Nucleic acids encoding a cellulose binding domain

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

TABLE OF CONTENTS 
1. INTRODUCTION 
2. BACKGROUND OF THE INVENTION 
2.1 Immobilization of Proteins 
2.2 Cellulose 
2.3 Cellulase 
2.4 Heat Shock Protein (HSP) 
3. SUMMARY OF THE INVENTION 
4. DESCRIPTION OF THE FIGURES 
5. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS 
6. EXAMPLE: EXPERIMENTAL PROCEDURE FOR CLONING OF THE PUTATIVE CELLULOSE 
BINDING DOMAIN 
6.1 Materials and Methods 
6.1.1 Bacterial Strains and Plasmids 
6.1.2 Materials 
6.1.3 Cloning of Putative Cellulose Binding Domains 
6.1.4 Expression of CBD 
7. EXAMPLE: PURIFICATION OF CBD PROTEIN 
7.1 Preparation of CBD Protein 
7.1.1 Purification of CBD Protein 
7.1.2 Determination of the CBD-Cellulose Dissociation Constant and the 
Cellulose Binding Capacity 
7.1.3 Determination of Binding to Other Polysaccharides 
7.2 Results 
7.2.1 Purification of the CBD for Binding Analyses 
7.2.2 Time course of the Binding of the CBD to Cellulose 
7.2.3 Analysis of the CBD-Cellulose Binding Affinity and Binding Capacity 
7.2.4 Binding Site Competition 
7.2.5 Effect of pH on the Dissociation Constant 
7.2.6 Binding of the CBD to Other Polysaccharides 
7.3 Results 
8. CONSTRUCTION OF CBD-ProtA GENE AND ITS EXPRESSION IN E. COLI 
8.1 Material and Methods 
8.1.1 Enzymes and Chemicals 
8.1.2 Plasmids and Bacteria 
8.1.3 Cloning of CBD-ProtA 
8.1.4 Expression and purification of CBD-ProtA 
8.1.5 Binding Analysis of CBD-ProtA 
8.1.6 Results 
9. CLONING OF CBD-HSP FUSION PROTEIN 
10. CONSTRUCTION OF NH.sub.2 -V.sub.H -V.sub.L -CBD-CO.sub.2 
CLAIMS

5. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS 
The present invention is directed to the identification of cellulose 
binding domain (CBD) protein that is capable of binding cellulose with 
high affinity and in a reversible manner. The CBD of the present invention 
may be used, for example, in the bio-immobilization of biologically active 
molecules to cellulose. The CBD of the present invention may be fused to a 
second protein to form a CBD fusion protein. The presence of a CBD protein 
in a CBD fusion protein allows for easy and selective purification of the 
CBD fusion protein by incubation with cellulose. Examples of second 
proteins include: Protein A, protein G, streptavidin, avidin, Taq 
polymerase and other polymerases, alkaline phosphatase, RNase, DNase, 
various restriction enzymes, peroxidates, glucanases such as endo-1,4-beta 
glucanase, endo-1,3-beta-glucanase, chitinases, and others, beta and alfa 
glucosidases, beta and alfa glucoronidases, amylase, transferases such as 
glucosyl-transferases, phospho-transferases, 
chloramphenicol-acetyl-transferase, beta-lactamase and other antibiotic 
modifying and degrading enzymes, luciferase, esterases, lipases, 
proteases, bacteriocines, antibiotics, enzyme inhibitors, different growth 
factors, hormones, receptors, membranal proteins, nuclear proteins, 
transcriptional and translational factors and nucleic acid modifying 
enzymes. Specifically, the CBD protein may be fused to an antibody or an 
antigenic determinant to form a CBD fusion product that is useful in 
diagnostic kits and in immunoassays. 
Thus, for example, bodily fluids can be tested for the presence of 
particular antibodies (e.g., heat shock protein (HSP) antibody) by making 
use of a CBD and an HSP epitope. Conversely, an HSP protein, a 
cross-reactive HSP-related protein, or antigenic portions thereof can be 
detected using a CBD-HSP antibody fusion protein. 
The term "CBD" or "CBD protein" or "cellulose binding domain protein" 
refers to a protein comprising the amino acid sequence shown in FIG. 1 and 
includes functional homologs and functional derivatives thereof, provided 
that the functional homolog or functional derivative possesses the 
capability of binding to cellulose with high affinity and in a reversible 
manner. The CBD of the present invention is provided substantially free of 
other proteins with which it is naturally associated, for instance, the 
balance of the major CpbA protein, discussed above. In addition, one or 
more predetermined amino acid residues in the polypeptide may be 
substituted, inserted, or deleted, for example, to produce a CBD having 
improved biological properties, or to vary expression levels. Some of the 
desired CBD proteins falling within the scope of the present invention may 
optionally possess covalent or non-covalent modifications of the naturally 
occurring molecule, including, but not limited to, glycosylation 
modifications. Through the use of recombinant DNA technology, the CBD 
proteins of the present invention having residue deletions, substitutions 
and/or insertions may be prepared by altering the underlying nucleic acid. 
The modifications or mutations that may be made in the DNA encoding the 
CBD of the present invention must not alter the reading frame and 
preferably will not create complementary regions that could produce 
secondary mRNA structure (See, European Patent Publication No. EP 75,444) 
The CBD protein of the present invention is one having at least 70% 
sequence similarity to the amino acid sequence shown in FIG. 1 [SEQ ID NO: 
2], preferably, at least 80% sequence similarity, more preferably, at 
least 90% sequence similarity, and most preferably, at least 95% sequence 
similarity. The term "X% sequence similarity" is not intended to be 
limited to sequences having a X% sequence similarity over the entire 
length of the protein. The 70% sequence similarity is also intended to 
include X% sequence similarity occurring in identified functional areas 
within the CBD protein of FIG. 1. An example of a functional area would be 
a defined set of amino acids having the ability to bind cellulose with 
high affinity and in a reversible manner. Such protein homologs may also 
be referred to herein as "CBD functional homologs." In one embodiment of 
the present invention, such a functional area may have about 100 amino 
acids. In another embodiment of the present invention, such a functional 
area may have about 50 amino acids. The most desirable CBD protein of the 
present invention is one comprised of the amino acid sequence shown in 
FIG. 1. 
The term "CBD functional derivative" as used herein refers to any 
"fragment" "variant" "analogue" or "chemical derivative" of the CBD 
protein amino acid sequence shown in FIG. 1 which retains the capability 
of binding to cellulose with high affinity and in a reversible manner and 
is preferably between about 2 and about 160 amino acids in length, more 
preferably between about 25 and about 125 amino acids in length and most 
preferably between about 50 and about 100 amino acids in length. 
The term "fragment" is used to indicate a CBD protein which is derived from 
the CBD protein shown in FIG. 1, and has a naturally occurring sequence. 
Such a fragment may be produced by proteolytic cleavage of the full-length 
protein. Alternatively, the fragment is obtained recombinantly by 
appropriately modifying the DNA sequence encoding the CBD protein to 
delete one or more amino acids at one or more sites of the C-terminal, 
N-terminal, and within the naturally occurring sequence. Fragments of the 
CBD protein can be screened for the ability to bind cellulose with high 
affinity and in a reversible manner to determine the identity or utility 
of a functional derivative. 
The term "variant" as used herein is defined as a molecule in which the 
amino acid sequence, glycosylation pattern, or other feature of a 
naturally occurring molecule has been modified covalently or noncovalently 
and is intended to include mutants. Some of the variants falling within 
this invention possess amino acid substitutions deletions, and/or 
insertions provided that the final construct possesses the desired ability 
of binding cellulose with high affinity and in a reversible manner. Amino 
acid substitutions in the CBD protein may be made on the basis of 
similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity 
and/or the amphipathic nature of the residues involved. For example, 
negatively charged amino acids include aspartic acid and glutamic acid; 
positively charged amino acids include lysine and arginine; amino acids 
with uncharged polar head groups or nonpolar head groups having similar 
hydrophilicity values include the following: leucine, isoleucine, valine; 
glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, 
tyrosine. Also included within the definition of variant are those 
proteins having additional amino acids at one or more sites of the 
C-terminal, N-terminal, and within the naturally occurring CBD sequence as 
long as the variant retains the capability of binding cellulose with high 
affinity and in a reversible manner. 
The term "chemical derivative" as used herein refers to a CBD protein 
produced by chemical modification of naturally occurring or variant CBD 
protein. Illustrative of an example of a chemical modification would be 
replacement of H by an alkyl, acyl, or amino group. 
The phrase "binding cellulose with high affinity" as used herein refers to 
the ability of the CBD protein to bind to cellulose with a K.sub.d in 
.mu.M ranging from about 1.5 to about 0.8, preferably from about 1.4 to 
about 0.8. More preferably, the high affinity binding refers to the 
ability of the instant CBD to bind to crystalline cellulose with a K.sub.d 
of about 1.1 or less, most preferably about 1.0 or less. 
The phrase "binding cellulose in a reversible manner" as used herein refers 
to the ability of the CBD protein to be released from the cellulose-CBD 
protein binding complex by releasing agents or solutions, such as 6M urea, 
6M guanidine-HCl and other denaturing reagents, including nonionic 
surfactants. Preferably, however, those denaturing reagents are used which 
allow the released CBD or fusion product to be reconstituted. For example, 
the CBD may be reconstituted from the treatment with 6M urea or 6M 
quanidine-Hcl by subjecting the denatured protein to renaturing conditions 
described in Sections 6.1.4, 7.1.1, 7.2.1, and 8.1.4, below. 
