PATENT DOCUMENT

Abstract:
The present invention relates to isolated  Porphorymonas gingivalis  polypeptides and nucleotides. The polypeptides include;
       an amino acid sequence selected from the group consisting of SEQ. ID. NO. 265 to SEQ. ID. NO. 528, SEQ. ID. NO. 531 and SEQ. ID. NO. 532; or   an amino acid sequence at least 85%, preferably at least 95%, identical to an amino acid sequence selected from the group consisting of SEQ. ID. NO. 265 to SEQ. ID. NO. 528, SEQ. ID. NO. 531 and SEQ. ID. NO. 532; or   at least 40 amino acids having a contiguous sequence of at least 40 amino acids identical to a contiguous amino acid sequence selected from the group consisting of SEQ. ID. NO. 265 to SEQ. ID. NO. 528, SEQ. ID. NO. 531 and SEQ. ID. NO. 532.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of application Ser. No. 09/581,286, filed Jun. 28, 2000 now abandoned, which is a 317 of PCT/AU98/01023 filed Dec. 10, 1998, the entire content of which is hereby incorporated by reference in this application. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to  P. gingivalis  nucleotide sequences,  P. gingivalis  polypeptides and probes for detection of  P. gingivalis.  The  P. gingivalis  polypeptides and nucleotides can be used in compositions for use in raising an immune response in a subject against  P. gingivalis  and treating or preventing or reducing the severity of the condition known as periodontitis. 
     BACKGROUND OF THE INVENTION 
     Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth&#39;s supporting structures. Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem. One bacterium that has attracted considerable interest is  P. gingivalis  as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas  P. gingivalis  is rarely recovered, and then in low numbers, from healthy sites. A proportional increase in the level of  P. gingivalis  in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease. The progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of  P. gingivalis.  These findings in both animals and humans suggest a major role for  P. gingivalis  in the development of adult periodontitis. 
       P. gingivalis  is a black-pigmented, anaerobic, asaccharolytic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids. The microorganism has an absolute growth requirement for iron, preferentially in the form of haeme or its Fe(III) oxidation product haemin and when grown under conditions of excess haemin is highly virulent in experimental animals. A number of virulence factors have been implicated in the pathogenicity of  P. gingivalis  including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes. 
     In order to develop an efficacious and safe vaccine to prevent, eliminate or reduce  P. gingivalis  colonisation it is necessary to identify and produce antigens that are involved in virulence that have utility as immunogens possibly through the generation of specific antibodies. Whilst it is possible to attempt to isolate antigens directly from cultures of  P. gingivalis  this is often difficult. For example as mentioned above,  P. gingivalis  is a strict anaerobe and can be difficult to isolate and grow. It is also known that, for a number of organisms, when cultured in vitro that many virulence genes are down regulated and the encoded proteins are no longer expressed. If conventional chemistry techniques were applied to purify vaccine candidates potentially important (protective) molecules may not be identified. With DNA sequencing, as the gene is present (but not transcribed) even when the organism is grown in vitro it can be identified, cloned and produced as a recombinant DNA protein. Similarly, a protective antigen or therapeutic target may be transiently expressed by the organism in vitro or produced in low levels making the identification of these molecules extremely difficult by conventional methods. 
     With serological identification of therapeutic targets one is limited to those responses which are detectable using standard methods such as Western Blotting or ELISA. The limitation here is the both the level of response that is generated by the animal or human and determining whether this response is protective, damaging or irrelevant. No such limitation is present with a sequencing approach to the identification of potential therapeutic or prophylactic targets. 
     It is also well known that  P. gingivalis  produces a range of broadly active proteases (University of Melbourne International Patent Application No PCT/AU96/00673, U.S. Pat. Nos. 5,475,097 and 5,523,390), which make the identification of intact proteins difficult because of their degradation by these proteases. 
     SUMMARY OF THE INVENTION 
     The present inventors have attempted to isolate  P. gingivalis  nucleotide sequences which can be used for recombinant production of  P. gingivalis  polypeptides and to develop nucleotide probes specific for  P. gingivalis.  The DNA sequences listed below have been selected from a large number of  P. gingivalis  sequences according to their indicative potential as vaccine candidates. This intuitive step involved comparison of the deduced protein sequence from the  P. gingivalis  DNA sequences to the known protein sequence databases. Some of the characteristics used to select useful vaccine candidates include; the expected cellular location, such as outer membrane proteins or secreted proteins, particular functional activities of similar proteins such as those with an enzymatic or proteolytic activity, proteins involved in essential metabolic pathways that when inactivated or blocked may be deleterious or lethal to the organism, proteins that might be expected to play a role in the pathogenesis of the organism e.g. red cell lysis, cell agglutination or cell receptors and proteins which are paralogues to proteins with proven vaccine efficacy. 
     In a first aspect the present invention consists an isolated antigenic  Porphorymonas gingivalis  polypeptide, the polypeptide comprising;
         an amino acid sequence selected from the group consisting of SEQ. ID. NO. 265 to SEQ. ID. NO. 528, SEQ. ID. NO. 531 and SEQ. ID. NO. 532; or   an amino acid sequence at least 85%, preferably at least 95%, identical to an amino acid sequence selected from the group consisting of SEQ. ID. NO. 265 to SEQ. ID. NO. 528, SEQ. ID. NO. 531 and SEQ. ID. NO. 532; or   at least 40 amino acids having a contiguous sequence of at least 40 amino acids identical to a contiguous amino acid sequence selected from the group consisting of SEQ. ID. NO. 265 to SEQ. ID. NO. 528, SEQ. ID. NO. 531 and SEQ. ID. NO. 532.       

     In an embodiment of the present invention the polypeptide comprises;
         an amino acid sequence selected from the group consisting of SEQ. ID. NO. 386 to SEQ. ID. NO. 528 and SEQ. ID. NO. 532; or   an amino acid sequence at least 85%, preferably at least 95%, identical to an amino acid sequence selected from the group consisting of SEQ. ID. NO. 386 to SEQ. ID. NO. 528 and SEQ. ID. NO. 532; or   at least 40 amino acids having a contiguous sequence of at least 40 amino acids identical to a contiguous amino acid sequence selected from the group consisting of SEQ. ID. NO. 386 to SEQ. ID. NO. 528 and SEQ. ID. NO. 532.       

     As used herein % identity for polypeptides is to be calculated using the alignment algorithm of Needleman and Munsch (9) using a standard protein lo scoring matrix (Blosum 50). 
     In a preferred embodiment of the present invention the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ. ID. NO. 386, SEQ. ID. NO. 424, SEQ. ID. NO. 425, SEQ. ID. NO. 434, SEQ. ID. NO. 447, SEQ. ID. NO. 458, SEQ. ID. NO. 475, SEQ. ID. NO. 498, SEQ. ID. NO. 499, SEQ. ID. NO. 500, SEQ. ID. NO. 501, SEQ. ID. NO. 387, SEQ. ID. NO. 400, SEQ. ID. NO. 411, SEQ. ID. NO. 419, SEQ. ID. NO. 420, SEQ. ID. NO. 427, SEQ. ID. NO. 429, SEQ. ID. NO. 433, SEQ. ID. NO. 437, SEQ. ID. NO. 438, SEQ. ID. NO. 443, SEQ. ID. NO. 444, SEQ. ID. NO. 448, SEQ. ID. NO. 449, SEQ. ID. NO. 452, SEQ. ID. NO. 455, SEQ. ID. NO. 457, SEQ. ID. NO. 459, SEQ. ID. NO. 461, SEQ. ID. NO. 462, SEQ. ID. NO. 463, SEQ. ID. NO. 467, SEQ. ID. NO. 468, SEQ. ID. NO. 469, SEQ. ID. NO. 482, SEQ. ID. NO. 484, SEQ. ID. NO. 485, SEQ. ID. NO. 494, SEQ. ID. NO. 508, SEQ. ID. NO. 509, SEQ. ID. NO. 510, SEQ. ID. NO. 520, SEQ. ID. NO. 521, SEQ. ID. NO. 522, SEQ. ID. NO. 525, SEQ. ID. NO. 526, SEQ. ID. NO. 528, SEQ. ID. NO. 389, SEQ. ID. NO. 390 and SEQ. ID. NO. 391. 
     In another preferred embodiment of the present invention the polypeptide comprises an amino acid sequence selected from the group consisting of residue 422 to residue 531 of SEQ. ID. NO. 303, residue 534 to residue 582 of SEQ. ID. NO. 303, residue 127 to residue 232 of SEQ. ID. NO. 301, residue 240 to residue 259 of SEQ. ID. NO. 301, residue 139 to residue 156 of SEQ. ID. NO. 295, residue 160 to residue 178 of SEQ. ID. NO. 295, residue 180 to residue 207 of SEQ. ID. NO. 295, residue 221 to residue 257 of SEQ. ID. NO. 295, residue 259 to residue 323 of SEQ. ID. NO. 295, residue 885 to residue 985 of SEQ. ID. NO. 299, residue 147 to residue 259 of SEQ. ID. NO. 363, residue 140 to residue 252 of SEQ. ID. NO. 344, residue 247 to residue 356 of SEQ. ID. NO. 353, residue 359 to residue 391 of SEQ. ID. NO. 353, residue 120 to residue 254 of SEQ. ID. NO. 300, residue 287 to residue 311 of SEQ. ID. NO. 286, residue 313 to residue 352 of SEQ. ID. NO. 286, residue 354 to residue 401 of SEQ. ID. NO. 286, residue 208 to residue 252 of SEQ. ID. NO. 287, residue 259 to residue 373 of SEQ. ID. NO. 287, residue 5 to residue 120 of SEQ. ID. NO. 293, residue 123 to residue 139 of SEQ. ID. NO. 293, residue 233 to residue 339 of SEQ. ID. NO. 265, residue 67 to residue 228 of SEQ. ID. NO. 278, residue 130 to residue 172 of SEQ. ID. NO. 274, residue 174 to residue 238 of SEQ. ID. NO. 274, residue 99 to residue 112 of SEQ. ID. NO. 274, residue 114 to residue 128 of SEQ. ID. NO. 274, residue 26 to residue 69 of SEQ. ID. NO. 285, residue 71 to residue 128 of SEQ. ID. NO. 285, residue 130 to residue 146 of SEQ. ID. NO. 285, residue 620 to residue 636 of SEQ. ID. NO. 327, residue 638 to residue 775 of SEQ. ID. NO. 327, residue 397 to residue 505 of SEQ. ID. NO. 301, residue 528 to residue 545 of SEQ. ID. NO. 301, residue 556 to residue 612 of SEQ. ID. NO. 301, residue 614 to residue 631 of SEQ. ID. NO. 301, residue 633 to residue 650 of SEQ. ID. NO. 301, residue 553 to residue 687 of SEQ. ID. NO. 299, residue 305 to residue 447 of SEQ. ID. NO. 289, residue 1 to residue 52 of SEQ. ID. NO. 364, residue 65 to residue 74 of SEQ. ID. NO. 364, residue 486 to residue 604 of SEQ. ID. NO. 275, residue 158 to residue 267 of SEQ. ID. NO. 272, residue 270 to residue 282 of SEQ. ID. NO. 272, residue 163 to residue 237 of SEQ. ID. NO. 273, residue 240 to residue 251 of SEQ. ID. NO. 273, residue 213 to residue 344 of SEQ. ID. NO. 282, residue 183 to residue 324 of SEQ. ID. NO. 292, residue 327 to residue 341 of SEQ. ID. NO. 292, residue 352 to residue 372 of SEQ. ID. NO. 292, residue 141 to residue 166 of SEQ. ID. NO. 271, residue 168 to residue 232 of SEQ. ID. NO. 271, residue 1 to residue 13 of SEQ. ID. NO. 302, residue 15 to residue 28 of SEQ. ID. NO. 302, residue 30 to residue 72 of SEQ. ID. NO. 302, residue 476 to residue 529 of SEQ. ID. NO. 277, residue 41 to residue 146 of SEQ. ID. NO. 299, residue 149 to residue 162 of SEQ. ID. NO. 299, residue 166 to residue 177 of SEQ. ID. NO. 299, residue 192 to residue 203 of SEQ. ID. NO. 299, residue 71 to residue 343 of SEQ. ID. NO. 290, residue 346 to residue 363 of SEQ. ID. NO. 290, residue 36 to residue 240 of SEQ. ID. NO. 331, residue 242 to residue 270 of SEQ. ID. NO. 331, residue 1 to residue 192 of SEQ. ID. NO. 375, residue 266 to residue 290 of SEQ. ID. NO. 375, residue 23 to residue 216 of SEQ. ID. NO. 279, residue 220 to residue 270 of SEQ. ID. NO. 279, residue 285 to residue 386 of SEQ. ID. NO. 279, residue 84 to residue 234 of SEQ. ID. NO. 297, residue 248 to residue 259 of SEQ. ID. NO. 297, residue 261 to residue 269 of SEQ. ID. NO. 297, residue 275 to residue 402 of SEQ. ID. NO. 294, residue 1 to residue 171 of SEQ. ID. NO. 298, residue 403 to residue 417 of SEQ. ID. NO. 307, residue 420 to residue 453 of SEQ. ID. NO. 307, residue 456 to residue 464 of SEQ. ID. NO. 307, residue 468 to residue 690 of SEQ. ID. NO. 307, residue 1 to residue 285 of SEQ. ID. NO. 304, residue 287 to residue 315 of SEQ. ID. NO. 304, residue 318 to residue 336 of SEQ. ID. NO. 304, residue 255 to residue 269 of SEQ. ID. NO. 342, residue 271 to residue 337 of SEQ. ID. NO. 342, residue 347 to residue 467 of SEQ. ID. NO. 281, residue 116 to residue 136 of SEQ. ID. NO. 375, residue 138 to residue 357 of SEQ. ID. NO. 375, residue 133 to residue 423 of SEQ. ID. NO. 364, residue 141 to residue 299 of SEQ. ID. NO. 305, residue 202 to residue 365 of SEQ. ID. NO. 296, residue 134 to residue 426 of SEQ. ID. NO. 288, residue 1 to residue 218 of SEQ. ID. NO. 276, residue 1 to residue 246 of SEQ. ID. NO. 280, residue 444 to residue 608 of SEQ. ID. NO. 364, residue lo to residue 686 of SEQ. ID. NO. 283, residue 1 to residue 148 of SEQ. ID. NO. 296, residue 1 to residue 191 of SEQ. ID. NO. 287, residue 193 to residue 204 of SEQ. ID. NO. 287, residue 209 to residue 373 of SEQ. ID. NO. 287, residue 211 to residue 470 of SEQ. ID. NO. 284, residue 472 to residue 482 of SEQ. ID. NO. 284, residue 133 to residue 144 of SEQ. ID. NO. 281, residue 146 to residue 336 of SEQ. ID. NO. 281, residue 1 to residue 264 of SEQ. ID. NO. 303, residue 265 to residue 295 of SEQ. ID. NO. 303, residue 297 to residue 326 of SEQ. ID. NO. 303, residue 328 to residue 338 of SEQ. ID. NO. 303, residue 247 to residue 356 of SEQ. ID. NO. 353, residue 358 to residue 391 of SEQ. ID. NO. 353, residue 257 to residue 288 of SEQ. ID. NO. 298, residue 290 to residue 385 of SEQ. ID. NO. 298, residue 245 to residue 256 of SEQ. ID. NO. 298, residue 422 to residue 802 of SEQ. ID. NO. 303, residue 803 to residue 814 of SEQ. ID. NO. 303, residue 139 to residue 156 of SEQ. ID. NO. 295, residue 160 to residue 340 of SEQ. ID. NO. 295, residue 145 to residue 361 of SEQ. ID. NO. 282, residue 363 to residue 387 of SEQ. ID. NO. 282, residue 398 to residue 471 of SEQ. ID. NO. 282, residue 573 to residue 679 of SEQ. ID. NO. 320, residue 27 to residue 168 of SEQ. ID. NO. 291, residue 170 to residue 183 of SEQ. ID. NO. 291, residue 185 to residue 415 of SEQ. ID. NO. 291, residue 1 to residue 301 of SEQ. ID. NO. 364, residue 114 to residue 702 of SEQ. ID. NO. 337, residue 377 to residue 412 of SEQ. ID. NO. 321, residue 413 to residue 772 of SEQ. ID. NO. 321, residue 14 to residue 454 of SEQ. ID. NO. 265, residue 129 to residue 614 of SEQ. ID. NO. 268, residue 1 to residue 930 of SEQ. ID. NO. 300, residue 932 to residue 1046 of SEQ. ID. NO. 300, residue 1 to residue 301 of SEQ. ID. NO. 364, residue 1 to residue 42 of SEQ. ID. NO. 381, residue 44 to residue 973 of SEQ. ID. NO. 381, residue 1 to residue 93 of SEQ. ID. NO. 358, residue 95 to residue 179 of SEQ. ID. NO. 358, residue 181 to residue 227 of SEQ. ID. NO. 358, residue 114 to residue 702 of SEQ. ID. NO. 337, residue 1 to residue 659 of SEQ. ID. NO. 355, residue 661 to residue 907 of SEQ. ID. NO. 355, residue 1 to residue 131 of SEQ. ID. NO. 370, residue 133 to residue 601 of SEQ. ID. NO. 370, residue 1 to residue 813 of SEQ. ID. NO. 344, residue 377 to residue 412 of SEQ. ID. NO. 321, residue 413 to residue 772 of SEQ. ID. NO. 321, and residue 189 to residue 614 of SEQ. ID. NO. 364. 
     In a second aspect the present invention consists in a n isolated antigenic  Porphorymonas gingivalis  polypeptide, the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ. ID. NO. 386 to SEQ. ID. NO. 528 and SEQ. ID. NO. 532 less the leader sequence set out in Table 3. 
     In a third aspect the present invention consists in an isolated DNA molecule, the DNA molecule comprising a nucleotide sequence which encodes the polypeptide of the first aspect the present invention or a sequence which hybridises thereto under stringent conditions. 
     It is preferred that the isolated DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 264, SEQ. ID. NO. 529 and SEQ. ID. NO. 530. 
     In a fourth aspect the present invention consists in a recombinant expression vector comprising the DNA molecule of the second aspect of the present invention operably linked to a transcription regulatory element. 
     The present invention also provides a cell comprising this recombinant expression vector. 
     In a further aspect the present invention consists in a method for producing a  P. gingivalis  polypeptide comprising culturing the cell under conditions that permit expression of the polypeptide. 
     In yet a further aspect the present invention provides a composition for use in raising an immune response directed against  P. gingivalis  in a subject, the composition comprising an effective amount of at least one polypeptide of the first aspect of the present invention, or at least one DNA molecule of the second aspect of the present invention, or both,and a pharmaceutically acceptable carrier. It is preferred that the pharmaceutically acceptable carrier is an adjuvant. In other aspects the present invention provides methods of treating  P. gingivalis  infection in subject comprising the administration of the composition to the subject such that treatment of  P. gingivalis  infection occurs. The treatment may be prophylactic or therapeutic. 
     In yet another aspect the present invention provides an antibody raised against a polypeptide of the first aspect the invention. The antibody may be polyclonal or monoclonal. The present invention also provides compositions including these antibodies. It is preferred that these compositions are adapted for oral use and may be, for example, dentifrices, mouthwashes, etc. 
     In a still further aspect the present invention provides a nucleotide probe comprising at least 18 nucleotides and having a contiguous sequence of at least 18 nucleotides identical to a contiguous nucleotide sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 121, SEQ. ID. NO. 529, and sequences complementary thereto. It is preferred that the probe further comprises a detectable label. 
     The present invention also provides a method for detecting the presence of  P. gingivalis  nucleic acid in a sample comprising:
         (a) contacting a sample with the nucleotide probe under conditions in which a hybrid can form between the probe and a  P. gingivalis  nucleic acid in the sample; and   (b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a  P. gingivalis  nucleic acid in the sample.       

