305310315 320 
ValThrSerGlyAlaAlaIleThrLeuAsnThrSerSerGlyValThr 
325330335 
GlyLeuThrAlaLeuAsnThrAsnThr SerGlyAlaAlaGlnThrVal 
340345350 
ThrAlaGlyAlaGlyGlnAsnLeuThrAlaThrThrAlaAlaGlnAla 
3553 60365 
AlaAsnAsnValAlaValAspGlyGlyAlaAsnValThrValAlaSer 
370375380 
ThrGlyValThrSerGlyThrThrThrValGl yAlaAsnSerAlaAla 
385390395400 
SerGlyThrValSerValSerValAlaAsnSerSerThrThrThrThr 
405 410415 
GlyAlaIleAlaValThrGlyGlyThrAlaValThrValAlaGlnThr 
420425430 
AlaGlyAsnAlaValAsnT hrThrLeuThrGlnAlaAspValThrVal 
435440445 
ThrGlyAsnSerSerThrThrAlaValThrValThrGlnThrAlaAla 
450 455460 
AlaThrAlaGlyAlaThrValAlaGlyArgValAsnGlyAlaValThr 
465470475480 
IleThrAspSerAlaAla AlaSerAlaThrThrAlaGlyLysIleAla 
485490495 
ThrValThrLeuGlySerPheGlyAlaAlaThrIleAspSerSerAla 
50 0505510 
LeuThrThrValAsnLeuSerGlyThrGlyThrSerLeuGlyIleGly 
515520525 
ArgGlyAlaLeuThr AlaThrProThrAlaAsnThrLeuThrLeuAsn 
530535540 
ValAsnGlyLeuThrThrThrGlyAlaIleThrAspSerGluAlaAla 
5455 50555560 
AlaAspAspGlyPheThrThrIleAsnIleAlaGlySerThrAlaSer 
565570575 
SerThrIl eAlaSerLeuValAlaAlaAspAlaThrThrLeuAsnIle 
580585590 
SerGlyAspAlaArgValThrIleThrSerHisThrAlaAlaAlaLeu 
595600605 
ThrGlyIleThrValThrAsnSerValGlyAlaThrLeuGlyAlaGlu 
610615620 
LeuAlaThrGlyL euValPheThrGlyGlyAlaGlyAlaAspSerIle 
625630635640 
LeuLeuGlyAlaThrThrLysAlaIleValMetGlyAlaGlyAspAsp 
645650655 
ThrValThrValSerSerAlaThrLeuGlyAlaGlyGlySerValAsn 
660665670 
GlyGlyAspGlyThrAspValLeuValAlaAsnValAsnGlySerSer 
675680685 
PheSerAlaAspProAlaPheGlyGlyPheGluThrLeuArgValAla 
690695700 
GlyAlaAlaAlaGlnGlySerHisAsnAlaAsnGlyPheThrAlaLeu 
705710715720 
GlnLeuGlyAlaThrAlaGlyAlaThrThrPheThrAsnValAlaVal 
725730735 
AsnValGlyLeuThrValLeuAlaAlaProThrGlyThrThrThr Val 
740745750 
ThrLeuAlaAsnAlaThrGlyThrSerAspValPheAsnLeuThrLeu 
755760765 
SerSerSerAlaAlaLeuAlaAlaGlyThrValAlaLeuAlaGlyVal 
770775780 
GluThrValAsnIleAlaAlaThrAspThrAsnThrThrAlaHisVal 
785790795800 
AspThrLeuThrLeuGlnAlaThrSerAlaLysSerIleValValThr 
805810 815 
GlyAsnAlaGlyLeuAsnLeuThrAsnThrGlyAsnThrAlaValThr 
820825830 
SerPheAspAlaSerAlaValThrGlyThrGlySerAl aValThrPhe 
835840845 
ValSerAlaAsnThrThrValGlyGluValValThrIleArgGlyGly 
850855860 
AlaGlyAlaAspSerLeuThrGlySerAlaThrAlaAsnAspThrIle 
865870875880 
IleGlyGlyAlaGlyAlaAspThrLeuValTyrThrG lyGlyThrAsp 
885890895 
ThrPheThrGlyGlyThrGlyAlaAspIlePheAspIleAsnAlaIle 
900905 910 
GlyThrSerThrAlaPheValThrIleThrAspAlaAlaValGlyAsp 
915920925 
LysLeuAspLeuValGlyIleSerThrAsnGly AlaIleAlaAspGly 
930935940 
AlaPheGlyAlaAlaValThrLeuGlyAlaAlaAlaThrLeuAlaGln 
945950955 960 
TyrLeuAspAlaAlaAlaAlaGlyAspGlySerGlyThrSerValAla 
965970975 
LysTrpPheGlnPheGlyGlyAspThr TyrValValValAspSerSer 
980985990 
AlaGlyAlaThrPheValSerGlyAlaAspAlaValIleLysLeuThr 
9951 0001005 
GlyLeuValThrLeuThrThrSerAlaPheAlaThrGluValLeuThr 
101010151020 
LeuAla 
1025 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
XaaCysThrSerAspGlnAspGluGlnPheIleProLysGlyCysSer 
15 1015 
Xaa 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 110 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ArgSerProHisProGlyIleAs nAspValTyrAlaMetHisLysGly 
151015 
SerIleTyrHisGlyMetCysMetThrValAlaValAspGluValSer 
20 2530 
LysAspArgThrThrTyrArgAlaHisArgAlaThrSerPheThrLys 
354045 
TrpGluArgProPheGlyAsp GluTrpGluGlyPheHisGlyLeuHis 
505560 
GlyAsnAsnThrThrIleIleProAspLeuGluLysTyrValAlaGln 
6570 7580 
TyrLysThrSerMetMetGluProMetSerIleLysSerValProHis 
859095 
ProSerIleLeuAlaPh eTyrAsnGluThrAspLeuSerGly 
100105110 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 330 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA to mRNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CGATCTCCACATCCCGGAATAAATGACGTCTACGCTATGCACAAAGGCTCCATCTATCAC60 
GGGATGTCCATGACGGTCGCTGTGGACGAGGTATCCAAGGACAGGACGACGTACAGGGCC120 
CATCGCGCTAC CAGCTTCACGAAATGGGAACGACCCTTTGGGGATGAGTGGGAGGGCTTT180 
CACGGATTGCACGGAAACAACACCACCATTATTCCAGACCTGGAGAAATACGTCGCCCAG240 
TACAAGACGAGCATGATGGAACCGATGAGCATCAAATCCGTACCCCATCCAAGCA TCCTG300 
GCCTTCTACAATGAGACAGACTTATCAGGG330