The term "CBD fusion protein" as used herein refers to the joining together 
of at least two proteins, a CBD protein and a second protein. In some 
embodiments of the present invention, the second protein may be fused or 
joined to a third protein. In the present invention, examples of second 
proteins include enzymes, such as nucleic acid modification enzymes, 
proteases, hormones or hormone precursors, polypeptides, peptides, 
antibodies, antigens, antigenic epitopes and variants thereof. In some 
preferred embodiments of the present invention, the second protein is 
Protein A; in other preferred embodiments of the present invention, the 
second protein is an HSP protein. One preferred embodiment of the present 
invention is a fusion protein comprised of CBD protein, Protein A or 
anti-HSP recombinant IgG. The CBD fusion protein of the present invention 
may comprise an enzymatic or chemical cleavage site upstream and 
preferably adjacent the N-terminus of the second protein and/or an 
enzymatic or chemical cleavage site downstream and preferably adjacent the 
C-terminus of the second protein thereby providing a means for recovering 
the second protein from the CBD fusion protein through use of a cleaving 
agent. 
The term "CBD fusion protein-cellulose binding complex" as used herein 
refers to the complex formed when cellulose binds the CBD protein of a CBD 
fusion protein. 
"Nucleic acid" refers to a nucleotide sequence comprising a series of 
nucleic acids in a 5' to 3' phosphate diester linkage that may be either 
an RNA or a DNA sequence. If the nucleic acid is DNA, the nucleotide 
sequence is either single or double stranded. CBD protein encoding nucleic 
acid is RNA or DNA that encodes a CBD protein capable of binding cellulose 
with high affinity, is complementary to nucleic acid sequence encoding 
such CBD protein, or hybridizes to nucleic acid sequence encoding such CBD 
protein and remains stably bound to it under stringent conditions. 
The phrase "nucleic acid encoding the CBD protein of the present invention" 
includes nucleic acid of genomic, cDNA, synthetic, and semi-synthetic 
origin which, by virtue of its origin or manipulation, is not associated 
with any portion of the polynucleotide to which it is associated in 
nature, and may be linked to a polynucleotide other than that to which it 
is linked in nature, and includes single or double stranded polymers of 
ribonucleotides, deoxyribonucleotides, nucleotide analogs, or combinations 
thereof, as long as the CBD being encoded retains its ability to bind 
cellulose with high affinity. The phrase also includes various 
modifications known in the art, including but not limited to radioactive 
and chemical labels, methylation, caps, internucleotide modifications such 
those with charged linkages (e.g., phosphorothioates, phosphorodithioates, 
etc.) and uncharged linkages (e.g., methyl phosphonates, phosphotriesters, 
phosphoramidites, carbamites, etc.), as well as those containing pendant 
moieties, intercalators, chelators, etc. as long as the CBD encoded by the 
nucleic acid retains the ability to bind cellulose with high affinity and 
in a reversible manner. 
CBD encoding nucleic acid may be used to construct recombinant expression 
vectors capable of expressing the CBD protein or the CBD fusion protein of 
the present invention. A nucleic acid construct is capable of expressing a 
protein if it contains nucleotide sequences containing transcriptional and 
translational regulatory information and such sequences are "operably 
linked" to nucleotide coding sequences. "Operably linked" refers to a 
linkage in which the regulatory DNA sequences and the DNA sequence to be 
expressed are connected in such a way as to permit transcription and 
ultimately translation. 
In constructing the CBD fusion protein expression vector, the nucleic acid 
encoding the CBD protein will be linked or joined to the nucleic acid 
encoding the second protein such that the open reading frame of the CBD 
protein and the second protein is intact, allowing translation of the CBD 
fusion protein to occur. CBD nucleic acid may be obtained from a variety 
of cell sources that produce cellulose binding domains that bind with high 
affinity and in a reversible manner or that produce CBD encoding mRNA. The 
preferred source of CBD encoding nucleic acid is Clostridium 
cellulovorans. The CBD encoding nucleic acid may be obtained as described 
in Section 6.1. 
The nucleic acid encoding the CBD protein of the present invention may be 
obtained from isolated and purified RNA from cell sources or by genomic 
cloning. Either cDNA or genomic libraries of clones may be prepared using 
techniques well known in the art and may be screened for particular CBD 
encoding nucleic acid with nucleotide probes that are substantially 
complementary to any portion of the gene. If detection of CBD protein 
encoding conserved nucleotide regions is desired, nucleotide probes should 
be based on CBD nucleotide sequences conserved from species to species. If 
detection of CBD protein encoding unique nucleotide regions is desired, 
nucleotide probes should be based on unique CBD nucleotide sequences. 
Alternatively, cDNA or genomic DNA may be used as templates for PCR 
cloning with suitable oligonucleotide primers. Full length clones, i.e., 
those containing the entire coding region of the desired CBD protein may 
be selected for constructing expression vectors, or overlapping cDNAs can 
be ligated together to form a complete coding sequence. Alternatively, 
CBD-encoding DNAs may be synthesized in whole or in part by chemical 
synthesis using techniques deemed to be standard in the art. 
Many vectors are available, and selection of the appropriate vector will 
depend on 1) whether it is to be used for nucleic acid amplification or 
for nucleic acid expression, 2) the size of the nucleic acid to be 
inserted into the vector, and 3) the host cell to be transformed with the 
vector. Each vector contains various components depending on its function 
(amplification of nucleic acid or expression of nucleic acid) and the host 
cell for which it is compatible. 
The term "host cell" refers to those cells capable of growth in culture and 
capable of expressing a CBD protein or CBD fusion protein. The host cells 
of the present invention encompass cells in in vitro culture and include 
procaryotic, eucaryotic, and insect cells. A host cell strain may be 
chosen which modulates the expression of the inserted sequences, or 
modifies and processes the gene product in the specific fashion desired. 
Expression from certain promoters can be elevated in the presence of 
certain inducers (e.g., zinc and cadmium ions for metallothionine 
promoters. Therefore expression of the CBD protein or CBD fusion protein 
may be controlled. The ability to control expression will be important if 
the CBD protein or CBD fusion protein is lethal to a host cell. 
Modifications (e.g., phosphorylation) and processing (e.g., cleavage) of 
protein products are important for the function of the protein. Different 
host cells have characteristic and specific mechanisms for the 
post-translational processing and modification of protein. Appropriate 
cell lines or host systems can be chosen to ensure the correct 
modification and processing of the CBD protein or CBD fusion protein 
expressed. Preferably, the host cell should secrete minimal amounts of 
proteolytic enzymes. 
In the present invention, a host cell is provided comprised of nucleic acid 
encoding the CBD protein or CBD fusion protein of the present invention 
that is capable of binding to cellulose with high affinity. The preferred 
host cell for cloning and expression of the CBD proteins of the present 
invention is a prokaryotic cell. Procaryotes are particularly useful for 
rapid production of large amounts of nucleic acid, for production of 
single-stranded nucleic acid templates used for site-directed mutagenesis, 
for screening many mutants simultaneously, and for nucleic acid sequencing 
of the mutants generated. An example of a prokaryotic cell useful for 
cloning and expression of the CBD protein of the present invention is E. 
coli strain XL1-blue from Stratagene. Another example of a prokaryotic 
cell useful for cloning and expression of the CBD fusion protein is 
Staphylococcus aureus. 
Various expression vector/host systems may be utilized equally well by 
those skilled in the art for the recombinant expression of CBD proteins 
and CBD fusion proteins. Such systems include but are not limited to 
microorganisms such as bacteria transformed with recombinant bacteriophage 
DNA, plasmid DNA or cosmid DNA expression vectors containing the desired 
CBD coding sequence; yeast transformed with recombinant yeast expression 
vectors containing the desired CBD coding sequence; insect cell systems 
infected with recombinant virus expression vectors (e.g., baculovirus) 
containing the desired CBD coding sequence; plant cell systems infected 
with recombinant virus expression vectors (e.g., cauliflower mosaic virus 
CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid 
expression vectors (e.g., Ti plasmid) containing the desired CBD coding 
sequence; or animal cell systems infected with recombinant virus 
expression vectors (e.g. adenovirus, vaccinia virus) including cell lines 
engineered to contain multiple copies of the CBD nucleic acid either 
stably amplified (e.g., CHO/dhfr, CHO/glutamine synthetase) or unstably 
amplified in double-minute chromosomes (e.g., murine cell lines). 
Vector components generally include, but are not limited to, one or more of 
the following: a signal sequence, an origin of replication, one or more 
marker genes, an enhancer element, a promoter, and a transcription 
termination sequence. The expression elements of these vectors vary in 
their strength and specificities. Depending on the host/vector system 
utilized, any one of a number of suitable transcription and translation 
elements may be used. For example, when cloning in procaryotic cell 
systems, promoters isolated from the genome of procaryotic cells, (e.g., 
the bacterial tryptophane promoter) may be used. Promoters produced by 
recombinant DNA or synthetic techniques may also be used to provide for 
transcription of the inserted sequences. 
A signal sequence may be a component of the vector, or it may be a part of 
the CBD nucleic acid that is inserted into the vector. The signal sequence 
may be the naturally occurring CBD sequence or a non-naturally occurring 
sequence. The signal sequence should be one that is recognized and 
processed by the host cell. An origin of replication refers to the unique 
site of initiation of replication of a host organism. It is desirable for 
cloning and expression vectors to comprise a selection gene, also termed a 
selectable marker. This gene encodes a protein necessary for the survival 
or growth of transformed host cells grown in a selective culture medium. 
Host cells not transformed with the vector containing the selection gene 
will not survive in the culture medium. Typical selection genes encode 
proteins that confer resistance to antibiotics or other toxins, e.g. 
ampicillin; complement auxotrophic deficiencies; or supply critical 
nutrients not available from complex media. One example of a selection 
scheme utilizes a drug to arrest growth of a host cell. Those cells that 
are successfully transformed with a heterologous gene express a protein 
conferring drug resistance and thus survive the selection regimen. 
Expression and cloning vectors usually contain a promoter that is 
recognized by the host organism and is operably linked to the nucleic acid 
encoding the polypeptide of interest. Promoters are untranslated sequences 
located upstream (5') to the start codon of a structural gene (generally 
within about 100 to 1000 bp) that control the transcription and 
translation of a particular nucleic acid sequence, such as that encoding a 
CBD protein or CBD fusion protein, to which they are operably linked. Such 
promoters typically fall into two classes, inducible and constitutive. 