     DETAILED DESCRIPTION 
     Definitions 
     A purified or isolated polypeptide or a substantially pure preparation of a polypeptide are used interchangeably herein and, as used herein, mean a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 mg of the polypeptide. 
     A purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells. 
     A purified or isolated or a substantially pure nucleic acid, e.g., a substantially pure DNA, (are terms used interchangeably herein) is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional  P. gingivalis  DNA sequence. 
     A “contig” as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism. 
     An “open reading frame”, also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon. 
     As used herein, a “coding sequence” is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus. A coding sequence can include but is not limited to messenger RNA synthetic DNA, and recombinant nucleic acid sequences. 
     A “complement” of a nucleic acid as used herein refers to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence. 
     A “gene product” is a protein or structural RNA which is specifically encoded by a gene. 
     As used herein, the term “probe” refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label. A label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like. Similarly, a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a “capture ligand”. Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernible to one of ordinary skill in the art using routine experimentation. 
     Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. 
     The terms peptides, proteins, and polypeptides are used interchangeably herein. 
     An “immunogenic component” as used herein is a moiety, such as an  P. gingivalis  polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal. 
     An “antigenic component” as used herein is a moiety, such as  P. gingivalis  polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex. 
     As used herein, the term “cell-specific promoter” means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue. The term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well. 
     As used herein, the term “control sequence” refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers. The term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences. 
     As used herein, the term “operably linked” refers to sequences joined or ligated to function in their intended manner. For example, a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell. 
     A “sample” as used herein refers to a biological sample, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. 
     The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present). The disclosure of these texts are incorporated herein by reference. 
     Pharmaceutically Acceptable Carriers 
     The antibodies, polypeptides and DNA of the present invention can be included in compositions which include a carrier or diluent. These compositions include pharmaceutical compositions where the carrier or diluent will be pharmaceutically acceptable. Pharmaceutically acceptable carriers or diluents include those used in compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. They are non-toxic to recipients at the dosages and concentrations employed. Representative examples of pharmaceutically acceptable carriers or diluents include, but are not limited to; water, isotonic solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline) and can also contain one or more of, mannitol, lactose, trehalose, dextrose, glycerol, ethanol or polypeptides (such as human serum albumin). The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. 
     As will be well understood by those skilled in the art alterations may be made to the amino acid sequences set out in the Sequence Listings. These alterations may be deletions, insertions, or substitutions of amino acid residues. The altered polypeptides can be either naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by performing site-directed metagenesis on the encoding DNA). It is intended that such altered polypeptides which have at least 85%, preferably at least 95% identity with the sequences set out in the Sequence Listing are within the scope of the present invention. Antibodies raised against these altered polypeptides will also bind to the polypeptides having one of the sequences set out in the Sequence Listings. The level of % identity is to be calculated as set out above. 
     Protein sequences are homologous if they are related by divergence from a common ancestor. Consequently, a species homologue of the protein will be the equivalent protein which occurs naturally in another species. Within any one species a homologue may exist as numerous allelic variants, and these will be considered homologues of the protein. Allelic variants and species homologues can be obtained by following standard techniques known to those skilled in the art. 
     An allelic variant will be a variant that is naturally occurring within an individual organism. 
     Mutants, Variants and Homology—Nucleic Acids 
     Mutant polynucleotides will possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed metagenesis on the DNA). It is thus apparent that polynucleotides of the invention can be either naturally occurring or recombinant (that is to say prepared using recombinant DNA techniques). 
     An allelic variant will be a variant that is naturally occurring within an individual organism. 
     Nucleotide sequences are homologous if they are related by divergence from a common ancestor. Consequently, a species homologue of the polynucleotide will be the equivalent polynucleotide which occurs naturally in another species. Within any one species a homologue may exist as numerous allelic variants, and these will be considered homologues of the polynucleotide. Allelic variants and species homologues can be obtained by following standard techniques known to those skilled in the art. 
     Antibody Production 
     Antibodies, either polyclonal or monoclonal, which are specific for a polypeptide of the present invention can be produced by a person skilled in the art using standard techniques such as, but not limited to, those described by Harlow et al. Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press (1988), and D. Catty (editor), Antibodies: A Practical Approach, IRL Press (1988). 
     Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a protein. For the production of polyclonal antibodies, a number of host animals are acceptable for the generation of antibodies by immunization with one or more injections of a polypeptide preparation, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response in the host animal, depending on the host species, including but not limited to Freund&#39;s (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole lympet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and  Corynebacterium parvum.    
     A monoclonal antibody to an epitope of a protein may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 493-497), and the more recent human B-cell hybridoma technique (Kesber et al. 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al. 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). In addition, techniques developed for the production of “chimeric antibodies” by splicing the genes from antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity may be used (Morrison et al. 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985 Nature 31:452-454). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce 4-specific single chain antibodies. 
     Recombinant human or humanized versions of monoclonal antibodies are a preferred embodiment for human therapeutic applications. Humanized antibodies may be prepared according to procedures in the literature (e.g. Jones et al. 1986, Nature 321:522-25; Reichman et al. 1988 Nature 332:323-27; Verhoeyen et al. 1988, Science 239:1534-36). The recently described “gene conversion metagenesis” strategy for the production of humanized monoclonal antibody may also be employed in the production of humanized antibodies (Carter et al. 1992 Proc. Natl. Acad. Sci. U.S.A. 89:4285-89). Alternatively, techniques for generating the recombinant phase library of random combinations of heavy and light regions may be used to prepare recombinant antibodies (e.g. Huse et al. 1989 Science 246:1275-81). 
     Antibody fragments which contain the idiotype of the molecule such as Fu F(ab1) and F(ab2) may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab) E2 fragment which can be produced by pepsin digestion of the intact antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragment, and the two Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Alternatively, Fab expression libraries may be constructed (Huse et al. 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragment with the desired specificity to a protein. 
     Adjuvants 
     “Adjuvant” means a composition comprised of one or more substances that enhances the immunogenicity and efficacy of a vaccine composition. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil;  Corynebacterium -derived adjuvants such as  Corynebacterium parvum; Propionibacterium -derived adjuvants such as  Propionibacterium  acne;  Mycobacterium bovis  ( Bacillus  Calmetic and Guerinn or BCG); interleukins such as interleukin 2 and interleukin-12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A; dextran sulfate; DEAE-Dextran or DHAE-Dextran with aluminium phosphate; carboxypolymethylene such as Carbopol&#39; EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); vaccinia or animal posvirus proteins; sub-viral particle adjuvants such as cholera toxin, or mixtures thereof. 
     As used herein, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO 4  at 50° C.; (2) employ during hybridisation 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° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt&#39;s solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS 
     As will be understood the present invention includes within its scope DNA vaccination. Further information regarding DNA vaccination may be found in Donnelly et al, Journal of Immunological Methods 176(1994) 145-152, the disclosure of which is incorporated herein by reference. 
     Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer, or group of elements or integers. 
     Preparation of the  P. gingivalis  Library for Sequencing. 
     To determine the DNA sequence of  P. gingivalis  genomic DNA was isolated from  P. gingivalis  strain W50 (ATCC 53978) essentially by the method described by Mamur J. (J. Mol. Biol. 3, 208-218, 1961). Cloning of DNA fragments was performed essentially as described by Fleischmann et al., (Science; 269, 496-512, 1995)(2). Briefly, purified genomic DNA from  P. gingivalis  was nebulized to fragment the DNA and was treated with Bal3l nuclease to create blunt ends then run twice through preparative 1% agarose gels. DNA fragments of 1.6-2.0 kb were excised from the gel and the DNA recovered. This DNA was then ligated to the vector pUC18 (SmaI digested and dephosphorylated; Pharmacia) and electrophoresed through a 1% preparative agarose gel. The fragment comprising linear vector plus one insert was excised, purified and this process repeated to reduce any vector without insert contamination. The recovered vector plus insert DNA was blunt-ended with T4 DNA polymerase, then a final ligation to produce circular DNA was performed. Aliquots of Epicurian Coli Electroporation-Competent Cells (Stratagene) were transformed with the ligated DNA and plated out on SOB agar antibiotic diffusion plates containing X-gal and incubated at 37° C. overnight. Colonies with inserts appeared white and those without inserts (vector alone) appeared blue. Plates were stored at 4° C. until the white clones were picked and expanded for the extraction of plasmid DNA for sequencing. 
     DNA Sequencing 
     Plasmid DNA was prepared by picking bacterial colonies into 1.5 ml of LB, TB or SOB broth supplemented with 50-100 ug/ml Ampicillin in 96 deep well plates. Plasmid DNA was isolated using the QIAprep Spin or QIAprep 96 Turbo miniprep kits (QIAGEN GmbH, Germany). DNA was eluted into a 96 well gridded array and stored at −20 C. 
     Sequencing reactions were performed using ABI PRISM Dye Terminator and ABI PRISM BIGDye Terminator Cycle Sequencing Ready Reaction kits with AmpliTaq DNA polymerase FS (PE Applied Biosystems, Foster City, Calif.) using the M13 Universal forward and reverse sequencing primers. Sequence reactions were conducted on either a Perkin-Elmer GeneAmp 9700 (PE Applied Biosystems) or Hybaid PCR Express (Hybaid, UK) thermal cyclers. Sequencing reactions were analysed on ABI PRISM 377 DNA sequencers (PE Applied Biosystems). 
     The sequences obtained are set out below. The relationship between these sequences is set out in Table 1. The initiation codon was calculated using a combination of sequence homology alignment (FASTA), signal sequence prediction (PSORT, SignalP) or ORF prediction (GeneMark). 
                                                                 TABLE 1                   Reference table indicating the relationships of each sequence ID to       the selected proteins.                DNA                       sequence   Amino acid           of   sequence of       Amino acid       Protein   complete   complete   DNA sequence   sequence of       name   ORF   ORF   of protein   protein                    PG1   1   265   122   386       PG10   2   266   123   387       PG100   3   267   124   388       PG101   4   268       PG102   5   269   125, 126   389, 390       PG104   6   270   127   391       PG105   7   271   128   392       PG106   8   272   129   393       PG107   9   273   130, 131, 132   394, 395, 396       PG108   10   274   133   397       PG109   11   275   134, 135   398, 399       PG11   12   276   136   400       PG110   13   277   137   401       PG111   14   278       PG112   15   279   138, 139   402, 403       PG113   16   280   140   404       PG114   17   281   141   405       PG115   18   282   142   406       PG116   19   283   143   407       PG117   20   284   144   408       PG118   21   285   145   409       PG119   22   286   146   410       PG12   23   287   147   411       PG120   24   288   148   412       PG121   25   289   149   413       PG122   26   290   150   414       PG123   27   291   151   415       PG124   28   292   152   416       PG125   29   293   153   417       PG126   30   294   154   418       PG13   31   295   155   419       PG14   32   296   156   420       PG15   33   297   157   421       PG16   34   298   158   422       PG18   35   299   159   423       PG2   36   300   160, 161   424, 425       PG21   37   301   162   426       PG22   38   302   163   427       PG23   39   303   164   428       PG24   40   304   165   429       PG25   41   305   166   430       PG27   42   306   167   431       PG28   43   307   168   432       PG29   44   308   169   433       PG3   45   309   170   434       PG30   46   310   171   435       PG31   47   311   172   436       PG32   48   312   173   437       PG33   49   313   174   438       PG34   50   314   175, 176   439, 440       PG35   51   315   177   441       PG36   52   316   178   442       PG37   53   317   179, 180   443, 444       PG38   54   318   181   445       PG39   55   319   182   446       PG4   56   320   183   447       PG40   57   321   184   448       PG41   58   322   185   449       PG42   59   323   186   450       PG43   60   324   187   451       PG44   61   325   188   452       PG45   62   326   189   453       PG46   63   327   190   454       PG47   64   328   191   455       PG48   65   329   192   456       PG49   66   330   193   457       PG5   67   331   194   458       PG50   68   332   195   459       PG51   69   333   196   460       PG52   70   334   197   461       PG53   71   335   198   462       PG54   72   336   199   463       PG55   73   337   200   464       PG56   74   338   201, 202   465, 466       PG57   75   339   203, 204, 205   467, 468, 469       PG58   76   340   206, 207   470, 471       PG59   77   341   208, 209, 210   472, 473, 474       PG6   78   342   211   475       PG60   79   343   212   476       PG61   80   344   213   477       PG62   81   345   214   478       PG63   82   346   215   479       PG64   83   347   216   480       PG65   84   348   217   481       PG66   85   349   218   482       PG67   86   350   219   483       PG68   87   351   220, 221   484, 485       PG69   88   352   222   486       PG7   89   353   223   487       PG70   90   354   224   488       PG71   91   355   225   489       PG72   92   356   226   490       PG73   93   357   227   491       PG74   94   358   228   492       PG75   95   359   229   493       PG76   96   360   230   494       PG77   97   361   231   495       PG78   98   362   232   496       PG79   99   363   233   497       PG8   100   364   234, 235, 236,   498, 499, 500, 501                   237       PG80   101   365   238   502       PG81   102   366   102   366       PG82   103   367   239   503       PG83   104   368   240   504       PG84   105   369   241, 242   505, 506       PG85   106   370   243   507       PG86   107   371   244, 245   508, 509       PG87   108   372   246   510       PG88   109   373   247, 248, 249   511, 512, 513       PG89   110   374   250   514       PG9   111   375   251, 252, 253   515, 516, 517       PG90   112   376   254, 255   518, 519       PG91   113   377   256   520       PG92   114   378   257   521       PG93   115   379   258   522       PG94   116   380   259   523       PG95   117   381   260   524       PG96   118   382   261   525       PG97   119   383   262   526       PG98   120   384   263   527       PG99   121   385   264   528       PG127   529   531   530   532                    
DNA Sequence Analysis
 