Inducible promoters are promoters that initiate increased levels of 
transcription from nucleic acid under their control in response to some 
change in culture conditions, e.g., the presence or absence of a nutrient 
or a change in temperature. At this time, a large number of promoters 
recognized by a variety of potential host cells are well known. These 
promoters are operably linked to nucleic acid encoding the polypeptide of 
interest by removing the promoter from the source nucleic acid by 
restriction enzyme digestion and inserting the isolated promoter sequence 
into the vector. Both the naturally occurring promoter sequence and many 
heterologous promoters may be used to direct amplification and/or 
expression of the polypeptide of interest. However, heterologous promoters 
are preferred, as they generally permit greater transcription and higher 
yields of expressed polypeptide of interest as compared to the naturally 
occurring promoter. In general, plasmid vectors containing promoters and 
control sequences which are derived from species compatible with the host 
cell are used with these hosts. The vector ordinarily carries a 
replication site as well as marker sequences which are capable of 
providing phenotypic selection in transformed cells. For example, E. coli 
is typically transformed using pBR322, a plasmid derived from an E. coli 
species (Bolivar, et al., Gene 2: 95 [1977]). The pBR322 plasmid contains 
genes for ampicillin and tetracycline resistance and thus provides easy 
means for identifying transformed cells. The pBR322 plasmid, or other 
microbial plasmid, must also contain or be modified to contain promoters 
and other control elements commonly used in recombinant DNA construction. 
Promoters suitable for use with prokaryotic hosts illustratively include 
the .beta.-lactamase and lactose promoter systems (Chang et al., "Nature", 
275:615 [1978]; and Goeddel et al., "Nature" 281:544 [1979]), alkaline 
phosphatase, the tryptophan (trp) promoter system (Goeddel "Nucleic Acids 
Res."8:4057 [1980] and EPO Appln. Publ. No. 36,776) and hybrid promoters 
such as the tac promoter (H. de Boer et al., "Proc. Natl. Acad. Sci. USA" 
80:21-25 [1983]). However, other functional bacterial promoters are 
suitable. Their nucleotide sequences are generally known, thereby enabling 
a skilled worker operably to ligate them to nucleic acid encoding CBD 
(Siebenlist et al., "Cell" 20:269 [1980]) using linkers or adapters to 
supply any required restriction sites. Promoters for use in bacterial 
systems also will contain a Shine-Dalgarno (S. D.) sequence operably 
linked to the nucleic acid encoding CBD. 
Expression vectors used in prokaryotic host cells will also contain 
sequences necessary for the termination of transcription and for 
stabilizing the mRNA. 
Construction of suitable vectors containing one or more of the above listed 
components and including the desired coding and control sequences employs 
standard ligation techniques. Isolated plasmids or nucleic acid fragments 
are cleaved, tailored, and religated in the form desired to generate the 
plasmids required. 
Particularly useful in the practice of this invention are expression 
vectors that provide for the expression of prokaryotic cells of nucleic 
acid encoding the CBD protein. In general, expression involves the use of 
an expression vector that is able to replicate efficiently in a host cell, 
such that the host cell accumulates many copies of the expression vector 
and, in turn, synthesizes high levels of a desired polypeptide encoded by 
the expression vector. 
Host cells are transfected and preferably transformed with the 
above-described expression or cloning vectors of this invention and 
cultured in conventional nutrient media modified as appropriate for 
inducing promoters, selecting transformants, or amplifying the genes 
encoding the desired sequences. 
"Transformation" means introducing nucleic acid into an organism so that 
the nucleic acid is replicable, either as an extrachromosomal element or 
by chromosomal integration. Unless indicated otherwise, the method used 
herein for transformation of the host cells is the method of calcium 
treatment using calcium chloride as described by Cohen, F. N. et al., 
Proc. Natl. Acad. Sci. (USA),69:2110 (1972). 
For analysis to confirm correct sequences in plasmids constructed, the 
ligation mixtures are used to transform E. coli K12 strain 294 (ATCC 
31446) and successful transformants selected by ampicillin or tetracycline 
resistance where appropriate. Plasmids from the transformants are 
prepared, analyzed by restriction and/or sequenced by the method of 
Messing et al., Nucleic Acids Res. 9: 309 (1981) or by the method of Maxam 
et al., Methods in Enzymology 65:499 (1980). 
Host cells may be transformed with the expression vectors of this invention 
and cultured in conventional nutrient media modified as is appropriate for 
inducing promoters, selecting transformants or amplifying genes. The 
culture conditions, such as temperature, pH and the like, are those 
previously used with the host cell selected for expression, and will be 
apparent to the ordinarily skilled artisan. 
Prokaryotic cells used to produce the polypeptide of this invention are 
cultured in suitable media as described generally in Sambrook, et al. 
(1989) Electrophoresis buffers in Molecular Cloning (Nolan, C. ed.), Cold 
Spring Harbor Laboratory Press, NY, pp. B.23-24; Sambrook et al. (1989) 
Bacterial Media in Molecular Cloning (Nolan, C. ed.), Cold Spring Harbor 
Laboratory Press, NY, pp. A.1-4. 
The selection of host cells producing a CBD protein or CBD fusion protein 
of the present invention may be identified by at least four general 
approaches: 
(a) DNA-DNA, DNA-RNA or RNA antisense RNA hybridization: the presence of 
CBD proteins of the present invention can be detected by nucleic acid 
hybridization using hybridization probes and/or primers for PCR reactions 
comprising nucleotides that are homologous to the CBD coding sequence; 
(b) the presence or absence of "marker" gene functions: the selection of 
host cells having nucleic acid encoding CBD protein of the present 
invention can be identified and selected based upon the presence or 
absence or certain marker gene functions, e.g., resistance to antibiotics. 
For example, if the CBD coding sequence is inserted within a marker gene 
sequence of the cloning or expression vector, recombinants containing that 
coding sequence can be identified by the absence of the marker gene 
function. Alternatively, a marker gene can be placed in tandem with the 
CBD nucleic acid sequence under the control of the same or different 
promoter used to control the expression of the CBD coding sequence. 
Expression of the marker in response to induction or selection indicates 
expression of the CBD coding of the CBD coding sequence. 
(c) assessing the level of transcription as measured by the expression of 
CBD protein or CBD fusion protein mRNA transcripts in the host cell: 
transcriptional activity of the CBD coding region can be assessed by 
hybridization assays. For example, polyadenylated RNA can be isolated and 
analyzed by Northern blot using a probe homologous to the CBD coding 
sequence or particular portions thereof. Alternatively, total nucleic 
acids of the host cell may be extracted and assayed for hybridization to 
such probes; and 
(d) detection of the CBD protein or CBD fusion protein as measured by 
immunoassay and, by the ability of the protein to bind cellulose with high 
affinity and in a reversible manner. The expression of CBD proteins can be 
assessed immunologically, for example by Western blots or by immunoassays 
such as RIAs. The expression of CBD protein can be assayed by the ability 
of the expressed protein to bind cellulose with high affinity and in a 
reversible manner. 
The expressions "cell" and "cell culture" are used interchangeably and all 
such designations include progeny and ancestors. It is also understood 
that all progeny may not be precisely identical in DNA content, due to 
deliberate or inadvertent mutations. Mutant progeny that have the same 
function or biological activity as screened for in the cell are included. 
The phrase "stringent conditions" as used herein refers to those 
hybridizing conditions that (1) employ low ionic strength and high 
temperature for washing, for example, 0.015M NaCl/0.0015M sodium 
citrate/0.1% SDS at 50.degree. C.; (2) employ during hybridization a 
denaturing agent such as formamide, for example, 50% (vol/vol) formamide 
with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM 
sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate 
at 42.degree. C.; or (3) employ 50% formamide, 5.times. SSC (0.75M NaCl, 
0.075M Sodium pyrophosphate, 5.times. Denhardt's solution, sonicated 
salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 
42.degree. C. with washes at 42.degree. C. in 0.2.times. SSC and 0.1% SDS. 
The term "recovery" as used herein refers to the ability of the cellulose 
CBD protein complex to release the CBD protein from the cellulose-CBD 
protein binding complex under certain conditions, which conditions include 
the use of releasing agents, for example, denaturing reagents, such as 6M 
urea or 6M guanidine-HCl. Any releasing agent that has the ability of 
releasing the CBD protein from the cellulose-CBD protein binding complex 
can be used to recover the CBD protein. Preferably, the CBD is only 
temporarily denatured and not irreversibly degraded by treatment with the 
releasing agent. Thus, the CBD is recovered by reconstituting the eluted 
protein, as described in Section 7.1.1, 7.2.1, or 8.1.4. 
The use of the phrase "cleaving agents" as used herein refers to a reagent 
used to cleave the CBD protein or CBD fusion protein specifically so as to 
release or excise certain components, such as the second protein of a CBD 
fusion protein, as desired. Suitable cleaving agents herein include 
enzymes, such as endoproteases, prohormone convertases, e.g., PC1, PC2, 
furin, Kex2, subtilisn, or its mutants; and chemical agents, such as 
organic and inorganic acids, hydroxylamine, N-bromosuccinimide, and 
cyanogen bromide. Hydrolysis of peptide bonds catalyzed by a variety of 
proteolytic enzymes is taught in The Enzymes, 3rd Edition, Boyer, Ed., 
Academic Press, Vol. III [1971]; Meth. Enzymology., Vol. XIX, Perlman and 
Lorand, Ed., New York: Academic Press [1970]; Meth. Enzymol., Vol. XLV, 
Lorand, Ed. New York: Academic Press [1976]; Drapeau, J. Biol. Chem., 253: 
pg. 5899-5901 [1978] and Drapeau, Meth. Enzymology., 47: 89-9[1977]. For 
an extensive listing of chemical agents, see Witcop in Advances in Protein 
Chemistry., Anfinsen et al., ed., vol. 16 pg. 221-321, Academic Press, New 
York [1961], including Table III on p. 226. Other cleavage agents suitable 
herein are deemed to be understood by those skilled in the art keeping in 
mind the desired junction for cleavage and whether the reagent can act on 
reduced or oxidized forms of CBD fusion proteins. Conditions used for 
cleavage of the CBD fusion protein will depend on the cleavage agent 
employed, and the conditions will be readily apparent to one skilled in 
the art given the cleavage agent employed. 
The CBD fusion protein of the present invention is designed and constructed 
to comprise the codon(s) necessary to achieve cleavage by the desired 
cleaving agent at desired positions, i.e. upstream, preferably adjacent 
the N-terminus of the second protein of the CBD fusion protein or 
downstream, and preferably adjacent the C-terminus of the second protein 
or both if the second protein of the fusion protein is an internal amino 
acid of the fusion protein. 
The term "glycosylation" and grammatical derivatives as used herein refers 
to the post-translational modification process of adding a series of sugar 
residues to proteins to produce glycoproteins. Glycosylation can occur in 
the cytosol, the endoplasmic reticulum, or the Glogi apparatus of 
mammalian cells. Alternatively, glycosylation can be accomplished by 
synthetic methods, for example by providing an appropriate glycosyl donor. 
See, e.g., Kahne, et al. J. Am. Chem. Soc., vol. 111: pg. 6881-2 [1989]. 
This invention also relates to diagnostic detection of proteins of interest 
in test samples, especially in biological samples, such as tissue extracts 
or biological fluids, such as serum or urine through use of the CBD fusion 
protein of the present invention. The biological samples are preferably of 
mammalian origin and most preferably of human origin. A preferred protein 
of interest to be detected in a mammalian biological sample is an HSP 
protein, an HSP antibody, cross-reactive HSP-related proteins, or 
antigenic portions thereof. The presence of the HSP antibody in a 
mammalian biological sample, for example, may be predictive or indicative 
of insulin-dependent diabetes mellitus (IDDM). In one embodiment of the 
present invention, the CBD Protein A fusion protein is comprised of a 
third protein, an IgG antibody, for example, IgG anti-HSP, which is used 
to detect the presence of an antigen, for example HSP, in biological 
samples using a variety of immunoassay formats well known in the art. 
Alternatively, the second protein of the CBD fusion protein is comprised 
of an antigenic determinant, an epitope, useful in the detection of 
antibodies that recognize the antigenic determinant. A preferred epitope 
is the HSP protein. 
Protein A is a protein found in the cell wall of Staphylococcus aureus that 
binds to the Fc portion of Ig molecules and thus precipitates the 
antibodies. Protein A has utility in immunoassays, such as an RIA or 
ELISA, where it is used to isolate antibodies or antigen-antibody 
complexes. 
In the present invention, Protein A is a preferred second protein of a CBD 
fusion protein. A CBD-Protein A fusion protein has utility in diagnostic 
immunoassays that detect the presence of or measure the quantity or 
concentration of an antibody or an anitbody-antigen complex. 
A CBD-Protein A fusion protein of the present invention also has utility in 
a diagnostic kit comprised of cellulose and a CBD-fusion protein wherein 
the CBD fusion protein component retains its ability to bind both 
cellulose and IgG of a second component, for example, an antibody-antigen 
complex or an antibody. The CBD fusion protein of the present invention 
also has utility as a means for affinity purification of antibodies or 
antigenic determinants, i.e. epitopes. A preferred antigenic determinant 
of the present invention is the HSP protein, related protein or antigenic 
portion thereof. Preferred second proteins of a CBD fusion protein include 
HSP protein or anti-HSP IgG. In the present invention, CBD-HSP epitope 
fusion proteins find utility in immunoassays designed to measure 
quantities of HSP antibody found in the serum of human mammals. 
The "antibody" as used herein is meant to include polyclonal antibodies, 
monoclonal antibodies (MAbs), humanized or chimeric antibodies, single 
chain antibodies, anti-idiotypic (anti-Id) antibodies, and epitope-binding 
fragments of any of the above. An epitope refers to an antigenic 
determinant of an antigenic molecule. 
The term IgG refers to a class of antibodies. IgG is a tetramer containing 
two light chains and two heavy chains that represents 80% of all 
immunoglobulins. 
The term "detectable label" as used herein refers to any label which 
provides directly or indirectly a detectable signal and includes, for 
example, enzymes, radiolabeled molecules, fluoresors, particles, 
chemiluminesors, enzyme substrates or co-factors, enzyme inhibitors, 
magnetic particles. Examples of enzymes useful as detectable labels in the 
present invention include alkaline phosphatase and horse radish 
peroxidase. A variety of methods are available for linking the detectable 
labels to proteins of interest and include for example the use of a 
bifunctional agent, such as 4,4'-difluoro-3,3'-dinitro-phenylsulfone, for 
attaching an enzyme, for example, horse radish peroxidase, to a protein of 
interest. The attached enzyme is then allowed to react with a substrate 
yielding a reaction product which is detectable. 
Falling within the scope of the present invention is a signal amplification 
method wherein the use of a detectable label comprised of a CBD protein 
allows for detection of femtogram quantities of the substance of interest. 
In this method, a first CBD protein is part of a CBD fusion product that 
is incubated with a cellulose fiber under conditions suitable for 
formation of a cellulose-CBD fusion product binding complex. Excess 
labeled CBD, for example, CBD fused or bound to an enzyme, such as horse 
radish peroxidase or alkaline phosphatase, or a fluoresor or chemical 
stain, is incubated with the cellulose-CBD fusion product binding complex 
under conditions suitable to allow binding of the excess labeled CBD. The 
binding of excess labeled CBD to the cellulose-CBD fusion product binding 
complex in effect allows for detection of very low quantities of 
substances of interest. In the signal amplification method of the present 
invention, the preferred cellulose fiber is a pebble-milled cellulose 
fiber. The pebble-milled cellulose fiber can be stained with a variety of 
chemical dyes or mixed with calcofluor that bind the cellulose and produce 
an intense bright blue fluorescence upon uv illumination. 
Also falling within the scope of the present invention is the use of the 
Streptavidin/biotin detection system. Biotin is capable of forming a tight 
and, essentially, irreversible complex with Streptavidin. In this aspect 
of the present invention, a CBD fusion product comprised of a CBD protein 
fused to Streptavidin is provided. A nucleotide or protein is biotinylated 
through techniques deemed to be routine to those skilled in the art in 
order to form a biotinylated chimeric probe capable of binding a substance 
of interest. The biotinylated probe is incubated with a substance of 
interest, the CBD-Streptavidin is incubated with the biotinylated probe 
and a detectable label is used to measure the biotinylated probe and 
consequently the substance of interest. The detectable label may be as 
described above in the signal amplification method, or any label, such as 
a fluoresor or chemical stain. 
6. EXAMPLE: EXPERIMENTAL PROCEDURE FOR CLONING OF THE PUTATIVE CELLULOSE 
BINDING DOMAIN 
6.1 Materials and Methods 
6.1.1 Bacterial Strains and Plasmids 
E. coli Xl 1 Blue strain was from StrateGene (La Jolla, Calif.), and was 
used for all cloning experiments. E. coli BL21 (DE3) and pET-8c were as 
described (Studier et al., J. Mol. Biol. vol. 189, pp. 113-130 [1986]). 
6.1.2 Materials 
PC buffer, pH 7, contained 50 mM KH.sub.2 PO.sub.4, 10 mM Na.sub.3 3C.sub.6 
H.sub.5 O.sub.7 (sodium citrate), and 1 mM NaN.sub.3. TEDG buffer (Chang 
et al., J. Bacteriol. vol. 172, pp. 3257-3263 [1990]) contained 10 mM 
Tris, pH 7, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 5% v/v glycerol. 
Although Tris has a low buffer capacity at pH 7, the buffer was suitable 
because hydrogen ions were neither produced nor used. Restriction 
endonucleases were from BRL (Bethesda, Md.). All other chemicals used were 
of the highest purity commercially available. AVICEL (microcrystalline 
cellulose) PH101 (lot #1117) was from FMC Corp. (Philadelphia, Pa.). 
Absorbant cotton was from the Seamless Rubber Co. (New Haven, Conn.). 
Cellulin fiber was from Weyerhaeuser (Tacoma, Wash.). Granular chitin from 
crab shells is available from Sigma. All other binding substrates were 
purchased from Sigma Chemical Co., St. Louis, Mo. Each of the 
polysaccharides was washed twice with PC buffer before use. Masses of the 
polysaccharides refer to their dry weights as supplied from the 
manufacturer, except for nigeran, cellulin, and cotton. These three 
binding substrates had large particle sizes which interfered with the 
assay. Nigeran was recrystallized by dissolving the solid in hot water, 
filtering, and cooling on ice. The sizes of the cellulin and cotton fibers 
were reduced by processing with a Gifford-Wood mini-mill for five minutes. 
6.1.3 Cloning of Putative Cellulose Binding Domains 
DNA primers complementary to the regions of cbpA (Shoseyov et al., PNAS USA 
vol. 89, pp. 3483-3487 [1992]) flanking the putative CBD (CbpA residues 
28-189) were synthesized by a Gene Assembler Plus (Pharmacia). The forward 
primer contained an NcoI restriction site (recognition sequence: CCATGG) 
with the ATG in-frame with the gene fragment, to act as a translational 
start codon when cloned into the pET-8c vector cloning site. The reverse 
primer contained a stop codon and a BamHI site. Polymerase Chain Reaction 
(PCR) was performed using 20 pmol each primer, 200 .mu.M dNTPs, and 1 ng 
cbpA DNA [cloned into vector pGEMEX-1 (Promega) as in (Shoseyov et al., 
1992, supra)] as a template, in a total volume of 100 .mu.l. Taq 
polymerase was from Amersham, using buffer conditions recommended by the 
manufacturer. PCR was carried out for 40 cycles as described (Innis et 
al., Optimization of PCRs In: PCR Protocols Ed. Innis et al., Pub. 