     DNA files in FASTA format were converted to GCG format files and imported into a database. The DNA files were translated into amino acid files using the program Flip obtained from ANGIS(Australian Genomic Information Service, University of Sydney, Australia). A series of bioinformatic analyses were performed on the proteins in order to select potential vaccine candidates. The programs used were FASTA homology searching (1), PSORT (2,3), SignalP (4), TopPred (5), and GeneMark (6). The proteins and their bioinformatic results were stored in the custom written database for search and retrieval of proteins with the desired characteristics 
     The FASTA homology results for these proteins were then examined for any alignment with a protein suggesting surface location or vaccine efficacy. All proteins were searched for homology against a non-redundant bacterial protein database compiled by ANGIS using the FASTA algorithm. The settings used for the FASTA searches were Ktup=2, gap creation penalty=−12, gap extension penalty=−2, width for deriving alignment in opt=16 and the Blosum 50 scoring matrix. Individual FASTA search results were examined for significant homology by statistical probability and amino acid alignments. The results are set out in Table 2. 
     Protein files were then trimmed to the first, second, third, fourth and fifth methionine residues using a protein trimming program (ANGIS). The trimmed proteins were then subjected to PSORT analysis for the detection of signal sequences and the prediction of cell location. Proteins exhibiting a PSORT probability of outer membrane &gt;0.8 were considered to indicate surface localisation. A second signal sequence detection program SignalP was also performed and, in certain instances, this program detected signals not identified with PSORT. All proteins identified by other methods were also analysed by PSORT and SignalP. Previously, the C-terminal amino acid of bacterial outer membrane proteins has been shown to be important for the assembly of the protein on the outer membrane (7). A typical structure definition for outer membrane proteins has been determined as the presence of a signal sequence at the N-terminus and a tyrosine or phenylalanine at the C-terminus. A number of the selected proteins exhibit this characteristic structure. The program TopPred was used to determine the presence and number of membrane spanning domains (MSDs) and the presence of such sequences indicates a preference to be attached to membranes such as the outer membrane. The results of PSORT, SignalP and TopPred analyses with the C-terminal amino acids of the selected proteins are set out in Table 3. 
     The 70 amino acids from the C-terminus of a number of  P. gingivalis  outer membrane proteins share 50-100% protein sequence identity. These proteins included RGP1, RGP2, KGP, HagA, HagC, HagD, prtH and prtT. This conserved motif may be involved in the attachment or sorting of proteins to the outer membrane. The protein data set was searched using FASTA homology as described above and a number of novel proteins were identified which demonstrate similar motifs at their C-termini. The results are listed in Table 4 
     The TonBIII box is a 30 amino acid motif present within TonB outer membrane receptors in a wide variety of bacteria. The TonBIII box of  P. gingivalis  (8) was used to search the protein data set for homology by FASTA as described above. Those proteins demonstrating significant homology are listed in Table 5. 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 FASTA protein homology results of complete ORFs against a non-redundant protein database. 
               
             
          
           
               
                   
                   
                 Genbank 
                 Length 
                 Length of 
                 FASTA homology results 
               
             
          
           
               
                 Protein 
                   
                 accession 
                 of 
                 
                   P. gingivalis 
                 
                 Identity 
                   
                   
               
               
                 name 
                 Homology description 
                 number 
                 homolog 
                 protein 
                 % 
                 Overlap 
                 E value 
               
               
                   
               
               
                 PG1 
                 48 kD outer membrane protein,  Actinobacillus   
                 U24492 
                 449aa 
                 451aa 
                 32 
                 454aa 
                 1.40E−42 
               
               
                   
                 
                   pleuropneumoniae 
                 
               
               
                 PG2 
                 Outer membrane protein (susC),  Bacteroides   
                 L49338 
                 1038aa  
                 1017aa, 1014aa 
                 28 
                 1099aa  
                 4.60E−32 
               
               
                   
                 
                   thetaiotaomicron 
                 
               
               
                 PG3 
                 Outer membrane porin F adhesin,  Pseudomonas   
                 U19743 
                 317aa 
                 223aa 
                 35 
                 187aa 
                 1.10E−10 
               
               
                   
                 
                   fluorescens 
                 
               
               
                 PG4 
                 Outer membrane protein A,  Escherichia fergusonii   
                 M63352 
                 243aa 
                 672aa 
                 48 
                  88aa 
                 4.10E−10 
               
               
                 PG5 
                 Adhesin protein (AdcA),  Streptococcus pneumoniae   
                 Z71552 
                 423aa 
                 315aa 
                 25 
                 279aa 
                 9.40E−15 
               
               
                 PG6 
                 Hemolysin A (phyA),  Prevotella melaninogenica   
                 U27587 
                 332aa 
                 324aa 
                 60 
                 306aa 
                 3.00E−74 
               
               
                 PG7 
                 Hemolysin (tlyC),  Serpulina hyodysenteriae   
                 X73141 
                 268aa 
                 404aa 
                 33 
                 266aa 
                 1.40E−24 
               
               
                 PG8 
                 Heme uptake protein A,  Bacteriodes fragilis   
                 X97122 
                 431aa 
                 598aa, 550aa, 
                 79 
                 417aa 
                 6.70E−121 
               
               
                   
                   
                   
                   
                 458aa, 426aa 
               
               
                 PG9 
                 Internalin A (inlA),  Lysteria monocytogenes   
                 M67471 
                 744aa 
                 1266aa, 1232aa, 
                 38 
                 340aa 
                 7.30E−23 
               
               
                   
                   
                   
                   
                 1174aa 
               
               
                 PG10 
                 Macrophage infectivity potentiator (MIP), 
                 U92214 
                 234aa 
                 195aa 
                 50 
                 201aa 
                 4.70E−31 
               
               
                   
                   Legionella oakridgensis . 
               
               
                 PG11 
                 Haemagglutinin (phg),  Prevotella intermedia   
                 AF017417 
                 309aa 
                 313aa 
                 44 
                 309aa 
                 3.60E−44 
               
               
                 PG12 
                 Outer membrane lipoprotein,  Haemophilus   
                 M68502 
                 274aa 
                 271aa 
                 36 
                 254aa 
                 9.60E−27 
               
               
                   
                 
                   influenzae 
                 
               
               
                 PG13 
                 Ferric receptor (cfrA),  Campylobacter coli   
                 U80812 
                 696aa 
                 757aa 
                 24 
                 625aa 
                 1.20E−18 
               
               
                 PG14 
                 36 kD antigen,  Helicobacter pylori   
                 U86610 
                 329aa 
                 331aa 
                 37 
                 326aa 
                 1.10E−35 
               
               
                 PG15 
                 Outer membrane protein,  Erwinia amylovara   
                 X77921 
                 377aa 
                 267aa 
                 30 
                 253aa 
                 5.40E−08 
               
               
                 PG16 
                 C terminal protease,  Bartonella bacilliformis   
                 L37094 
                 434aa 
                 569aa 
                 36 
                 357aa 
                 3.00E−35 
               
               
                 PG18 
                 Protein-export membrane protein (secD), 
                 AE000652 
                 503aa 
                 981aa 
                 32 
                 611aa 
                 1.10E−36 
               
               
                   
                 
                   Helicobacter pylori 
                 
               
               
                 PG21 
                 Surface antigen gene,  Methanosarcina mazei   
                 X84710 
                 783aa 
                 821aa 
                 37 
                 331aa 
                 6.20E−33 
               
               
                 PG22 
                 Alpha-hemolysin gene,  Aeromonas hydrophila   
                 L36462 
                 85aa 
                 106aa 
                 57 
                  67aa 
                 2.60E−14 
               
               
                 PG23 
                 clpA/clpB protease,  Bacillus subtilis   
                 D26185 
                 810aa 
                 859aa 
                 45 
                 855aa 
                 7.10E−122 
               
               
                 PG24 
                 Putative hemolysin,  Streptococcus mutans   
                 AF051356 
                 445aa 
                 417aa 
                 29 
                 432aa 
                 1.80E−29 
               
               
                 PG25 
                 Cysteine protease,  Porphyromonas gingivalis   
                 U54691 
                 1723aa  
                 293aa 
                 42 
                 142aa 
                 1.10E−12 
               
               
                 PG27 
                 TonB linked adhesin,  Porphyromonas gingivalis   
                 Y07618 
                 1097aa  
                 312aa 
                 45 
                 360aa 
                 3.20E−41 
               
               
                 PG28 
                 Cysteine protease/hemagglutinin,  Porphyromonas   
                 S75942 
                 886aa 
                 843aa 
                 35 
                 838aa 
                 7.00E−90 
               
               
                   
                 
                   gingivalis 
                 
               
               
                 PG30 
                 Putative NlpD lipoprotein,  Aquifex aeolicus   
                 AE000754 
                 187aa 
                 337aa 
                 42 
                 142aa 
                 1.80E−12 
               
               
                 PG31 
                 Hemolysin (tlyC),  Serpulina hyodysenteriae   
                 X73141 
                 141aa 
                 151aa 
                 31 
                 123aa 
                 1.80E−07 
               
               
                 PG32 
                 Major outer membrane protein (oprF),  Pseudomonas   
                 M94078 
                 350aa 
                 391aa 
                 26 
                 382aa 
                 3.40E−07 
               
               
                   
                 
                   aeruginosa 
                 
               
               
                 PG33 
                 Major outer membrane protein (oprF),  Pseudomonas   
                 L21200 
                 317aa 
                 385aa 
                 32 
                 163aa 
                 2.30E−06 
               
               
                   
                 
                   fluorescens 
                 
               
               
                 PG34 
                 Putative membrane protein,  Rhodobacter capsulatus   
                 Q07396 
                 193aa 
                 190aa 
                 46 
                 190aa 
                 2.20E−36 
               
               
                 PG35 
                 Colcin 1 receptor,  Escherichia coli   
                 J04229 
                 663aa 
                 833aa 
                 25 
                 590aa 
                 2.40E−10 
               
               
                 PG36 
                 Outer membrane antigen (oma87),  Pasteurella   
                 U60439 
                 789aa 
                 891aa 
                 21 
                 894aa 
                 3.70E−10 
               
               
                   
                 
                   multocida 
                 
               
               
                 PG37 
                 Cationic outer membrane protein (ompH),  Yersinia   
                 M34854 
                 164aa 
                 174aa, 170aa 
                 27 
                 168aa 
                 4.30E−07 
               
               
                   
                 
                   enterocolitica 
                 
               
               
                 PG38 
                 Cationic outer membrane protein (ompH),  Yersinia   
                 M34854 
                 164aa 
                 163aa 
                 23 
                 160aa 
                 5.90E−05 
               
               
                   
                 
                   enterocolitica 
                 
               
               
                 PG39 
                 Outer membrane protein (susC)  Bacteroides   
                 L49338 
                 1038aa  
                 827aa 
                 24 
                 347aa 
                 1.50E−06 
               