Academic Press, San Diego, pp. 3-12 [1990]). The PCR product was purified 
by phenol/chloroform extraction followed by ethanol precipitation and a 
wash with 70% ethanol, then dried by vacuum and resuspended in 27 .mu.l 
distilled water. The DNA was then cleaved by NcoI and BamHI, and run on a 
2.5% low melting point agarose (Nuseieve GTG, FMC) gel in TBE buffer 
(Sambrook, et al. (1989) Electrophoresis buffers in Molecular Cloning 
(Nolan, C. ed.), Cold Spring Harbor Laboratory Press, NY, pp. B.23-24. DNA 
bands stained by ethidium bromide were visualized by long-wave ultraviolet 
light and cut from the gel. The vector, plasmid pET-8c, was prepared by 
cleaving 1 .mu.g of pET-8c DNA with NcoI/BamHI and cutting the linearized 
DNA band from the gel. Vector and insert DNAs were ligated by using 100 
ng of vector DNA and 300 ng of insert with a Takara Ligation kit. The 
ligated plasmids were used to transform competent E. coli XL1-Blue strain, 
which were then plated on LB plates (Sambrook et al. (1989) Bacterial 
Media in Molecular Cloning (Nolan, C. ed.), Cold Spring Harbor Laboratory 
Press, NY, pp. A.1-4.) containing 100 .mu.g/ml Ampicillin and 12.5 
.mu.g/ml tetracycline. After overnight incubation at 37.degree. C., 
colonies were selected and grown in liquid LB media with ampicillin and 
tetracycline. Plasmid DNA from each culture was rescued as described 
(Sambrook, et al. (1989) in Molecular Cloning (Nolan, C. ed.), Cold Spring 
Harbor Laboratory Press, NY) and cleaved with restriction enzymes to 
verify the insertion of the gene fragment. The insert sequence was 
confirmed by DNA sequencing using the same procedures as those reported in 
Shoseyov et al. (1992) PNAS USA 89:3483-3487. 
6.1.4 Expression of CBD 
The overexpression vector (pET-CBD) enables us to overproduce the 17 kDa 
CBD in E. coli strain BL21(DE3). CBD was accumulated to at least 70 
mg/liter in inclusion bodies. However, additional quantity of about 20 
mg/liter of water-soluble CBD could be recovered from the water-soluble 
sonic extract of the E. coli. The cleared extract was mixed with 
(SIGMACELL 20 (20 micron average particle size cellulose)) cellulose; then 
the CBD-cellulose complex was washed by 1M NaCl solution as well as 
distilled water to remove non-specific proteins, and then pure CBD was 
eluted by 6M guanidine-HCl. CBD was fully renatured by slow dialysis at 
room temperature and regained its ability to bind to cellulose (FIG. 10. 
lane 2). 
7. EXAMPLE: PURIFICATION OF CBD PROTEIN 
7.1 Preparation of CBD Protein 
7.1.1 Purification of CBD Protein 
Plasmid DNA containing the insert was used to transform E. coli BL21 (DE3). 
Plasmid-containing cultures were grown at 37.degree. C. in NZCYM 
(Sambrook, et al. (1989) in Molecular Cloning (Nolan, C. ed.), Cold Spring 
Harbor Laboratory Press, NY) medium containing ampicillin (100 .mu.g/ml) 
with shaking to Klett reading 160 (green filter). At this point, IPTG was 
added to a final concentration 1 mM. After 4 h, the cells were harvested 
by centrifugation, resuspended in 50 mM phosphate/12 mM citrate pH 7 (PC) 
buffer containing RNAse A at 10 .mu.g/ml and DNAse I at 1 .mu.g/ml, and 
lysed by sonication on ice with a Biosonic II sonicator at maximum power 
for 45 s followed by a 15 s cooling period, repeated a total of 4 times. 
The insoluble fraction of a 1 l cell culture was collected by 
centrifugation (30 min at 12,000 g, 4.degree. C.) and resuspended in 20 ml 
of 6M guanidine HCl. This was kept on ice for 30 min with occasional 
vortexing to disperse the pellet. Insoluble debris was removed by 
centrifugation (30 min at 12,000 g, 4.degree. C.). The soluble guanidine 
HCl extract was gradually diluted to 400 ml total volume with TEDG 
renaturation buffer over a two h period at 4.degree. C. Ammonium sulfate 
was added to 80% saturation After four h at 4.degree. C. precipitated 
proteins were collected by centrifugation (30 min at 12,000 g, 4.degree. 
C.), resuspended in 40 ml PC buffer, and dialyzed against PC buffer. 
Further purification of the CBD protein fragment of CbpA was carried out by 
affinity chromatography on cellulose: Three additions of 1.0 g AVICEL.RTM. 
PH101 microcrystalline cellulose were used to remove the CBD protein from 
the solution. After each addition, the suspension was allowed to come to 
equilibrium (1 h at room temperature with slow rotation). The cellulose 
was then collected by centrifugation and removed before the next addition. 
The three grams of cellulose were washed once by 1M NaCl/PC buffer and 
twice by PC buffer. Purified CBD was eluted from the cellulose by three 
washes with 10 ml 6M urea. The urea fractions were pooled and dialyzed 
against PC buffer (4.degree. C. to about ambient temperature). Protein 
concentration in the final purified fraction was analyzed by colorimetric 
methods using the MicroBCA protein assay kit (Pierce, Rockford, Ill.), 
using bovine serum albumin (BSA) standards. 
7.1.2 Determination of the CBD-Cellulose Dissociation Constant and the 
Cellulose Binding Capacity 
Samples of CBD protein (typically 0 to 100 .mu.g) were added to 1.5 ml 
capacity microfuge tubes containing PC buffer supplemented with 1 mg/ml 
BSA and the desired amount of cellulose (typically 1 mg added from a stock 
slurry containing 10 mg/ml cellulose and 1 mg/ml BSA in PC buffer). 
Potential competitors, e.g. cellobiose (4 mg/ml) or carboxymethylcellulose 
(CMC, 4 mg/ml) were included by adding 200 .mu.l of a 20 mg/ml stock 
solution in PC/BSA buffer. The final volume was always 1 ml. The pH of the 
buffer was 7.0 unless otherwise noted. For experiments at other pH values, 
the PC/BSA buffer was adjusted by the addition of concentrated HCl or 
NaOH. 
Assay tubes were mixed by slow vertical rotation (30 RPM) at 37.degree. C. 
for one h. The samples were then spun in a microfuge for one min to 
sediment the cellulose and cellulose-protein complexes. After removing the 
buffer, the pellet was washed by resuspension in 1 ml of PC buffer. The 
wash was separated out by centrifugation and discarded. Pellets were then 
resuspended in a final 1 ml PC buffer. (The centrifugation step would not 
be expected to perturb the equilibrium because the [C] and [PC] were 
concentrated to the same extent.) 
Of the original BSA in the assay tubes (ca. 1 mg/ml), only about 0.1 .mu.g 
would remain after the washing steps assuming no non-specific adsorption 
and a liquid volume of 10 .mu.l in the pellet. Any color development due 
to this residual BSA was accounted for by the 0 CBD control tubes. 
Aliquots (150 .mu.l) of this well-mixed suspension were taken for protein 
determinations with the MicroBCA kit. The manufacturer's instructions were 
followed, except that the sample volume was brought to 0.5 ml with PC 
buffer, to which 0.5 ml of BCA working reagent was added. Assay mixtures 
were incubated at 60.degree. C. for 30 min. The protein concentration was 
determined colorimetrically from the cleared supernatants at 562 nm in a 
Shimadzu 160 U spectrophotometer. Assay tubes to which no CBD protein was 
added were used to correct for a small amount of color development caused 
by the cellulose and residual BSA. The data were compared to BSA standards 
and adjusted to accommodate the dilutions that were made to determine the 
amount of protein bound to the cellulose in each sample. The practical 
detection limit of this assay was about 0.2 .mu.g/ml. After correction for 
dilutions, this corresponds to about 0.034 nmol of CBD bound to the 
cellulose in the assay tube. The free CBD protein concentration, [P], was 
determined by subtracting the bound protein concentration, [PC], from the 
total CBD added to the tube, [P].sub.t : 
EQU [P]=[P].sub.t -[PC] (1) 
The system was analyzed assuming a simple equilibrium interaction (Segel, 
1975): 
##EQU1## 
where the dissociation constant, K.sub.d, is defined as: 
##EQU2## 
The data were analyzed by double reciprocal plots of 1/[PC] versus 1/[P] at 
different fixed levels of cellulose (equation 4) and W: 
##EQU3## 
and by Scatchard plots of [PC]/[P] versus [PC]: 
##EQU4## 
It must be noted that the cellulose is not a soluble component, and that 
[C] represents the concentration of binding sites on the cellulose surface 
exposed to the buffer, per unit volume. Similarly, the [PC] represents the 
concentration of binding site-protein complexes per unit volume. Straight 
lines were fitted to the data points by the least-squares method using 
DeltaGraphProfessional plotting application (Deltapoint, Inc., Monterey, 
Calif.). Each point was the average of three independent protein assays 
from the same binding assay tube. Experiments were performed in duplicate. 
At least two different amounts of cellulose were used to determine the 
K.sub.d and PC.sub.max /g cellulose. These values were averaged to provide 
the listed values in Table I, FIG. 9. 
7.1.3 Determination of Binding to Other Polysaccharides 
Xylan, nigeran, SEPHADEX (cross-linked Dextran) G-75, and chitin were used 
in assays to determine whether they were substrates for CBD protein. In 
all cases, the methods used were the same as those used in determining the 
binding to cellulose. Chitin exhibited a very high background in the 
MicroBCA assay, which increased proportionally to the 60.degree. C. 
incubation time, so the color development time was reduced to 15 min. 