               
                   
                 
                   thetaiotaomicron 
                 
               
               
                 PG40 
                 Heme receptor (Hut A),  Vibrio cholera   
                 Q56644 
                 693aa 
                 772aa 
                 23 
                 722aa 
                 4.90E−09 
               
               
                 PG41 
                 Outer membrane protein (tolC),  Escherichia coli   
                 X54049 
                 495aa 
                 462aa 
                 22 
                 436aa 
                 4.60E−09 
               
               
                 PG42 
                 Neuraminidase,  Micromonospora viridifaciens   
                 D01045 
                 647aa 
                 492aa 
                 32 
                 375aa 
                 2.10E−22 
               
               
                 PG43 
                 Immunoreactive outer membrane protein (omp28), 
                 U30815 
                 250aa 
                 245aa 
                 24 
                 178aa 
                 0.0015 
               
               
                   
                 
                   Brucella melitensis 
                 
               
               
                 PG44 
                 Macrophage infectivity potentiator,  Legionella   
                 U92208 
                 242aa 
                 276aa 
                 35 
                 219aa 
                 9.10E−18 
               
               
                   
                 
                   israelensis 
                 
               
               
                 PG45 
                 Outer membrane protein,  Neisseria meningitidis   
                 AF021245 
                 797aa 
                 775aa 
                 21 
                 699aa 
                 0.0034 
               
               
                 PG46 
                 Outer membrane protein 85,  Neisseria gonorrhoeae   
                 UB1959 
                 792aa 
                 774aa 
                 31 
                 117aa 
                 0.00098 
               
               
                 PG47 
                 Outer membrane protein (susC)  Bacteroides   
                 L49338 
                 1038aa  
                 867aa 
                 20 
                 962aa 
                 1.00E−03 
               
               
                   
                 
                   thetaiotaomicron 
                 
               
               
                 PG48 
                 Immunoglobulin binding surface protein (sir22), 
                 X75750 
                 365aa 
                 431aa 
                 25 
                 269aa 
                 5.20E−05 
               
               
                   
                 
                   Streptococcus pyogenes 
                 
               
               
                 PG49 
                 Fimbrillin (orf2),  Porphyromonas gingivalis   
                 D42067 
                 453aa 
                 333aa 
                 23 
                 296aa 
                 0.062 
               
               
                 PG50 
                 Outer membrane protein (susC)  Bacteroides   
                 L49338 
                 1038aa  
                 848aa 
                 26 
                 579aa 
                 1.60E−11 
               
               
                   
                 
                   thetaiotaomicron 
                 
               
               
                 PG51 
                 PGaA antigen,  Porphyromonas gingivalis   
                 X95938 
                 202aa 
                 202aa 
                 54 
                 126aa 
                 1.20E−25 
               
               
                 PG52 
                 Alkaline protease secretion apparatus (aprF) 
                 X64558 
                 481aa 
                 455aa 
                 21 
                 427aa 
                 3.50E−06 
               
               
                   
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 PG53 
                 Protein export protein (tolC),  Salmonella enteritidis   
                 U25178 
                 491aa 
                 444aa 
                 23 
                 436aa 
                 6.20E−11 
               
               
                 PG54 
                 Protease I,  Achromobacter lyticus   
                 J5128 
                 653aa 
                 940aa 
                 24 
                 695aa 
                 1.50E−22 
               
               
                 PG55 
                 Fimbrillin (orf3),  Porphyromonas gingivalis   
                 D42067 
                 670aa 
                 670aa 
                 43 
                 688aa 
                 4.90E−106 
               
               
                 PG56 
                 Cysteine protease  Porphyromonas gingivalis   
                 U68468 
                 364aa 
                 1282aa, 1274aa 
                 25 
                 212aa 
                 0.00012 
               
               
                 PG57 
                 Cysteine protease,  Porphyromonas gingivalis   
                 U68468 
                 1358aa  
                 924aa, 922aa, 
                 31 
                 742aa 
                 1.40E−23 
               
               
                   
                   
                   
                   
                 921aa 
               
               
                 PG60 
                 Outer membrane protein 11,  Helicobacter pylori   
                 AE000562 
                 186aa 
                 547aa 
                 25 
                 183aa 
                 2.20E+00 
               
               
                 PG61 
                 Ferric pseudobactin M114 receptor protein (pbuA), 
                 X73412 
                 826aa 
                 749aa 
                 22 
                 585aa 
                 1.00E−05 
               
               
                   
                   Pseudomonas  sp. 
               
               
                 PG66 
                 Attachment and invasion protein (ail),  Salmonella   
                 AF007380 
                 165aa 
                 206aa 
                 21 
                 140aa 
                 1.90E+00 
               
               
                   
                 
                   typhimurium 
                 
               
               
                 PG68 
                 Serum opacity factor,  Streptococcus pyogenes   
                 U02290 
                 1025aa  
                 1225aa, 1224aa 
                 24 
                 176aa 
                 2.10E−01 
               
               
                 PG69 
                 Vacuolating cytotoxin (vacA),  Helicobacter pylori   
                 U63261 
                 160aa 
                 425aa 
                 32 
                 111aa 
                 1.20E+00 
               
               
                 PG70 
                 Outer membrane protein,  Neisseria gonorrhoea   
                 U52069 
                 174aa 
                 266aa 
                 22 
                 153aa 
                 6.90E+00 
               
               
                 PG71 
                 Gliding motility protein (gldA),  Flavobacterium   
                 AF007381 
                 578aa 
                 834aa 
                 23 
                 572aa 
                 3.90E−25 
               
               
                   
                 
                   johnsoniae 
                 
               
               
                 PG75 
                 Class 3 outer membrane porin (porB),  Neisseria   
                 U07191 
                 332aa 
                 391aa 
                 23 
                 239aa 
                 4.60E−01 
               
               
                   
                 
                   meningitidis 
                 
               
               
                 PG81 
                 Outer membrane protein (ompA),  Shigella   
                 V01344 
                 351aa 
                 &gt;235aa 
                 26 
                 186aa 
                 3.10E−01 
               
               
                   
                 
                   dysenteriae 
                 
               
               
                 PG82 
                 Outer membrane protein (alkL),  Pseudomonas   
                 X65936 
                 230aa 
                 434aa 
                 26 
                 136aa 
                 2.80E+00 
               
               
                   
                 
                   oleovorans 
                 
               
               
                 PG83 
                 Gliding motility protein (gldA),  Flavobacterium   
                 AF007381 
                 578aa 
                 926aa 
                 21 
                 639aa 
                 8.50E−09 
               
               
                   
                 
                   johnsoniae 
                 
               
               
                 PG87 
                 Hypothetical protein,  Mycobacterium tuberculosis   
                 AL021942 
                 877aa 
                 781aa 
                 29 
                 794aa 
                 2.20E−34 
               
               
                 PG89 
                 NADH-ubiquinone oxidoreductase,  Helicobacter   
                 AE000631 
                 512aa 
                 259aa 
                 24 
                 186aa 
                 3.90E−01 
               
               
                   
                 
                   pylori 
                 
               
               
                 PG91 
                 Neuraminidase (nanH),  Bacteroides fragilis   
                 D28493 
                 544aa 
                 540aa 
                 24 
                 251aa 
                 1.60E+00 
               
               
                 PG92 
                 Hypothetical protein,  Mycobacterium tuberculosis   
                 AL021942 
                 877aa 
                 771aa 
                 29 
                 770aa 
                 8.00E−30 
               
               
                 PG93 
                 Cytoadherence protein P1,  Mycoplasma pneumoniae   
                 X07191 
                 219aa 
                 776aa 
                 41 
                  63aa 
                 6.90E−01 
               
               
                 PG94 
                 Arginyl endopeptidase,  Porphyromonas gingivalis   
                 D26470 
                 991aa 
                 1157aa 
                 24 
                 328aa 
                 7.60E−08 
               
               
                 PG95 
                 Sensor protein (EVGS),  Escherichia coli   
                 D14008 
                 1197aa  
                 961aa 
                 28 
                 511aa 
                 2.60E−17 
               
               
                 PG105 
                 Plasma cell membrane glycoprotein, Human 
                 P22413 
                 873aa 
                 449aa 
                 34 
                 404aa 
                 5.60E−33 
               
               
                 PG106 
                 Hypothetical secreted protein,  Helicobacter pylori   
                 O24951 
                 242aa 
                 246aa 
                 30 
                 252aa 
                 7.80E−22 
               
               
                 PG107 
                 Cell division ATP binding protein,  Mycobacterium   
                 O32883 
                 229aa 
                 246aa, 241aa, 
                 46 
                 193aa 
                 1.20E−26 
               
               
                   
                 
                   leprae 
                 
                   
                   
                 232aa 
               
               
                 PG108 
                 ABC transporter,  Archaeoglobus fulgidis   
                 O29244 
                 228aa 
                 219aa 
                 51 
                 219aa 
                 3.80E−41 
               
               
                 PG109 
                 Proteinase IV,  Escherichia coli   
                 F64936 
                 618aa 
                 595aa, 589aa 
                 38 
                 597aa 
                 1.10E−57 
               
               
                 PG110 
                 Preprotein translocase,  Staphylococcus aureus   
                 O06446 
                 843aa 
                 523aa 
                 43 
                 521aa 
                 6.00E−71 
               
               
                 PG111 
                 ABC transporter,  Synechocystis  sp. 
                 P73758 
                 574aa 
                 &gt;720aa 
                 40 
                 579aa 
                 1.70E−73 
               
               
                 PG112 
                 Glycosyl transferase,  Erwinia amylovora   
                 Q46634 
                 351aa 
                 375aa, 362aa 
                 31 
                 363aa 
                 1.60E−32 
               
               
                 PG113 
                 Heat shock protein (dnak),  Treponema pallidum   
                 AE001203 
                 635aa 
                 640aa 
                 62 
                 644aa 
                 9.10E−138 
               
               
                 PG114 
                 Dihydrolipamide dehydrogenase,  Clostridium   
                 Q59299 
                 578aa 
                 449aa 
                 37 
                 450aa 
                 3.80E−54 
               
               
                   
                 
                   magnum 
                 
               
               
                 PG115 
                 Zinc protease,  Escherichia coli   
                 P31828 
                 931aa 
                 941aa 
                 27 
                 890aa 
                 6.60E−57 
               
               
                 PG116 
                 Heat shock protein (HTPG),  Escherichia coli   
                 P10413 
                 624aa 
                 684aa 
                 32 
                 627aa 
                 4.60E−48 
               
               
                 PG117 
                 Transcriptional regulator,  Aquifex aeolicus   
                 O66591 
                 506aa 
                 464aa 
                 39 
                 389aa 
                 2.40E−49 
               
               
                 PG118 
                 ABC transporter,  Bacillus subtilus   
                 H70019 
                 261aa 
                 250aa 
                 59 
                 251aa 
                 1.50E−60 
               
               
                 PG119 
                 ATP-dependent protease,  Aquifex aeolicus   
                 O66827 
                 444aa 
                 461aa 
                 46 
                 458aa 
                 1.60E−77 
               
               
                 PG120 
                 Nitrogen assimilation regulatory protein, 
                 P10576 
                 480aa 
                 457aa 
                 49 
                 242aa 
                 3.80E−45 
               
               
                   
                   Bradyrhizobium  sp. 
               
               
                 PG121 
                 Cobalamin synthesis protein,  Bacillus megaterium   
                 E1331323 
                 367aa 
                 602aa 
                 36 
                 324aa 
                 9.20E−37 
               
               
                 PG122 
                 Outer membrane integrity (tolA),  Haemophilus   
                 P71397 
                 819aa 
                 443aa 
                 37 
                 441aa 
                 1.90E−54 
               
               
                   
                 
                   influenzae 
                 
               
               
                 PG123 
                 Fimbrillin,  Porphyromonas gingivalis   
                 D1034032 
                 490aa 
                 479aa 
                 32 
                 480aa 
                 7.30E−48 
               
               
                 PG124 
                 Heat shock protein (dnaJ),  Leptospira interrogans   
                 AF007813 
                 369aa 
                 383aa 
                 46 
                 356aa 
                 2.30E−57 
               
               
                 PG125 
                 Cobalamin biosynthesis protein(CBIK),  Salmonella   
                 Q05592 
                 264aa 
                 293aa 
                 37 
                 259aa 
                 3.70E−26 
               
               
                   
                 
                   typhimurium 
                 
               
               
                 PG126 
                 ABC-type permease,  Pseudomonas aeruginosa   
                 O68878 
                 326aa 
                 356aa 
                 33 
                 333aa 
                 1.30E−30 
               
               
                 PG127 
                 Endonuclease excision repair protein (uvrB), 
                 X93486 
                 670aa 
                 678aa 
                 56 
                 675aa 
                 1.10E−134 
               
               
                   
                 
                   Pseudomonas aeruginosa 
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Results of PSORT, SignalP and TopPred analysis of the proteins. The signal present column indicates the presence of 
               
               
                 a signal sequence detected with either PSORT or SignalP. The terms in parentheses indicates the type of signal sequence as 
               
               
                 determined by PSORT. The cell location &amp; probability values are generated by PSORT and represent the probability of the 
               
               
                 protein being in the cell compartments outer membrane (OM), inner membrane (IM), periplasmic space (PC) or cytoplasm 
               
               
                 (C). The number of transmembrane domains (TMDs) was determined by TopPred and does not include uncleavable signal 
               
               
                 sequences. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                 C- 
                   
               
               
                   
                 Protein 
                   
                   
                   
                 SignalP 
                 PSORT 
                 Cell Location &amp; 
                 terminal 
                 Number 
               
               
                 Protein 
                 seqID 
                 Protein 
                   
                 Methionine 
                 cleavage 
                 cleavage 
                 probability 
                 Amino 
                 of 
               
             
          
           
               
                 name 
                 number 
                 Length 
                 Signal Present 
                 in ORF 
                 site 
                 site 
                 OM 
                 IM 
                 PS 
                 C 
                 Acid 
                 TMD&#39;s 
               
               
                   
               
             
          
           
               
                 PG1 
                 386 
                 451aa 
                 Y 
                 1 
                 24 
                 34 
                 0 
                 0 
                 0 
                 0.22 
                 N 
                 0 
               
               
                 PG2 
                 424 
                 1017aa  
                 Y 
                 1 
                 20 
                 20 
                 0.94 
                 0 
                 0.33 
                 0 
                 F 
                 3 
               
               
                 PG2 
                 425 
                 1014aa  
                 Y 
                 2 
                 17 
                 17 
                 0.94 
                 0 
                 0.29 
                 0 
                 F 
                 3 
               
               
                 PG3 
                 434 
                 223aa 
                 Y (lipoprotein) 
                 1 
                 — 
                 18 
                 0.79 
                 0.76 
                 0 
                 0 
                 K 
                 3 
               