Because of chitin's high background, only two widely different protein 
concentrations were used. 
7.2 Results 
7.2.1 Purification of the CBD for Binding Analyses 
In order to selectively produce the putative CBD region of CbpA (residues 
28-189), oligonucleotide primers were designed complementary to bases 67 
to 86 and 558 to 579 of cbpA (FIG. 1). As shown in FIG. 2, these primers 
were designed with mismatches to create an NcoI site and an ATG start 
codon on one end of the PCR product and a TAG stop codon followed by a 
BamHI site at the other end. This gene fragment was then cloned into the 
T7 RNA polymerase expression plasmid pET-8c, resulting in plasmid pET-CBD. 
See, Studier, F., and B. A. Moffatt (1986) J. Mol. Biol. 189: 113-130. The 
cloned gene fragment codes for a methionine at the N-terminus of the CBD, 
but the rest of the CBD aa sequence corresponds to residues 28 to 189 of 
CbpA. The protein fragment has a molecular weight of 17634. The insertion 
was verified by DNA sequencing. CBD protein was produced by E. coli BL21 
(DE3) cells harboring pET-CBD. After the addition of IPTG, this host 
strain produces T7 RNA polymerase, which recognizes the T7 promotor in the 
pET vector. The cbd gene fragment was under the control of this inducible 
promotor, and CBD protein was synthesized in large amounts after induction 
(FIG. 3). After a four h production period, the soluble extract from the 
lysed cells contained only small amounts of CBD protein, while most was 
found in the insoluble fraction. This protein was readily soluble in 
concentrated guanidine hydrochloride, and was renatured by slow dilution 
into TEDG buffer. It was found that protein prepared in this fashion binds 
to AVICEL (microcrystalline cellulose), verifying the putative CBD. 
Although this fraction is mostly CBD protein, the assays described require 
the protein to be highly pure. This purity is provided by a single 
cellulose-affinity binding step, as described in the Section 7.1.1. The 
affinity-purified CBD protein appears on acrylamide gels as a single band 
when stained with Coomasie brilliant blue. Approximately 70 mg of CBD 
protein can be recovered from the cells harvested from a 1 l culture. 
7.2.2 Time course of the Binding of the CBD to Cellulose 
The time course of the interaction of AVICEL (microcrystalline cellulose) 
with CBD (FIG. 4) discloses several features of the process: 
(a) At initial concentrations of 1.0 mg/ml Avicel.RTM. and 2.0 .mu.M CBD 
(i.e., [P].sub.o), a plateau value of 1.2 .mu.M complex (i.e., [PC]) is 
attained by 60 minutes. A separate experiment established that the maximum 
CBD binding capacity of the cellulose sample was 2.1 
.mu.moles.times.g.sup.-1, corresponding to an effective concentration of 
2.1 .mu.M total cellulose sites (i.e., [C].sub.o). Assuming that an 
equilibrium was established (verified below), K.sub.d defined as [P] 
[C]/[PC] is about 0.6 .mu.M. 
(b) The second-order rate constant for association (k.sub.1) calculated 
from the integrated rate equation for a reversible Bi Uni reaction 
(Capellos & Bielski, 1980; Wilkinson, 1980) is about 2.7.times.10.sup.4 
M.sup.-1 .times.min.sup.-1 (average value for points from 5-60 min). The 
rate constant for the dissociation of the complex (k.sub.-1) calculated as 
k.sup.1 K.sub.d was 1.6.times.10.sup.-2 .times.min.sup.-1 (t.sub.1/2 =43 
min). The relatively long t.sub.1/2 for complex dissociation permitted the 
C+PC pellet to be washed once without significant loss of bound CBD. 
(Resuspension and recentrifugation of the initial pellet was completed in 
less than 1 min. During this period, less than 3% of the bound CBD would 
be lost.) It was also observed that after prolonged incubation, the 
measured [PC] declined, dropping to a level of about 50% of the maximum 
value after 18 hours. This decline may be caused by gradual denaturation 
of the protein. To reduce artifacts resulting from these effects, we used 
the shortest incubation time for which equilibration appeared to be 
"complete". (Any further increase in binding beyond 60 min would be 
obscured by the experimental error.) 
7.2.3 Analysis of the CBD-Cellulose Binding Affinity and Binding Capacity 
FIG. 5 shows a typical diagnostic plot of the binding of pure CBD to AVICEL 
microcrystalline cellulose. Within experimental error, the plots were 
linear yielding a K.sub.d of about 0.6 .mu.M and a [PC].sub.max of 2.1 
.mu.moles CBD bound per gram AVICEL (microcrystalline cellulose). The 
latter value corresponds to approximately 37 mg of CBD protein per gram of 
AVICEL (microcrystalline cellulose). The linearity of the diagnostic plots 
suggests that only one type of CBD-cellulose interaction is occurring. 
The ability of CBD to bind cellulose types other than AVICEL 
(microcrystalline cellulose) was also investigated. Table 1, FIG. 9, shows 
the values for the K.sub.d and PC.sub.max found for each of the 
substrates. SIGMACELL 20 and 50 are described as microcrystalline forms of 
cellulose; these materials were also found to bind to CBD. Highly 
crystalline forms of cellulose such as absorbant cotton and CELLULON.RTM. 
fiber (crystalline cellulose from Acetobacter xylinum) were able to bind 
substantially more of the CBD (up to 6.4 .mu.mol CBD per gram of 
substrate). Fibrous and microgranular cellulose, however, which are more 
processed and thus contain less of the native crystalline form, were found 
to bind a smaller amount of the CBD. The CBD-cellulose dissociation 
constant was about the same for all forms of cellulose, while the 
PC.sub.max varied over a 30-fold range. 
7.2.4 Binding Site Competition 
To determine if soluble carbohydrates competed with AVICEL 
(microcrystalline cellulose) for the CBD protein, cellobiose (a .beta.-1,4 
linked glucose dimer) and CMC (a soluble derivative of cellulose) were 
included in some assays at 4 times the weight/volume of AVICEL 
(microcrystalline cellulose) (1 mg Avicel.RTM., 4 mg cellobiose or CMC per 
ml assay). As Table 1, FIG. 9, shows, no significant differences in the 
K.sub.d or PC.sub.max were observed, indicating these soluble 
carbohydrates did not affect the binding of the CBD to AVICEL 
(microcrystalline cellulose). 
7.2.5 Effect of pH on the Dissociation Constant 
C. cellulovorans is a neutrophilic organism, thriving only around pH 7 
(Sleat et al., 1984), so this pH was used for most of the binding assays. 
However, other experiments established that the K.sub.d and PC.sub.max did 
not vary significantly with changes in pH over the range 5.0 to 8.0. In 
addition, it was noted that PC buffers as acidic as pH 3.5 or as basic as 
9.5 would not remove the CBD from AVICEL (microcrystalline cellulose) 
during 1-min washes. 
7.2.6 Binding of the CBD to Other Polysaccharides 
Xylan, SEPHADEX (cross-linked Dextran) G-75, nigeran, and chitin were used 
to explore the binding specificity of the CBD. Of these, only chitin 
showed measurable binding of the CBD peptide Table 1, FIG. 9. The 
chitin-CBD K.sub.d and binding capacity were similar to the AVICEL 
(microcrystalline cellulose)-CBD values. 
7.3 Results 
Our results show that CbpA contains a domain responsible for cellulose 
binding, and that this domain could function independently from the rest 
of CbpA. Because the purification protocol employed denaturation and 
renaturation steps, the fact that the purified protein was functional 
indicates that the CBD protein sequence was sufficient for proper folding 
of the protein fragment. 
We have found non-specific binding of the CBD to the assay tubes to be a 
problem in performing equilibrium binding experiments, and have developed 
an assay in which the CBD and cellulose are equilibrated in the presence 
of excess BSA. The BSA effectively eliminates non-specific CBD 
interactions with the tube. After equilibrium is reached, the cellulose 
and cellulose-protein complexes are collected and washed, then assayed for 
bound proteins. As described earlier, the dissociation of the 
CBD-cellulose is slow so that no detectable amount is removed during a 
rapid wash step. 
The bound CBD was measured directly by the protein assay, and the free CBD 
was calculated by subtracting the bound CBD from the total CBD, as shown 
in equation 1, Section 7.1.2. This method has the advantage that any CBD 
molecules adsorbed non-specifically with low affinity to the cellulose 
would be removed by the wash step, resulting in data that more accurately 
reflect the specific, high affinity interaction between the CBD and the 
cellulose surface. As shown in FIG. 5, data gathered using this type of 
assay yields (within experimental error) linear diagnostic plots. Double 
reciprocal plots are a convenient and conventional way of determining 
binding affinities and capacities for reversible Bi uni systems. The 
validity of the assay is supported by the observation that PC.sub.max 
increases linearly with the amount of cellulose used, while K.sub.d is 
independent of the cellulose quantity. Table 1, FIG. 9, shows the results 
obtained with several forms of cellulose and other carbohydrates. The 
results indicate that cellulose types described as "crystalline" have a 
higher CBD binding capacity than highly processed celluloses that have 
lost much of their crystallinity. The fact that the PC.sub.max of 
cellulose samples vary widely with different cellulose types while the 
K.sub.d remains constant indicates that we have measured one type of 
strong protein-cellulose interaction occurring between the CBD and the 
cellulose. The lower PC.sub.max of highly processed celluloses reflects a 
smaller number of potential protein interaction sites in the sample, and 
seems to correlate with the crystallinity of the sample. This result would 
indicate, although not wishing to be limited by theory, that there is some 
special feature present in crystalline cellulose that makes it acceptable 
as a binding substrate, whereas amorphous cellulose is found lacking. 
To further characterize the substrate specificity of the CBD, we measured 
the effect of added soluble substrates (cellobiose or CMC) on cellulose 
binding. Excess cellobiose or CMC had no effect on the CBD-AVICEL 
(microcrystalline cellulose) K.sub.d or PC.sub.max, as shown in Table 1, 
FIG. 9. This lack of competition suggests that the CBD recognition site is 
specific for something more complex than a simple repeating glucose or 
cellobiose moiety, and further suggests that, perhaps, a particular 
three-dimensional arrangement of cellulose chains is needed. 