               
                 PG4 
                 447 
                 672aa 
                 Y (lipoprotein) 
                 1 
                 22 
                 22 
                 0.79 
                 0.7 
                 0 
                 0 
                 R 
                 0 
               
               
                 PG5 
                 458 
                 315aa 
                 Y 
                 1 
                 40 
                 35 
                 0 
                 0.25 
                 0 
                 0 
                 R 
                 0 
               
               
                 PG6 
                 475 
                 324aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.2 
                 S 
                 1 
               
               
                 PG7 
                 487 
                 404aa 
                 N 
                 1 
                 7 
                 — 
                 0 
                 0.42 
                 0 
                 0 
                 E 
                 3 
               
               
                 PG8 
                 498 
                 598aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.22 
                 N 
                 0 
               
               
                 PG8 
                 499 
                 550aa 
                 N 
                 2 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.25 
                 N 
                 0 
               
               
                 PG8 
                 500 
                 458aa 
                 N 
                 3 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.34 
                 N 
                 0 
               
               
                 PG8 
                 501 
                 426aa 
                 N 
                 4 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.24 
                 N 
                 0 
               
               
                 PG9 
                 515 
                 1266aa  
                 N 
                 1 
                 7 
                 — 
                 0 
                 0 
                 0 
                 0.22 
                 E 
                 1 
               
               
                 PG9 
                 516 
                 1232aa  
                 N 
                 2 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.39 
                 E 
                 1 
               
               
                 PG9 
                 517 
                 1174aa  
                 N 
                 3 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.47 
                 E 
                 1 
               
               
                 PG10 
                 387 
                 195aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.11 
                 K 
                 0 
               
               
                 PG11 
                 400 
                 313aa 
                 Y 
                 1 
                 22 
                 26 
                 0.24 
                 0 
                 0.93 
                 0 
                 R 
                 1 
               
               
                 PG12 
                 411 
                 271aa 
                 Y (lipoprotein) 
                 3 
                 27 
                 29 
                 0.79 
                 0.7 
                 0 
                 0 
                 R 
                 0 
               
               
                 PG13 
                 419 
                 757aa 
                 Y 
                 1 
                 23 
                 25 
                 0.94 
                 0 
                 0.29 
                 0 
                 N 
                 0 
               
               
                 PG14 
                 420 
                 331aa 
                 Y (uncleavable) 
                 1 
                 35 
                 26 
                 0 
                 0.58 
                 0 
                 0 
                 K 
                 1 
               
               
                 PG15 
                 421 
                 267aa 
                 Y 
                 2 
                 24 
                 18 
                 0 
                 0.11 
                 0 
                 0 
                 K 
                 1 
               
               
                 PG16 
                 422 
                 569aa 
                 Y (lipoprotein) 
                 1 
                 24 
                 18 
                 0.79 
                 0.7 
                 0 
                 0 
                 G 
                 0 
               
               
                 PG18 
                 423 
                 981aa 
                 Y 
                 1 
                 30 
                   
                 0 
                 0.56 
                 0 
                 0 
                 K 
                 11  
               
               
                 PG21 
                 426 
                 821aa 
                 Y 
                 2 
                 24 
                 27 
                 0.34 
                 0 
                 0.37 
                 0 
                 G 
                 1 
               
               
                 PG22 
                 427 
                 106aa 
                 Y (uncleavable) 
                 1 
                 41 
                 41 
                 0 
                 0.29 
                 0 
                 0 
                 P 
                 0 
               
               
                 PG23 
                 428 
                 859aa 
                 N 
                 1 
                 — 
                   
                 0 
                 0.12 
                 0 
                 0 
                 A 
                 1 
               
               
                 PG24 
                 429 
                 417aa 
                 Y 
                 1 
                 19 
                 19 
                 0 
                 0.44 
                 0 
                 0 
                 N 
                 3 
               
               
                 PG25 
                 430 
                 293aa 
                 Y 
                 1 
                 27 
                 28 
                 0.2 
                 0 
                 0.62 
                 0 
                 R 
                 0 
               
               
                 PG27 
                 431 
                 312aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.28 
                 Q 
                 1 
               
               
                 PG28 
                 432 
                 843aa 
                 Y 
                 1 
                 21 
                 21 
                 0.93 
                 0 
                 0.24 
                 0 
                 H 
                 1 
               
               
                 PG29 
                 433 
                 290aa 
                 Y 
                 1 
                 18 
                 16 
                 0.28 
                 0 
                 0.94 
                 0 
                 K 
                 1 
               
               
                 PG30 
                 435 
                 337aa 
                 Y 
                 1 
                 21 
                 21 
                 0.24 
                 0 
                 0.4 
                 0 
                 K 
                 0 
               
               
                 PG31 
                 436 
                 151aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.3 
                 T 
                 0 
               
               
                 PG32 
                 437 
                 391aa 
                 Y 
                 1 
                 20 
                 20 
                 0.62 
                 0 
                 0.13 
                 0 
                 K 
                 0 
               
               
                 PG33 
                 438 
                 385aa 
                 Y 
                 1 
                 26 
                 26 
                 0.81 
                 0 
                 0.31 
                 0 
                 E 
                 1 
               
               
                 PG34 
                 439 
                 190aa 
                 Y 
                 1 
                 — 
                 13 
                 0 
                 0.5 
                 0 
                 0 
                 A 
                 5 
               
               
                 PG34 
                 440 
                 186aa 
                 Y (uncleavable) 
                 2 
                 — 
                 47 
                 0 
                 0.5 
                 0 
                 0 
                 A 
                 4 
               
               
                 PG35 
                 441 
                 833aa 
                 Y 
                 1 
                 22 
                 22 
                 0.94 
                 0 
                 0.37 
                 0 
                 F 
                 1 
               
               
                 PG36 
                 442 
                 891aa 
                 Y (uncleavable) 
                 1 
                 — 
                 40 
                 0 
                 0.31 
                 0 
                 0 
                 F 
                 2 
               
               
                 PG37 
                 443 
                 174aa 
                 Y (uncleavable) 
                 1 
                 28 
                 24 
                 0 
                 0.35 
                 0 
                 0 
                 K 
                 0 
               
               
                 PG37 
                 444 
                 170aa 
                 Y (uncleavable) 
                 2 
                 24 
                 20 
                 0 
                 0.35 
                 0 
                 0 
                 K 
                 0 
               
               
                 PG38 
                 445 
                 163aa 
                 Y 
                 1 
                 18 
                 18 
                 0.21 
                 0 
                 0.93 
                 0 
                 K 
                 1 
               
               
                 PG39 
                 446 
                 827aa 
                 Y 
                 1 
                 36 
                 36 
                 0.93 
                 0 
                 0.25 
                 0 
                 F 
                 3 
               
               
                 PG40 
                 448 
                 772aa 
                 Y 
                 2 
                 19 
                 19 
                 0.94 
                 0 
                 0.32 
                 0 
                 F 
                 4 
               
               
                 PG41 
                 449 
                 462aa 
                 Y 
                 2 
                 27 
                 27 
                 0.25 
                 0 
                 0.54 
                 0 
                 Q 
                 2 
               
               
                 PG42 
                 450 
                 492aa 
                 Y 
                 5 
                 30 
                 — 
                 0 
                 0 
                 0.00 
                 0.13 
                 Q 
                 2 
               
               
                 PG43 
                 451 
                 245aa 
                 Y (uncleavable) 
                 2 
                 28 
                 22 
                 0 
                 0.38 
                 0 
                 0 
                 K 
                 1 
               
               
                 PG44 
                 452 
                 276aa 
                 Y 
                 1 
                 19 
                 24 
                 0.15 
                 0 
                 0.89 
                 0 
                 K 
                 0 
               
               
                 PG45 
                 453 
                 775aa 
                 Y (lipoprotein) 
                 1 
                 19 
                 23 
                 0.79 
                 0.7 
                 0 
                 0 
                 F 
                 4 
               
               
                 PG46 
                 454 
                 774aa 
                 Y 
                 1 
                 27 
                 27 
                 0.73 
                 0 
                 0.22 
                 0 
                 F 
                 2 
               
               
                 PG47 
                 455 
                 867aa 
                 Y 
                 1 
                 24 
                 24 
                 0.94 
                 0 
                 0.38 
                 0 
                 F 
                 2 
               
               
                 PG48 
                 456 
                 431aa 
                 Y 
                 1 
                 24 
                 24 
                 0 
                 0.1 
                 0 
                 0 
                 R 
                 1 
               
               
                 PG49 
                 457 
                 333aa 
                 Y (uncleavable) 
                 1 
                 24 
                 18 
                 0 
                 0.12 
                 0 
                 0 
                 I 
                 0 
               
               
                 PG50 
                 459 
                 848aa 
                 Y 
                 1 
                 21 
                 21 
                 0.94 
                 0 
                 0.34 
                 0 
                 F 
                 3 
               
               
                 PG51 
                 460 
                 202aa 
                 Y 
                 1 
                 26 
                 25 
                 0.2 
                 0 
                 0.61 
                 0 
                 S 
                 0 
               
               
                 PG52 
                 461 
                 455aa 
                 Y (uncleavable) 
                 1 
                 23 
                 21 
                 0 
                 0.18 
                 0 
                 0 
                 F 
                 1 
               
               
                 PG53 
                 462 
                 444aa 
                 Y 
                 1 
                 14 
                 17 
                 0.36 
                 0 
                 0.22 
                 0 
                 D 
                 2 
               
               
                 PG54 
                 463 
                 940aa 
                 Y 
                 1 
                 27 
                 20 
                 0.86 
                 0 
                 0.25 
                 0 
                 Q 
                 5 
               
               
                 PG55 
                 464 
                 670aa 
                 Y (lipoprotein) 
                 1 
                 23 
                 23 
                 0.79 
                 0.7 
                 0 
                 0 
                 K 
                 2 
               
               
                 PG56 
                 465 
                 1282aa  
                 Y (uncleavable) 
                 1 
                 — 
                 21 
                 0 
                 0.04 
                 0 
                 0 
                 K 
                 4 
               
               
                 PG56 
                 466 
                 1274aa  
                 N 
                 2 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.27 
                 K 
                 5 
               
               
                 PG57 
                 467 
                 925aa 
                 Y 
                 1 
                 28 
                 24 
                 0.53 
                 0 
                 0.2 
                 0 
                 P 
                 3 
               
               
                 PG57 
                 468 
                 922aa 
                 Y 
                 2 
                 25 
                 21 
                 0.53 
                 0 
                 0.2 
                 0 
                 P 
                 3 
               
               
                 PG57 
                 469 
                 921aa 
                 Y 
                 3 
                 24 
                 20 
                 0.53 
                 0 
                 0.2 
                 0 
                 P 
                 3 
               
               
                 PG58 
                 470 
                 593aa 
                 Y 
                 1 
                 24 
                 24 
                 0.82 
                 0 
                 0.19 
                 0 
                 F 
                 1 
               
               
                 PG58 
                 471 
                 589aa 
                 Y 
                 2 
                 20 
                 20 
                 0.82 
                 0 
                 0.19 
                 0 
                 F 
                 1 
               
               
                 PG59 
                 472 
                 346aa 
                 Y 
                 1 
                 37 
                 — 
                 0 
                 0.18 
                 0 
                 0 
                 F 
                 1 
               
               
                 PG59 
                 473 
                 345aa 
                 Y 
                 2 
                 36 
                 56 
                 0.92 
                 0 
                 0.15 
                 0 
                 F 
                 1 
               
               
                 PG59 
                 474 
                 330aa 
                 Y 
                 3 
                 21 
                 41 
                 0.93 
                 0 
                 0.25 
                 0 
                 F 
                 1 
               
               
                 PG60 
                 476 
                 547aa 
                 Y 
                 1 
                 28 
                 28 
                 0.93 
                 0 
                 0.25 
                 0 
                 F 
                 0 
               
               
                 PG61 
                 477 
                 749aa 
                 Y 
                 2 
                 21 
                 21 
                 0.94 
                 0 
                 0.29 
                 0 
                 F 
                 3 
               
               
                 PG62 
                 478 
                 494aa 
                 Y 
                 1 
                 21 
                 21 
                 0.93 
                 0 
                 0.24 
                 0 
                 F 
                 2 
               
               
                 PG63 
                 479 
                 294aa 
                 Y 
                 1 
                 20 
                 20 
                 0.93 
                 0 
                 0.24 
                 0 
                 F 
                 1 
               
               
                 PG64 
                 480 
                 204aa 
                 Y 
                 1 
                 20 
                 20 
                 0.93 
                 0 
                 0.19 
                 0 
                 F 
                 1 
               
               
                 PG65 
                 481 
                 243aa 
                 Y 
                 1 
                 18 
                 18 
                 0.93 
                 0 
                 0.25 
                 0 
                 F 
                 1 
               
               
                 PG66 
                 482 
                 206aa 
                 Y 
                 1 
                 21 
                 21 
                 0.94 
                 0 
                 0.3 
                 0 
                 F 
                 1 
               
               
                 PG67 
                 483 
                 950aa 
                 Y 
                 1 
                 28 
                 36 
                 0.93 
                 0 
                 0.27 
                 0 
                 Y 
                 4 
               
               
                 PG68 
                 484 
                 1226aa  
                 Y 
                 1 
                 25 
                 25 
                 0.91 
                 0 
                 0.31 
                 0 
                 Y 
                 0 
               
               
                 PG68 
                 485 
                 1225aa  
                 Y 
                 2 
                 24 
                 24 
                 0.91 
                 0 
                 0.31 
                 0 
                 Y 
                 0 
               
               
                 PG69 
                 486 
                 425aa 
                 Y 
                 1 
                 29 
                 29 
                 0.93 
                 0 
                 0.21 
                 0 
                 F 
                 1 
               
               
                 PG70 
                 488 
                 260aa 
                 Y 
                 1 
                 18 
                 24 
                 0.93 
                 0 
                 0.24 
                 0 
                 F 
                 0 
               
               
                 PG71 
                 489 
                 834aa 
                 Y 
                 2 
                 20 
                 20 
                 0.94 
                 0 
                 0.31 
                 0 
                 N 
                 2 
               
               
                 PG72 
                 490 
                 399aa 
                 Y 
                 1 
                 27 
                 27 
                 0.94 
                 0 
                 0.32 
                 0 
                 H 
                 2 
               
               
                 PG73 
                 491 
                 382aa 
                 Y 
                 2 
                 20 
                 20 
                 0.94 
                 0 
                 0.3 
                 0 
                 L 
                 1 
               
               
                 PG74 
                 492 
                 222aa 
                 Y 
                 1 
                 24 
                 24 
                 0.94 
                 0 
                 0.32 
                 0 
                 L 
                 0 
               
               
                 PG75 
                 493 
                 391aa 
                 Y 
                 1 
                 26 
                 26 
                 0.94 
                 0 
                 0.3 
                 0 
                 H 
                 1 
               
               
                 PG76 
                 494 
                 446aa 
                 Y 
                 1 
                 21 
                 22 
                 0.94 
                 0 
                 0.32 
                 0 
                 V 
                 3 
               
               
                 PG77 
                 495 
                 308aa 
                 Y 
                 2 
                 28 
                 28 
                 0.94 
                 0 
                 0.38 
                 0 
                 K 
                 0 
               
               
                 PG78 
                 496 
                 314aa 
                 Y 
                 1 
                 23 
                 23 
                 0.94 
                 0 
                 0.29 
                 0 
                 D 
                 0 
               
               
                 PG79 
                 497 
                 285aa 
                 Y 
                 1 
                 — 
                 32 
                 0.93 
                 0 
                 0.26 
                 0 
                 G 
                 2 
               
               
                 PG80 
                 502 
                 240aa 
                 Y 
                 1 
                 19 
                 19 
                 0.93 
                 0 
                 0.22 
                 0 
                 N 
                 2 
               
               
                 PG81 
                 366 
                 &gt;235aa  
                 Y 
                 1 
                 28 
                 20 
                 0.93 
                 0 
                 0.21 
                 0 
                 Q 
                 1 
               
               
                 PG82 
                 503 
                 434aa 
                 Y 
                 1 
                 30 
                 24 
                 0.93 
                 0 
                 0.2 
                 0 
                 N 
                 3 
               
               
                 PG83 
                 504 
                 926aa 
                 Y 
                 1 
                 23 
                 57 
                 0.93 
                 0 
                 0.21 
                 0 
                 S 
                 1 
               
               
                 PG84 
                 505 
                 400aa 
                 Y 
                 1 
                 25 
                 25 
                 0.93 
                 0 
                 0.25 
                 0 
                 N 
                 1 
               
               
                 PG84 
                 506 
                 398aa 
                 Y 
                 2 
                 23 
                 23 
                 0.93 
                 0 
                 0.25 
                 0 
                 N 
                 1 
               
               
                 PG85 
                 507 
                 581aa 
                 Y 
                 1 
                 20 
                 20 
                 0.93 
                 0 
                 0.46 
                 0 
                 L 
                 2 
               
               
                 PG86 
                 508 
                 239aa 
                 Y 
                 1 
                 44 
                 — 
                 0 
                 0 
                 0 
                 0.12 
                 H 
                 0 
               
               
                 PG86 
                 509 
                 211aa 
                 Y 
                 2 
                 16 
                 46 
                 0.91 
                 0 
                 0.03 
                 0 
                 H 
                 0 
               
               
                 PG87 
                 510 
                 781aa 
                 Y 
                 1 
                 26 
                 47 
                 0.89 
                 0 
                 0.21 
                 0 
                 N 
                 2 
               
               
                 PG88 
                 511 
                 271aa 
                 Y 
                 2 
                 28 
                 19 
                 0.89 
                 0 
                 0.25 
                 0 
                 P 
                 0 
               
               
                 PG88 
                 512 
                 270aa 
                 Y 
                 3 
                 27 
                 18 
                 0.89 
                 0 
                 0.25 
                 0 
                 P 
                 0 
               
               
                 PG88 
                 513 
                 267aa 
                 Y 
                 4 
                 24 
                 15 
                 0.89 
                 0 
                 0.23 
                 0 
                 P 
                 0 
               
               
                 PG89 
                 514 
                 259aa 
                 Y 
                 2 
                 23 
                 25 
                 0.88 
                 0 
                 0.35 
                 0 
                 N 
                 1 
               
               
                 PG90 
                 518 
                 229aa 
                 Y 
                 1 
                 22 
                 21 
                 0.85 
                 0 
                 0.44 
                 0 
                 K 
                 0 
               
               
                 PG90 
                 519 
                 228aa 
                 Y 
                 2 
                 21 
                 20 
                 0.85 
                 0 
                 0.44 
                 0 
                 K 
                 0 
               
               
                 PG91 
                 520 
                 540aa 
                 Y 
                 1 
                 25 
                 25 
                 0.85 
                 0 
                 0.30 
                 0 
                 E 
                 0 
               
               
                 PG92 
                 521 
                 771aa 
                 Y 
                 2 
                 19 
                 19 
                 0.85 
                 0 
                 0.3 
                 0 
                 R 
                 3 
               
               
                 PG93 
                 522 
                 776aa 
                 Y 
                 1 
                 25 
                 25 
                 0.85 
                 0 
                 0.37 
                 0 
                 R 
                 4 
               
               
                 PG94 
                 523 
                 1157aa  
                 Y 
                 1 
                 23 
                 28 
                 0.8 
                 0 
                 0.25 
                 0 
                 Q 
                 5 
               
               
                 PG95 
                 524 
                 961aa 
                 Y (lipoprotein) 
                 1 
                 — 
                 19 
                 0.79 
                 0.87 
                 0 
                 0 
                 V 
                 1 
               
               
                 PG96 
                 525 
                 563aa 
                 Y 
                 1 
                 23 
                 23 
                 0.40 
                 0 
                 0.33 
                 0 
                 K 
                 0 
               
               
                 PG97 
                 526 
                 437aa 
                 Y 
                 1 
                 23 
                 23 
                 0.32 
                 0 
                 0.65 
                 0 
                 Q 
                 0 
               
               
                 PG98 
                 527 
                 318aa 
                 Y (lipoprotein) 
                 1 
                 19 
                 19 
                 0.79 
                 0.7 
                 0 
                 0 
                 L 
                 1 
               
               
                 PG99 
                 528 
                 461aa 
                 Y (uncleavable) 
                 1 
                 22 
                 20 
                 0 
                 0 
                 0.3 
                 0 
                 R 
                 0 
               
               
                 PG100 
                 388 
                 279aa 
                 Y 
                 1 
                 20 
                 18 
                 0.26 
                 0 
                 0.54 
                 0 
                 I 
                 0 
               
               
                 PG101 
                 268 
                 &gt;157aa  
                 N 
                   
                 — 
                 — 
                   
                   
                   
                   
                 R 
                 1 
               
               
                   
                   
                   
                 (ORF incomplete) 
               
               
                 PG102 
                 389 
                 562aa 
                 Y 
                 1 
                 29 
                 29 
                 0.19 
                 0 
                 0.4 
                 0 
                 S 
                 3 
               
               
                 PG102 
                 390 
                 558aa 
                 Y 
                 2 
                 25 
                 25 
                 0.26 
                 0 
                 0.46 
                 0 
                 S 
                 3 
               
               
                 PG104 
                 391 
                 391aa 
                 Y 
                 1 
                 17 
                 17 
                 0.62 
                 0 
                 0.22 
                   
                 R 
                 0 
               
               
                 PG105 
                 392 
                 449aa 
                 Y 
                 1 
                 22 
                 19 
                 0.31 
                 0 
                 0.91 
                 0 
                 P 
                 3 
               
               
                 PG106 
                 393 
                 246aa 
                 Y 
                 2 
                 41 
                 49 
                 0 
                 0 
                 0 
                 0.02 
                 L 
                 0 
               
               
                 PG107 
                 394 
                 246aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.32 
                 D 
                 1 
               
               
                 PG107 
                 395 
                 241aa 
                 N 
                 2 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.3 
                 D 
                 1 
               
               
                 PG107 
                 396 
                 232aa 
                 N 
                 3 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.21 
                 D 
                 1 
               
               
                 PG108 
                 397 
                 219aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.19 
                 R 
                 1 
               
               
                 PG109 
                 398 
                 595aa 
                 Y 
                 1 
                 35 
                 37 
                 0.26 
                 0 
                 0.93 
                 0 
                 Y 
                 3 
               
               
                 PG109 
                 399 
                 589aa 
                 Y 
                 2 
                 29 
                 31 
                 0.27 
                 0 
                 0.93 
                 0 
                 Y 
                 3 
               
               
                 PG110 
                 401 
                 &gt;523aa  
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.38 
                 in- 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 complete 
                   
               
               
                 PG111 
                 278 
                 &gt;720aa  
                 N 
                 — 
                 — 
                 — 
                   
                   
                   
                   
                 G 
                 1 
               
               
                   
                   
                   
                 (ORF incomplete) 
               
               
                 PG112 
                 402 
                 375aa 
                 Y 
                 1 
                 — 
                 43 
                 0 
                 0.12 
                 0 
                 0 
                 N 
                 1 
               
               
                 PG112 
                 403 
                 362aa 
                 Y 
                 2 
                 — 
                 30 
                 0 
                 0 
                 0.12 
                 0 
                 N 
                 1 
               
               
                 PG113 
                 404 
                 640aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.25 
                 K 
                 1 
               
               
                 PG114 
                 405 
                 449aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0.12 
                 0 
                 0 
                 G 
                 4 
               
               
                 PG115 
                 406 
                 941aa 
                 Y 
                 1 
                 23 
                 22 
                 0.13 
                 0 
                 0.92 
                 0 
                 Q 
                 2 
               
               
                 PG116 
                 407 
                 684aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0.12 
                 0 
                 0 
                 L 
                 2 
               
               
                 PG117 
                 408 
                 464aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0.19 
                 0 
                 0 
                 L 
                 1 
               
               
                 PG118 
                 409 
                 250aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.27 
                 E 
                 1 
               
               
                 PG119 
                 410 
                 461aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0.28 
                 0 
                 0 
                 E 
                 2 
               
               
                 PG120 
                 412 
                 457aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.21 
                 E 
                 0 
               
               
                 PG121 
                 413 
                 602aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.31 
                 E 
                 3 
               
               
                 PG122 
                 414 
                 443aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.14 
                 Q 
                 4 
               
               
                 PG123 
                 415 
                 479aa 
                 Y 
                 2 
                 22 
                 22 
                 0.26 
                 0 
                 0.94 
                 0 
                 K 
                 0 
               
               
                 PG124 
                 416 
                 383aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.29 
                 D 
                 2 
               
               
                 PG125 
                 417 
                 293aa 
                 Y 
                 1 
                 23 
                 15 
                 0.18 
                 0 
                 0.93 
                 0 
                 R 
                 1 
               
               
                 PG126 
                 418 
                 356aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0.52 
                 0 
                 0 
                 D 
                 9 
               
               
                 PG127 
                 532 
                 678aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.28 
                 A 
                 2 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Percentage identity and percentage similarity of various 
               
               
                 proteins with the 70 amino acids from the C-terminal of the 
               
               
                   P. gingivalis  arginine protease 1 (RGP1), arginine 
               
               
                 protease 2 (RGP2), and the cysteine protease/hemagglutinin 
               
               
                 (prtT). 
               
             
          
           
               
                 Protein 
                 Percent identity 
                 Percent similarity 
               
             
          
           
               
                 name 
                 RGP1 
                 RGP2 
                 prtT 
                 RGP1 
                 RGP2 
                 prtT 
               
               
                   
               
             
          
           
               
                 PG21 
                 17 
                 29 
                 21 
                 40 
                 57 
                 49 
               
               
                 PG25 
                 43 
                 41 
                 9 
                 64 
                 73 
                 14 
               
               
                 PG27 
                 41 
                 33 
                 7 
                 73 
                 74 
                 11 
               
               
                 PG28 
                 21 
                 26 
                 34 
                 49 
                 57 
                 74 
               
               
                 PG54 
                 19 
                 13 
                 16 
                 40 
                 43 
                 33 
               
               
                 PG57 
                 11 
                 14 
                 19 
                 20 
                 24 
                 34 
               
               
                 PG91 
                 31 
                 21 
                 39 
                 57 
                 53 
                 74 
               
               
                 PG96 
                 0 
                 13 
                 20 
                 0 
                 24 
                 43 
               
               
                 PG97 
                 10 
                 26 
                 33 
                 14 
                 47 
                 61 
               
               
                 PG98 
                 16 
                 20 
                 0 
                 47 
                 54 
                 0 
               
               
                 PG99 
                 19 
                 0 
                 26 
                 41 
                 0 
                 54 
               
               
                 PG100 
                 20 
                 21 
                 24 
                 39 
                 57 
                 41 
               
               
                 PG101 
                 11 
                 16 
                 27 
                 17 
                 39 
                 60 
               
               
                 PG102 
                 27 
                 20 
                 31 
                 50 
                 61 
                 61 
               
               
                 PG104 
                 16 
                 23 
                 26 
                 46 
                 44 
                 49 
               
               
                   
               
             
          
         
       
     
                                       TABLE 5                   Percentage identity and percentage similarity of various proteins with       the TonBIII box of  P. gingivalis .                Protein       Percent           name   Percent identity   similarity                       PG2   46   71           PG13   57   93           PG35   50   96           PG47   39   71           PG50   54   93                        
Cloning, Expression and Purification of Recombinant  P. gingivalis  Genes.
 