The specificity of the CBD for crystalline cellulose prompts a 
consideration of chitinases, which are known to bind tightly to chitin, a 
polymer of N-acetylglucosamine in .beta.-1,4 linkage. Like cellulose, 
chitin comes in a variety of forms, depending on the source and 
purification method used in its isolation (Cabib (1988) Methods Enzymol. 
161:460-462; Blackwell (1988) Methods Enzymol. 161:435-442). The chitin 
used for affinity purification of chitinases is .alpha.-chitin, in which 
the chains are arranged in an antiparallel fashion. This form of chitin is 
crystalline with a structure similar to that of native crystalline 
cellulose (often referred to as cellulose I). Cellulose I is the form in 
which the cellulose chains are arranged in parallel bundles, as opposed to 
cellulose II, in which the chains are in an antiparallel configuration. 
Processing of cellulose I under harsh conditions causes its disruption 
resulting in cellulose II. Both forms are crystalline, due to extensive 
hydrogen bond formation. Since our isolated CBD binds to less processed 
forms of cellulose, i.e., largely cellulose I, we were interested to see 
if the CBD would bind to .alpha.-chitin, which has a similar crystal 
structure, although of opposite strand orientation. Surprisingly, we found 
that the CBD did accept chitin as a binding substrate with a K.sub.d very 
similar to that for cellulose. Xylan (.beta.-1,4 xylose), nigeran 
(alternating a-1,4 and a-1,3 glucose), and SEPHADEX (cross-linked Dextran) 
G-75 (a-1,6 glucose with a-1,3 branches) (Coutinho et al. (1992) Mol. 
Microbiol. 6:1243-1252) were also examined, but the CBD did not show 
measurable binding to any of them under the conditions of the assay. Since 
chitin is the only one of these substrates that is crystalline, we feel 
that this demonstrates the importance of crystallinity in the substrate. 
Although this is certainly a part of the benefit of tight substrate 
binding, recent studies (Din et al. (1991) Bio/Technol. 9:1096-1099) have 
shown that binding to cellulose by isolated, non-enzymatic CBDs causes 
disruption of the cellulose fibers in a non-hydrolytic fashion. It is 
thought that the protein-cellulose binding lowers the degree of 
inter-chain hydrogen bonding near the surface of the crystal, which is 
followed by extensive hydration of these cellulose chains. The end result 
is a decrease in crystallinity. This process, termed "amorphogenesis," 
renders more of the cellulose fiber accessible to the 
endo-.beta.-1,4-glucanases. As we have discovered, the CBD exhibits no 
such amorphogenic effect. 
8. CONSTRUCTION OF CBD-ProtA GENE AND ITS EXPRESSION IN E. COLI 
8.1 Material and Methods 
8.1.1 Enzymes and Chemicals 
Chemicals were purchased from Sigma Chemicals Inc. unless stated otherwise. 
Restriction enzymes were purchased from New England Biolabs, Inc. Taq 
polymerase was purchased from Promega, Inc. 
8.1.2 Plasmids and Bacteria 
The plasmid pCB1 carrying cbpA (Shoseyov et al. 1992) was used to amplify 
cbd by PCR methods. The expression vector pRIT2 (Nilsson, et al (1985) 
EMBO J. 4(4):1075-1080) was used for the construction of the fusion genes. 
Initial transformations were conducted using E. coli strain XL1-blue 
(Strategene). Expression of CBD-ProtA was conducted in the temperature 
sensitive repressor containing strain, 2097. Also, E. coli strain N4830-1 
carries the temperature-sensitive repressor ci857 and is equivalent to E. 
coli strain 2097. The E. coli strain N4830-1 is available from Pharmacia, 
Inc. 
8.1.3 Cloning of CBD-ProtA 
CBD was PCR amplified using cbpA gene as a template. Primers A (N-terminal 
primer [SEQ ID NO: 14]; '5-GGGGGAATTCCATGGCAGCGACAT-'3) contain EcoRI 
site, primer B (C-terminal primer [SEQ ID NO: 15]; 
'5-GGGGGGATCCTATGGTGCT-'3) contain a stop codon followed by a BamHI site. 
The primers were designed and synthesized in a way that enable force 
cloning of EcoRI/BamHI 500 bp DNA fragment into the plasmid pRIT2 "in 
frame" fused to the C-terminal part of Protein A gene. PCR conditions were 
as described by Innis and Gelfand (1990) with the following modifications: 
2 ng template DNA (cbpA) and 1 mM MgCl.sub.2 were used in the reaction 
mixture. The reaction was conducted using a programmable thermal 
controller (M&J Research Inc.). Standard DNA manipulations were conducted 
according to Sambrook et al (1989) in Molecular Cloning (Nolan, C. ed.), 
Cold Spring Harbor Laboratory Press, NY. The PCR amplified product was 
digested with EcoRI and BamHI, and the expected 500 bp DNA fragment (FIG. 
1) was isolated from 1.5% agarose gel using QIAEX gel extraction kit 
(Qiagen Inc.). The EcoRI/BamHI fragment was ligated into EcoRI/BamHI 
linearized pRIT2 using T4 Ligase. The ligation mixture was used to 
transform XL1-Blue competent cells and transformed colonies were selected 
on LB agar plates containing 100 mg/l ampicillin. Successful construct 
containing the DNA insert was designated pCBD-ProtA1. 
8.1.4 Expression and purification of CBD-ProtA 
Expression of the fusion protein was conducted as described elsewhere 
(Nilsson, et al (1985) EMBO J. 4(4):1075-1080).40 ml LB containing 50 mg/l 
ampicillin was inoculated with 400 ul of overnight culture of E. coli 
strain 2097 containing pCBD-ProtA1. The culture was grown to 100 Klett 
(green filter) at 30.degree. C. and then shifted to 42.degree. C. for 45 
minutes and then grown for additional 2 hours at 40.degree. C. The cells 
were harvested by centrifugation at 2,000 g for 10 min. and resuspended in 
10 ml of 20 mM Tris-HCl buffer pH 7. The cells were lysed using W-385 
sonicator (Heat Systems-Ultrasonic, Inc.) at maximum power for 1 minute 
followed by a 30-second cooling period, repeated 3 times. The lysate was 
cleared by centrifugation (4,000 g for 10 min) and 500 mg of cellulose 
particles (Sigmacell 20, 20 microns average particle size) were added. The 
suspension was incubated for ten minutes at RT and centrifuged (2,000 g 
for 10 minutes). The supernatant was removed and the pellet was washed 
once with 5 ml of 1M NaCl to remove non-specific binding proteins and 
twice with 10 ml of deionized water. CBD-ProtA was remove from the 
cellulose by 5 ml 6M guanidine-HCl. After centrifugation (2,000 g, 10 min) 
the solution was then dialysed against 20 mM Tris-HCl buffer (pH 7). 
Proteins were analyzed on 12.5% SDS-PAGE according to Laemmli (1970). 
8.1.5 Binding Analysis of CBD-ProtA 
The binding of CBD-ProtA to IgG was determined as follows: A 100 .mu.l 
suspension of rabbit IgG (H+L)-Sepharose (8 mg/ml, Bio-Makor Inc.) that 
was prewashed with PBS (Phosphate Buffer Saline pH 7.4) was mixed with 1 
ml of isolated CBD-ProtA (50 .mu.g/ml). The mixture was incubated for 1 
hour at 8.degree. C. and then centrifuged (2,000 g, 5 minutes). The 
supernatant was removed and the pellet was washed twice with 500 .mu.l of 
PBS. Then, the CBD-ProtA was eluted with 200 .mu.l of 1M acetic acid. The 
pellet was mixed with 15 .mu.l of sample application buffer (SAB; 125 mM 
Tris-HCl pH 6.8, 4% SDS, 20% glycerol and 0.002% bromophenol blue), 15 
.mu.l samples of the different fractions were mixed with 15 .mu.l of SAB, 
then boiled for 5 minutes and analyzed on SDS-PAGE. 
The binding of CBD-ProtA to cellulose was determined as follows: 20 mg of 
cellulose SIGMACELL 20 (20 micron average particle size cellulose) were 
mixed with 200 .mu.l of isolated CBD-ProtA (50 .mu.g/ml). The mixture was 
incubated for 15 min at RT and centrifuged at 2,000 g for 5 min. The 
pellet was washed with 200 .mu.l 1M acetic acid and the pellet was 
resuspended with 40 .mu.l of SAB. The cellulose suspension was boiled 
along with the 15 .mu.l of the acetic acid wash (mixed with 15 .mu.l SAB) 
and analyzed on SDS-PAGE. 
8.1.6 Results 
It was shown that CBD-ProtA can be expressed in E. coli, and purified using 
cellulose in a one-step purification. The fusion protein has the expected 
size, 45 kDa (FIG. 8). CBD-protA retained its affinity to cellulose as 
well as to IgG. It was shown that 1M acetic acid releases the 
CBD-ProtA:IgG bond but not the CBD-ProtA:cellulose bond. 
Using this expression vector, we produced 6 mg of CBD-ProtA per 1 liter of 
culture. Our experience with the T7 overexpression system enabled us to 
produce more then 10 times that amount (70 mg/l) of pure CBD (FIG. 10). 
9. CLONING OF CBD-HSP FUSION PROTEIN 
An example for the cloning of CBD-HSP fusion protein: PCR primers for the 
amplification of HSP gene are prepared using the plasmide SJ60 as a 
template. The vector was described by Jindal et al. (1989) Mol. Cell. 
Biol. 9:2279. The primers will contain KpnI site at the N terminal of HSP 
and stop codon followed by a BamHI site at the C terminal. 
Forward primer [SEQ ID NO: 16]: 5'-ACGGTACCACTTCGGTTACCCACAGTC-3' 
Reverse primer [SEQ ID NO: 17]: 5'-GGGGATCCTACATGCCACCTCCCATTAG-3' 
In order to enable translational fusion of the C terminal part of CBD to 
the N terminal part of HSP, we introduce a KpnI site at the 3' end of cbd 
gene. This introduction achieves PCR amplification of cbd using pET-CBD as 
a template and the following primers: 
Forward primer [SEQ ID NO: 4]: 5'-GTATACCAGCCATGGCAGCG-3' 
Reverse primer [SEQ ID NO: 18]: 5'-GTACATCTGGATCCTATGGTACCGT-3' 
The amplified DNA is digested with NcoI and BamHI and is then ligated into 
NcoI/BamHI predigested pET8c vector. The ligation mixture is then used to 
transform XL1Blue and the new plasmid designated as pET-CBDK. This plasmid 
is digested with KpnI and BamHI and the KpnI/BamHI restricted HSP-PCR 
amplified fragment is ligated; transformed into XL1Blue and after 
conformation of the construct will be used to transform BL21(DE3) for 
overproduction of CBD-HSP. 
10. CONSTRUCTION OF NH.sub.2 -V.sub.H -V.sub.L -CBD-CO.sub.2 
CBD fused to recombinant antibody is carried out by cloning any desired 
V.sub.H -V.sub.L using the "Recombinant Phase Antibody System" (Pharmacia 
Inc.). The resulting pCANTAB5 plasmid carrying the V.sub.H -V.sub.L is 
used as a template for PCR amplification using the following primers: 
Forward primer [SEQ ID NO: 19]: 5'-AGCCATGGCGGCCCAGC-3' 
Reverse primer [SEQ ID NO: 20]: 5'-GGGGTACCAACAGTTTGTGCGGCC-3' 
These primers introduce the NcoI site at the 5' of the V.sub.H -V.sub.L and 
KpnI site at the 3' end. The amplified fragment is digested (partially if 
necessary) with NcoI and KpnI and is used in the expression vector of 
C-terminal fusion of CBD, below. 
To enable translational fusion of the N terminal part of CBD to the C 
terminal part of V.sub.H -V.sub.L, we introduce a KpnI site at the 3' end 
of cbd gene. This introduction is achieved by PCR amplification of cbd 
using pET-CBD as a template and the following primers: 
Forward primer [SEQ ID NO: 21]: 5'-GGGCCATGGCAGGTACCTCATCA-3' 
Reverse primer [SEQ ID NO: 9]: 5'-GTACATCTGGATCCTATGGTGCTGT-3' 
These primers introduce KpnI sit at the 5' end of cbd gene after the NcoI 
site, and maintain the stop codon followed by BamHI site at the 3' end. 
The amplified DNA is digested with NcoI and BamHI and then is ligated into 
NcoI/BamHI predigested pETSc vector. The ligation mixture is used to 
transform XL1Blue and the new plasmid is designated as pET-KCBD. This 
plasmid is digested with NcoI and KpnI and the NcoI/KpnI restricted 
V.sub.H -V.sub.L amplified fragment is then ligated; transformed into 
SL1Blue and after conformation of the construct is used to transform 
BL21(DE3) for overproduction of V.sub.H -V.sub.L -CBD fusion protein. 
In view of the above-disclosure and what is generally known in the art, it 
would be apparent to one of ordinary skill that a wide variety of CBD 
fusion products can be prepared which comprise the CBD and second proteins 
of known sequence. 
Furthermore, these clearly demonstrate the usefulness of CBD fusion 
proteins for affinity purifications of proteins and enzymes using 
cellulose as the insoluble solid matrix. Furthermore, it is also 
contemplated that the CBD fusion products offer a wide range of potential 
applications that would be apparent to one of ordinary skill in view of 
the above disclosure, including affinity separation methods and use in 
diagnostic kits. 
The following microorganisms were deposited with the American Type Culture 
Collection (ATCC), 12301 Parklawn Drive, Rockville Md. on Apr. 12 1993, 
and have been assigned the indicated accession number: 
______________________________________ 
Microorganisms Accession Number 
______________________________________ 
E.coli pCBD-ProA1/2097 
69283 
E.coli pET-CBD/BL21 (DE3) 
69282 
E.coli pCBD-ProtA1 75443 
E.coli pET-CBD 75444.-- 
______________________________________ 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 21 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 486 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..486 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCAGCGACATCATCAATGTCAGTTGAATTTTACAACTCTAACAAATCA48 
AlaAlaThrSerSerMetSerValGluPheTyrAsnSerAsnLysSer 
1 51015 
GCACAAACAAACTCAATTACACCAATAATCAAAATTACTAACACATCT96 
AlaGlnThrAsnSerIleThrProIleIleLysIleThrAsnThrSer 
202530 
GACAGTGATTTAAATTTAAATGACGTAAAAGTTAGATATTATTACACA144 
AspSerAspLeuAsnLeuAsnAspValLysValArgTyrTyrTyrThr 
354045 
AGTGATGGTACACAAGGACAAACTTTCTGGTGTGACCATGCTGGTGCA192 
SerAspGlyThrGlnGlyGlnThrPheTrpCysAspHisAlaGlyAla 
50 5560 
TTATTAGGAAATAGCTATGTTGATAACACTAGCAAAGTGACAGCAAAC240 
LeuLeuGlyAsnSerTyrValAspAsnThrSerLysValThrAlaAsn 
65 707580 
TTCGTTAAAGAAACAGCAAGCCCAACATCAACCTATGATACATATGTT288 
PheValLysGluThrAlaSerProThrSerThrTyrAspThrTyrVal 
859095 
GAATTTGGATTTGCAAGCGGAGCAGCTACTCTTAAAAAAGGACAATTT336 
GluPheGlyPheAlaSerGlyAlaAlaThrLeuLysLysGlyGlnPhe 
100105110 
ATAACTATTCAAGGAAGAATAACAAAATCAGACTGGTCAAACTACACT384 
IleThrIleGlnGlyArgIleThrLysSerAspTrpSerAsnTyrThr 
115120125 
CAAACAAATGACTATTCATTTGATGCAAGTAGTTCAACACCAGTTGTA432 
GlnThrAsnAspTyrSerPheAspAlaSerSerSerThrProValVal 
130 135140 
AATCCAAAAGTTACAGGATATATAGGTGGAGCTAAAGTACTTGGTACA480 
AsnProLysValThrGlyTyrIleGlyGlyAlaLysValLeuGlyThr 
145 150155160 
GCACCA486 
AlaPro 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 162 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AlaAlaThrSerSerMetSerValGluPheTyrAsnSerAsnLysSer 
151015 
AlaGlnThrAsnS erIleThrProIleIleLysIleThrAsnThrSer 
202530 
AspSerAspLeuAsnLeuAsnAspValLysValArgTyrTyrTyrThr 
35 4045 
SerAspGlyThrGlnGlyGlnThrPheTrpCysAspHisAlaGlyAla 
505560 
LeuLeuGlyAsnSerTyrValAspAsnThrSerLysValTh rAlaAsn 
65707580 
PheValLysGluThrAlaSerProThrSerThrTyrAspThrTyrVal 
8590 95 
GluPheGlyPheAlaSerGlyAlaAlaThrLeuLysLysGlyGlnPhe 
100105110 
IleThrIleGlnGlyArgIleThrLysSerAspTrpSerAsnTyrThr 
115120125 
GlnThrAsnAspTyrSerPheAspAlaSerSerSerThrProValVal 
130135140 
AsnProLysValThrGlyTyrI leGlyGlyAlaLysValLeuGlyThr 
145150155160 
AlaPro 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 486 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TGGTGCTGTACCAAGTACTTTAGCTCCACCTATATATCCTGTAACTTTTGGATTTACAAC60 
TGGTGTTGAACTACTTGCATCAAATGAATAGTCATTTGTTTGAGTGTAGTTTGACCAGTC120 
TGATTTTGTTATTCTTCCTTGAATAGTTATAAATTGTCCTTTTTTAAGAGTAGCTGCTCC180 
GCTTGCAAATCCAAATTCAACATATGTATCATAGGTTGATGTTGGGCTTGCTGTTTCTTT240 
AACGAAGTTTGCTGTCACTTTGCTAGTGTTATCAACATAGCTA TTTCCTAATAATGCACC300 
AGCATGGTCACACCAGAAAGTTTGTCCTTGTGTACCATCACTTGTGTAATAATATCTAAC360 
TTTTACGTCATTTAAATTTAAATCACTGTCAGATGTGTTAGTAATTTTGATTATTGGTGT420 
AATTGAGTTTGTTTGTGC TGATTTGTTAGAGTTGTAAAATTCAACTGACATTGATGATGT480 
CGCTGC486 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GTATACCAGCCATGGCAGCG20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
( B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCAAGTATACCAGCTTTAGCAGCGACA27 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GGTACAGCACCAGGTCCAGATGTACCATC29 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
( A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GATGGTACATCTGGACCTGGTGCTGTACC29 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TGTCGCTGCTAAAGCTGGTATACTTGG27 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GTACATCTGGATCCTATGGTGCTGT25 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 12..23 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GTATACCAGCCATGGCAGCG ACA23 
MetAlaAlaThr 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetAlaAlaThr 
1 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..15 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GGTACAGCACCATAGGATCCAGATGTAC28 
GlyThrAlaPro 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B ) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
GlyThrAlaPro 
1 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GGGGGAATTCCATGGCAGCGACAT24 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
( ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GGGGGGATCCTATGGTGCT19 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ACGGTACCACTTCGGTTACCCACAGTC27 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GGGGATCCTACATGCCACCTCCCATTAG28 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C ) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GTACATCTGGATCCTATGGTACCGT25 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AGCCATGGCGGCCCAGC17 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
( B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GGGGTACCAACAGTTTGTGCGGCC24 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GGGCCATGGCAGGTACCTCATCA23