PG1
 
     Oligonucleotides to the 5′ and 3′ regions of the deduced protein were used to amplify the gene of interest from a preparation of  P. gingivalis  W50 genomic DNA using the TaqPlus Precision PCR System ( Stratagene) and a PTC-100 (MJ Research) thermal cycler or similar device. The 5′ oligonucleotide primer sequence was GCGCCATATGCTGGCCGAACCGGCC, (SEQ ID NO: 533) the 3′ oligonucleotide primer sequence was GCGCCTCGAGTCAATTCATTTCCTTATAGAG (SEQ ID NO: 534). The PCR fragment was purified, digested with Nde I, Xho I restriction enzymes (Promega) and ligated into the corresponding sites of the plasmid pProEx-1 (Gibco-BRL) and transformed into  E. coli  ER1793 cells (a gift from Elizabeth Raleigh, New England Biolabs). A resulting clone expressing the correct insert was selected and induced with or without 0.1 mM IPTG (Promega) for expression of the recombinant protein. Expression of the recombinant protein was determined by SDS-PAGE analysis and Western Blot using the one of the rabbit antisera described above or an anti-hexahistidine antibody (Clontech) that detects the hexahistidine tag that was fused to the  P. gingivalis  recombinant protein. PG1 was purified by disruption of the  E. coli  cells by sonication in binding buffer (Novagen) and solubilisation by the addition of sarkosyl (N-Lauroyl sarcosine) to a 1% final concentration. There after the preparation was diluted to 0.1% sarkosyl in binding buffer, bound to a Nickel-nitrilotriacetic acid column (Ni-NTA; Qiagen), after washing bound proteins were eluted with 1M imidazole in elution buffer (Novagen) according to the Qiagen recommendations with 0.1% sarkosyl added to all buffers. Following purification samples were dialysed against 500 mM NaCl, 20 mM Tris, 0.1% sarkosyl at pH7.4 to remove the imidazole, concentrated as required and stored at 40 C until used. Purity and antigenicity were assessed by SDS-PAGE and Western blot using selected antisera (from those described above) and the protein concentration was determined by the BCA assay (Pierce). 
     PG2 
     The methods used for PG2 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGAAAAGAATGACGC, (SEQ ID NO: 535) the 3′ oligonucleotide primer sequence was CGCGAGATCTGAAAGACAACTGAATACC (SEQ ID NO: 536) and the PCR product was cloned into pGex-stop RBS(IV) (Patent application WO9619496, J C Cox, S E Edwards, I Frazer and E A Webb. Variants of human papilloma virus antigens) using the BstZ 171 and Bgl II restriction sites. 2% sarkosyl was used to solubilise PG2 and 8M urea was added to the solublisation buffer and to all other buffers. Urea was removed from the purified protein by sequential dialysis (4M then 2M then 1M then 0.5M then 0M urea all in 50 mM Tris, 500 mM NaCl, 0.1% sarkosyl, pH7.4). Purified protein was stored at 4° C. until required. 
     PG3 
     The methods used for PG3 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGTATACATGAAGAAATCAAGTGTAG, (SEQ ID NO: 537) the 3′ oligonucleotide primer sequence was GCGCAGATCTCTTCAGCGTACCTTGCTGTG (SEQ ID NO: 538) and DNA was amplified with Pfu DNA polymerase (Stratagene). The PCR product was cloned directly into pCR-Blunt and transformed into  E. coli  Top10F′ (InVitrogen) before subcloning into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  BL21DE3 (Pharmacia Biotech). The following modifications were made to the purification of PG3 from the PG1 method. Cells expressing the recombinant protein were disrupted by sonication in binding buffer and the insoluble inclusion bodies concentrated by centrifugation. Inclusion bodies were then solubilised in 6M urea (Sigma) in binding buffer and eluted with 6M urea added to the elution buffer. In some instances 6M guanidine hydrochloride (Sigma) was used instead of urea for these steps. Urea (or guanidine hydrochloride when it was substituted) was removed from the purified protein by sequential dialysis against reducing levels of urea (3M then 1.5M then 0.5M then 0M urea all in 50 mM Tris, 500 mM NaCl, 8% glycerol, pH7.4). Purified protein was stored frozen at −800 C until required. Protein concentration was determined by the Coomassie Plus protein assay (Pierce). 
     PG4 
     The methods used for PG4 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CTTCTGTATACTTACAGCGGACATCATAAAATC, (SEQ ID NO: 539) the 3′ oligonucleotide primer sequence was TTCCAGGAGGGTACCACGCAACTCTTCTTCGAT (SEQ ID NO: 540) and DNA was amplified with the Tth XL PCR kit (Perkin Elmer). The PCR product was cloned into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Kpn I restriction sites and transformed into  E. coli  ER1793. 
     PG5 
     The methods used for PG5 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was TTGCAACATATGATCAGAACGATACTTTCA, (SEQ ID NO: 541) the 3′ oligonucleotide primer sequence was AGCAATCTCGAGCGGTTCATGAGCCAAAGC (SEQ ID NO: 542) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24 (Novagen) using the Nde I and Xho I restriction sites and transformed into  E. coli  BL21 (Pharmacia Biotech). Removal of urea was not proceeded past 1M urea as the protein was insoluble at lower concentrations of urea. Purified protein was stored at 40 C until required. 
     PG6 
     The methods used for PG6 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was TAAACATATGTGCCTCGAACCCATAATTGCTCCG, (SEQ ID NO: 543) the 3′ oligonucleotide primer sequence was CGTCCGCGGAAGCTTTGATCGGCCATTGCTACT (SEQ ID NO: 544) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Nde I and Hind III restriction sites and transformed into  E. coli  BL21. 
     PG8 
     The methods used for PG8 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGGAGTTCAAGATTGTG, (SEQ ID NO: 545) the 3′ oligonucleotide primer sequence was CGCGAGATCTGTTTTCTGAAAGCTTTTC (SEQ ID NO: 546) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pProEx-1 using the Nde I and Xho I restriction sites and transformed into  E. coli  ER1793. 
     PG8A 
     PG8A is a shortened version of PG8 and has the first 173 amino acids removed. The methods used for PG8A were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGGAAAACTTAAAGAAC, (SEQ ID NO:547) the 3′ oligonucleotide primer sequence was CGCGAGATCTGTTTTCTGAAAGCTTTTC (SEQ ID NO: 548) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. Prior to dialysis of the purified protein EDTA (Sigma) was added to a final concentration of 10 mM. 
     PG10 
     The methods used for PG10 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGATATCATGGATAAAGTGAGCTATGC, (SEQ ID NO: 549) the 3′ oligonucleotide primer sequence was CGCGAGATCTTTTGTTGATACTCAATAATTC (SEQ ID NO:550) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was digested with Eco RV and Bgl II and ligated into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. 
     PG11 
     The methods used for PG11 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGTATACATGAGAGCAAACATTTGGCAGATACTTTCCG, (SEQ ID NO: 551) the 3′ oligonucleotide primer sequence was GCGCAGATCTGCGCAAGCGCAGTATATCGCC (SEQ ID NO: 552) and DNA was amplified with Tli DNA polymerase (Promega). The PCR product was cloned into pCR-Blunt and transformed into  E. coli  Top10F′ before subcloning into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. PG11 was purified by solubilisation of  E. coli  cells with 2% sarkosyl in binding buffer (Qiagen) which was diluted to 0.1% sarkosyl in binding buffer, bound to a Nickel-nitrilotriacetic acid column (Ni-NTA; Qiagen), after washing bound proteins were eluted with 1M imidazole (0.7% CHAPS (Sigma) in elution buffer; Qiagen) according to the Qiagen recommendations. Following purification samples were dialysed against 500 mM NaCl, 20 mM Tris, 0.7% CHAPS, 20% glycerol (Sigma) at pH7.4 to remove the imidazole, concentrated as required and stored at 4° C. until used. 
     PG12 
     The methods used for PG12 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGTATACATGAATAGCAGACATCTGACAATCACAATCATTGCCGG, (SEQ ID NO: 553) the 3′ oligonucleotide primer sequence was GCGCAGATCTGCTGTTCTGTGAGTGCAGTTGTTTAAGTG (SEQ ID NO: 554) and DNA was amplified with Tli DNA polymerase. The PCR product was cloned into pCR-Blunt and transformed into  E. coli  Top10F′ cells before subcloning into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  BL21. Purification of the recombinant protein was essentially the same as PG11 except 0.5% DHPC (1,2-Diheptanoyl-sn-glycero-3-phosphocholine; Avanti) in 50 mM Tris, 50 mM NaCl, pH8.0 was used to solubilise the inclusion bodies instead of sarkosyl and the DHPC was diluted to 0.1% before addition to the Ni-NTA and 0.1% DHPC was added to all buffers. 
     PG13 
     The methods used for PG13 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCCATATGCGGACAAAAACTATCTTTTTTGCG, (SEQ ID NO: 555) the 3′ oligonucleotide primer sequence was GCGCCTCGAGGTTGTTGAATCGAATCGCTATTTGAGC (SEQ ID NO: 556) and DNA was amplified with Tli DNA polymerase. The PCR product was cloned the expression plasmid pET24b using the Nde I and Xho I restriction sites and transformed into  E. coli  BL21. Purification of the recombinant protein was essentially the same as PG3 using 6M urea and 1% NOG (n-octyl glucoside; Sigma) was added to the dialysis buffer. Removal of urea was not proceeded past 2M urea as the protein was insoluble at lower concentrations of urea. Purified protein was stored at 4° C. until required. 
     PG14 
     The methods used for PG12 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGGCGCCATGACGGACAACAAACAACGTAATATCG, (SEQ ID NO: 557) the 3′ oligonucleotide primer sequence was GCGCCTCGAGTTACTTGCGTATGATCACGGACATACCC (SEQ ID NO: 558) and DNA was amplified with Tli DNA polymerase. The PCR product was cloned the expression plasmid pProEx-1 using the Ehe I and Xho I restriction sites and transformed into  E. coli  BL21. Purification of the recombinant protein was essentially the same as PG12. 
     PG15 
     The methods used for PG15 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CAAAAGTATACTAATAAATATCATTCTCAA, (SEQ ID NO: 559) the 3′ oligonucleotide primer sequence was GCTTATGGTACCTTTGGTCTTATCTATTAT (SEQ ID NO: 560) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Kpn I restriction sites and transformed into  E. coli  ER1793. 
     PG22 
     The methods used for PG22 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was CCCCGGATCCGATGCGACTGATCAAGGC, (SEQ ID NO: 561) the 3′ oligonucleotide primer sequence was CCCCCTCGAGCGGAACGGGGTCATAGCC and DNA (SEQ ID NO: 562) was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pET24b using the Bam HI and Xho I restriction sites and transformed into  E. coli  BL21DE3. Once PG22 was purified dialysis was performed in the same manner as for PG1 but in the presence of 1M imidazole. 
     PG24 
     The methods used for PG24 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGAATTACCTGTACATAC, (SEQ ID NO: 563) the 3′ oligonucleotide primer sequence was CGCGGGATCCGTTCGATTGGTCGTCGATGG (SEQ ID NO: 564) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was digested with Bst Z171 and Bam HI and ligated into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. Due to the low level of expression of PG24 purification was not proceeded with except on small scale. 
     PG24A 
     A modified version of PG24 was also cloned and expressed. PG24A is the same as PG24 with the predicted N-terminal sequence removed. The methods used for PG24A were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGCATATGGAGATTGCTTTCCTTTCTTCG, (SEQ ID NO: 565) the 3′ oligonucleotide primer sequence was CGCGCTCGAGTTAGTTCGATTGGTCGTCG (SEQ ID NO: 566) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pProEx-1 using the Nde I and Xho I restriction sites and transformed into  E. coli  ER1793. Purification of the recombinant protein was essentially the same as PG3 except 8M urea was used to solubilise the inclusion bodies and in the buffers used for the Ni-NTA column purification. Urea was removed by sequential dialysis (4M then 2M, then 1M then 0.5M then 0M urea all in 50 mM Tris, 500 mM NaCl, 8% glycerol, pH7.4). Purified protein was stored frozen at −80° C. until required. 
     PG29 
     The methods used for PG29 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGATATCGCTAGCATGAAAAAGCTATTTCTC, (SEQ ID NO: 567) the 3′ oligonucleotide primer sequence was GCGCAGATCTCTCGAGTTTGCCATCGGATTGCGGATTG (SEQ ID NO: 568) and DNA was amplified with Pfu DNA polymerase being used. The PCR product was cloned into pCR-Blunt (InVitrogen) and transformed into  E. coli  Top10F′ before subcloning into the expression plasmid pGex-stop RBS(IV) using the EcoR V and Bgl II restriction sites and transformed into  E. coli  BL21. 6M urea was used throughout the purification process. 
     PG30 
     The methods used for PG30 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TACGGAATTCGTGACCCCCGTCAGAAATGTGCGC, (SEQ ID NO: 569) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCTTTGATCCTCAAGGCTTTGCCCGG (SEQ ID NO: 570) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates of PG30. 10 ml cultures of recombinant  E. coli  were grown to an OD of 2.0 (A 600 nm ) in terrific broth and the cells were induced with 0.5 mM IPTG and samples taken for analysis at 4 hours post induction. Purification was not done for these studies. 
     PG31 
     The methods used for PG31 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was CGGGGAATTCGCAAAAATCAATTTCTATGCTGAA, (SEQ ID NO: 571) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCTGTATGCAATAGGGAAAGCTCCGA (SEQ ID NO: 572) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E.coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG32 
     The methods used for PG32 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGCAGAATTCCAGGAGAATACTGTACCGGCAACG, (SEQ ID NO: 573) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTTGGAGCGAACGATTACAACAC (SEQ ID NO: 574) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG33 
     The methods used for PG33 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCAGAATTCCAAGAAGCTACTACACAGAACAAA, (SEQ ID NO: 575) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCTTCCGCTGCAGTCATTACTACAA (SEQ ID NO: 576) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG35 
     The methods used for PG35 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGAATTCATGAAACAACTAAACATTATCAGC, (SEQ ID NO: 577) the 3′ oligonucleotide primer sequence was GCGTGCGGCCGCGAAATTGATCTTTGTACCGACGA (SEQ ID NO: 578) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG36 
     The methods used for PG36 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was AAAGGAATTCTACAAAAAGATTATTGCCGTAGCA, (SEQ ID NO: 579) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAACTCCTGTCCGAGCACAAAGT (SEQ ID NO: 580) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG37 
     The methods used for PG37 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was TGGCGAATTCAAACGGTTTTTGATTTTGATCGGC, (SEQ ID NO: 581) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTTGCTAAAGCCCATCTTGCTCAG (SEQ ID NO: 582) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG38 
     The methods used for PG38 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CCTCGAATTCCAAAAGGTGGCAGTGGTAAACACT, (SEQ ID NO: 583) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTTGATTCCGAGTTTCGCTTTTAC (SEQ ID NO: 584) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG39 
     The methods used for PG39 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCTGGATCCCAAGGCGTCAGGGTATCGGGCTAT, (SEQ ID NO: 585) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAATTCGACGAGGAGACGCAGGT (SEQ ID NO: 586) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG40 
     The methods used for PG40 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCTGAATTCAAGACGGACAACGTCCCGACAGAT, (SEQ ID NO: 587) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAAGTTGACCATAACCTTACCCA (SEQ ID NO: 588) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG41 
     The methods used for PG41 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GACTGAATTCCAAAACGCCTCCGAAACGACGGTA, (SEQ ID NO: 589) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCTTGTTCGGGAATCCCCATGCCGTT (SEQ ID NO: 590) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG42 
     The methods used for PG42 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was GTTTGAATTCGCAAATAATACTCTTTTGGCGAAG, (SEQ ID NO: 591) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTGCCGGACATCGAAGAGATCGTC (SEQ ID NO: 592) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG43 
     The methods used for PG43 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGAATTCAAAAAAGAAAAACTTTGGATTGCG, (SEQ ID NO: 593) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTTCAAAGCGAAAGAAGCCTTAAC (SEQ ID NO: 594) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG44 
     The methods used for PG44 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCCGAATTCTGTAAGAAAAATGCTGACACTACC, (SEQ ID NO: 595) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTTTTTCCCGGGCTTGATCCCGAT (SEQ ID NO: 596) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG45 
     The methods used for PG45 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GACAGGATCCTGCTCCACCACAAAGAATCTGCCG, (SEQ ID NO: 597) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAAGGGATAGCCGACAGCCAAAT (SEQ ID NO: 598) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG46 
     The methods used for PG46 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CTCGGAATTCCGTTATGTGCCGGACGGTAGCAGA, (SEQ ID NO: 599) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAACGGATAGCCTACTGCAATGT (SEQ ID NO: 600) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG47 
     The methods used for PG47 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGCCGAATTCCAAACAGTGGTGACCGGTAAGGTGATCGATTCAGAA, (SEQ ID NO: 601) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAAGTTTACACGAATACCGGTAGACCAAGTGCGGCC (SEQ ID NO: 602) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG48 
     The methods used for PG48 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAAAAATCCAAGCAGGTACAGCGA, (SEQ ID NO: 603) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCTCGTAACCATAGTCTTGGGTTTTTG (SEQ ID NO: 604) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG49 
     The methods used for PG49 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GAACGGATCCAACGAGCCGGTGGAAGACAGATCC, (SEQ ID NO: 605) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTAATCTCGACTTCATACTTGTACCA (SEQ ID NO: 606) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG50 
     The methods used for PG50 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GCTGGGATCCGCGACAGACACTGAGTTCAAGTAC, (SEQ ID NO: 607) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAACTTCACTACCAAGCCCATGT (SEQ ID NO: 608) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG51 
     The methods used for PG51 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TCTTGAATTCGCGCAAAGTCTTTTCAGCACCGAA, (SEQ ID NO: 609) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCACTTTTTCGTGGGATCACTCTCTT (SEQ ID NO: 610) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG52 
     The methods used for PG52 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was AGAAGAATTCAAACGGACAATCCTCCTGACGGCA, (SEQ ID NO: 611) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGAAGTCTTTGCCCTGATAGAAATC (SEQ ID NO: 612) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG53 
     The methods used for PG53 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCTGAATTCGCGAATCCCCTTACGGGCCAATCG, (SEQ ID NO: 613) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCGTCCGAAAGGCAGCCGTAATAGG (SEQ ID NO: 614) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG54 
     The methods used for PG54 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGCTGAATTCCAGATTTCGTTCGGAGGGGAACCC, (SEQ ID NO: 615) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTGCTTCACGATCTTTTGGCTCA (SEQ ID NO: 616) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG55 
     The methods used for PG55 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGAGGGATCCGAGCTCTCTATTTGCGATGGCGAG, (SEQ ID NO: 617) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCTTACCTGACTTCTTGTCACGAAT (SEQ ID NO: 618) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG56 
     The methods used for PG56 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was AAATGGATCCCGAAAAATTTTGAGCTTTTTGATG, (SEQ ID NO: 619) the 3′ oligonucleotide primer sequence was CTATGCGGCCGCTTTGATTCGTAATTTTTCCGTATC (SEQ ID NO: 620) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG57 
     The methods used for PG57 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGGATCCCAAGAGATCTCAGGCATGAATGCA, (SEQ ID NO: 621) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCGGCCTCTTTATCTCTACCTTTTC (SEQ ID NO: 622) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG58 
     The methods used for PG58 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGGTGAATTCCAAACCCCACGAAATACAGAAACC, (SEQ ID NO: 623) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAAGTCCAGCTAAAACCGGCGAA (SEQ ID NO: 624) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG59 
     The methods used for PG59 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAACAAGAGAAGCAGGTGTTTCAT, (SEQ ID NO: 625) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAGATGCTCTTATCGTCCAAACG (SEQ ID NO: 626) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG60 
     The methods used for PG60 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCGGAATTCCAGATGCTCAATACTCCTTTCGAG, (SEQ ID NO: 627) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAGAGGTAGGAGATATTGCAGAT (SEQ ID NO: 628) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG61 
     The methods used for PG61 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCAGAATTCCCCGTCTCCAACAGCGAGATAGAT, (SEQ ID NO: 629) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAATCGATTGTCAGACTACCCAG (SEQ ID NO: 630) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG62 
     The methods used for PG62 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAGCGGTTTCCGATGGTGCAGGGA, (SEQ ID NO: 631) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAGTGAAATCCGACACGCAGCTG (SEQ ID NO: 632) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG63 
     The methods used for PG63 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCAGAATTCCAAGAAGCAAACACTGCATCTGAC, (SEQ ID NO: 633) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAAGTGTACGCAACACCCACGCC (SEQ ID NO: 634) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG64 
     The methods used for PG64 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAGAGTCGTCCTGCTCTTAGACTG, (SEQ ID NO: 635) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAGCGAACACCGAGACCCACAAA (SEQ ID NO: 636) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG65 
     The methods used for PG65 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGGATCCATCGGACAAAGCCGCCCGGCACTT, (SEQ ID NO: 637) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTAAAGCGGTAACCTATGCCCACGAA (SEQ ID NO: 638) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG66 
     The methods used for PG66 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GTTTGAATTCCAAGACGTTATCAGACCATGGTCA, (SEQ ID NO: 639) the 35 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTAAAATGAGTGGAGAGCGTGGCCAT (SEQ ID NO: 640) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG67 
     The methods used for PG67 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GAACGAGCTCGCGGAACGTCCTATGGCCGGAGCA, (SEQ ID NO: 641) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATACCAAGTATTCGTGATGGGACG (SEQ ID NO: 642) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Sac I and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG68 
     The methods used for PG68 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GCTTGCGGCCGCCCTTATGAAAGATTTGCAGAT, (SEQ ID NO: 643) the 3′ oligonucleotide primer sequence was GGTGCTCGAGTATACTCAACAAGCACCTTATGCAC (SEQ ID NO: 644) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Not I and Xho I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG69 
     The methods used for PG69 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAGGAAGGGGAGGGGAGTGCCCGA, (SEQ ID NO: 645) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAAGCTGTAGCGGGCTTTGAACCA (SEQ ID NO; 646) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG70 
     The methods used for PG70 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGGTGGATCCTCGCAAATGCTCTTCTCAGAGAAT, (SEQ ID NO: 647) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTAAACGAAATATCGATACCAACATC (SEQ ID NO: 648) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG71 
     The methods used for PG71 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAGAACAATACCCTCGATGTACAC, (SEQ ID NO: 649) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATFGCCGGTAGGATTTCCTTGTCC (SEQ ID NO: 650) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG72 
     The methods used for PG72 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCGGAGAGCGACTGGAGACGGACAGC, (SEQ ID NO: 651) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATGATTGCCTTTCAGAAAAGCTAT (SEQ ID NO: 652) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed TGCTGAATTCGGAGAGCGACTGGAGACGGACAGC (SEQ ID NO: 653) into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG73 
     The methods used for PG73 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGGTGAATTCCAACAGACAGGACCGGCCGAACGC, (SEQ ID NO: 654) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTAAGAAAGGTATCTGATAGATCAG (SEQ ID NO: 655) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG74 
     The methods used for PG74 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAAGAAAATAATACAGAAAAGTCA, (SEQ ID NO: 656) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGAGGTTTAATCCTATGCCAATACT (SEQ ID NO: 657) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG75 
     The methods used for PG75 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCGGGATCCGCTCAGGAGCAACTGAATGTGGTA, (SEQ ID NO: 658) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGTGGAACAAATTGCGCAATCCATC (SEQ ID NO: 659) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG76 
     The methods used for PG76 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCAGAATTCGGAAACGCACAGAGCTTTTGGGAA, (SEQ ID NO: 660) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTACCTGCACCTTATGACTGAATAC (SEQ ID NO; 661) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG77 
     The methods used for PG77 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAAGAGAAAAAGGATAGTCTCTCT, (SEQ ID NO: 662) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCTTICTFATCGCCATAGAATACAGG (SEQ ID NO: 663) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG78 
     The methods used for PG78 were essentially the same as for PG30 with lo the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCAGAATTCCAGGATTCTTCCCACGGTAGCAAT, (SEQ ID NO: 664) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATCATGATAGTAAAGACTGGTTCT (SEQ ID NO: 665) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG79 
     The methods used for PG79 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was TGCTGAATTCGTAGTGACGCTGCTCGTAATTGTC, (SEQ ID NO: 666) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGCCGTCCTGCCTTTCTGCCTGACG (SEQ ID NO; 667) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG80 
     The methods used for PG80 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAATTCCAAAACGTGCAGTTGCACTACGAT, (SEQ ID NO: 668) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGTTGAAAGTCCATTTGACCGCAAG (SEQ ID NO: 669) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG81 
     The methods used for PG81 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GTTTGAATTCCAGGATTTTCTCTATGAAATAGGA, (SEQ ID NO: 670) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTGTTTATTACAAAAAGTCTTACG (SEQ ID NO: 671) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG82 
     The methods used for PG82 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GAACGAATTCCAGAACAACAACTTTACCGAGTCG, (SEQ ID NO: 672) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGTTCAGTTTCAGCTTTTTAAACCA (SEQ ID NO: 673) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG84 
     The methods used for PG84 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGGATCCCAGAATGATGACATCTTCGAAGAT, SEQ ID NO: 674) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATTGCGTCCCCGGCCACTACGTCC (SEQ ID NO: 675) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG85 
     The methods used for PG85 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGGTGAATTCGTACCAACGGACAGCACGGAATCG, (SEQ ID NO: 676) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCAGATTGGTGCTATAAGAAAGGTA (SEQ ID NO: 677) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG86 
     The methods used for PG86 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGGATCCCAAACGCATGATCATCTCATCGAA, (SEQ ID NO: 678) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGTGGTTCAGGCCGTGGGCAAATCT (SEQ ID NO: 679) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG87 
     The methods used for PG87 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCGGAATTCCAGAGCTATGTGGACTACGTCGAT, (SEQ ID NO: 680) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATTACTGTGATTAGCGCGACGCTG (SEQ ID NO: 681) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG88 
     The methods used for PG88 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCAGAATTCGCCGAATCGAAGTCTGTCTCTTTC, (SEQ ID NO: 682) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCGGCAAGTAACGCTTTAGTGGGGA (SEQ ID NO: 683) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG89 
     The methods used for PG89 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAATCGAAGTTAAAGATCAAGAGC, (SEQ ID NO: 684) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTATTAGTCCAAAGACCCACGGTAAA (SEQ ID NO: 685) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG90 
     The methods used for PG90 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCAGAATTCCAAACAACGACGAACAGTAGCCGG, (SEQ ID NO: 686) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTTTTGTGATACTGTTTGGGC (SEQ ID NO: 687) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG91 
     The methods used for PG91 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAGACGATGGGAGGAGATGATGTC, (SEQ ID NO: 688) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTCCACGATGAGCTTCTCTACGAA (SEQ ID NO: 689) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG92 
     The methods used for PG92 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAATTCGCCGATGCACAAAGCTCTGTCTCT, (SEQ ID NO: 690) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTCGAGGACGATTGCTTAGTTCGTA (SEQ ID NO: 691) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG93 
     The methods used for PG93 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAGCTCCAAGAGGAAGGTATTTGGAATACC, (SEQ ID NO: 692) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGCGAATCACTGCGAAGCGAATTAG (SEQ ID NO: 693) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Sac I and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG94 
     The methods used for PG94 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAGCTCCAAGAGGAAGGTATTTGGAATACC, (SEQ ID NO: 694) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTGTCCTACCACGATCATTTTCTT (SEQ ID NO: 695) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG95 
     The methods used for PG95 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAGCTCTGTGGAAAAAAAGAAAAACACTCT, (SEQ ID NO: 696) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTAACTGTCTCCTTGTCGCTCCCCGG (SEQ ID NO: 697) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Sac I and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG96 
     The methods used for PG96 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAGCTCCAAACGCAAATGCAAGCAGACCGA, (SEQ ID NO: 698) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTTGAGAATTTTCATTGTCTCACG (SEQ ID NO: 699) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Sac I and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG97 
     The methods used for PG97 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCGGGATCCCAGTTTGTTCCGGCTCCCACCACA, (SEQ ID NO: 700) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCTGTTTGATGAGCTTAGTGGTATA (SEQ ID NO: 701) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG98 
     The methods used for PG98 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCAGAATTCCAAGAAAGAGTCGATGAAAAAGTA, (SEQ ID NO: 702) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTAGCTGTGTAACATTAAGTTTTTTATTGAT (SEQ ID NO: 703) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG99 
     The methods used for PG99 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCAAGGACAATTCTTCTTACAAACCT, (SEQ ID NO: 704) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTCGAATCACGACTTTTCTCACAAA (SEQ ID NO: 705) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG100 
     The methods used for PG100 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCAGAATTCCAGTCTTTGAGCACAATCAAAGTA, (SEQ ID NO: 706) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGATAGCCAGCTTGATGCTCTTAGC (SEQ ID NO: 707) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG101 
     The methods used for PG101 were essentially the same as for PG30 with the following exceptions. The 5′ oligonucleotide primer sequence was TGCTGAATTCAAAGGCAAGGGCGATCTGGTCGGG, (SEQ ID NO: 708) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTCTCTTCTCGAACTTGGCCGAGTA (SEQ ID NO; 709) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG102 
     The methods used for PG102 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAATTCCAGATGGATATTGGTGGAGACGAT, (SEQ ID NO: 710) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCTCTACAATGATTTTTTCCACGAA (SEQ ID NO: 711) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     PG104 
     The methods used for PG104 were essentially the same as for PG30 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GAACGGATCCAACGTGTCTGCTCAGTCACCCCGA, (SEQ ID NO: 712) the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTCTGAGCGATACTTTTGCACGTAT (SEQ ID NO: 713) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
     Animal Antisera and Human Patient Sera. 
     Various antisera were raised for detecting the expression and refolding of the recombinant  P. gingivalis  proteins. A whole cell antisera was raised by injecting New Zealand White rabbits with 3 doses of sonicated  P. gingivalis  (strain W50) containing approximately 2 mg of protein. The first dose was given in Freunds complete adjuvant (FCA) and the second and third doses were given in Freunds incomplete adjuvant (IFA) at 3 week intervals. Doses (1 ml) were given intramuscularly into the hind legs and rabbits bled 7 days after the last dose, the blood clotted and serum removed and stored at −20° C. until required. A second rabbit antisera was produced in a similar manner but using a sarkosyl insoluble fraction (each dose was 0.69 mg of protein) derived from  P. gingivalis  W50 according to the method of Doidg and Trust T. et al 1994 as the immunogen. A third rabbit antisera was produced in a similar manner to the first only the sarkosyl soluble fraction (1 mg of protein per dose) derived from  P. gingivalis  W50 cells according to the method of Doidg P. and Trust T J. (1994 Infect Immun 62:4526-33) was used as the immunogen. 
     A “protected rat serum” pool was also used in these studies and was obtained from rats immunised with formalin killed whole  P. gingivalis  cells in FIA (strain ATCC 33277; 2 doses of 2×10 9  cells, 3 weeks apart). Rats were then challenged 2 weeks after their last dose with live  P. gingivalis  cells (strain 33277) given orally as previously described (Klaussen B. et al. 1991, Oral Microbiol Immunol 6:193-201) and the serum obtained from these rats 6 weeks after the final challenge inoculation at the time of sacrifice. 
     Human sera were obtained from adult patients undergoing treatment or assessment for periodontitis at an outpatient clinic. These patients had at least 6 teeth with 6 mm attachment loss and had  P. gingivalis  present in their sub-gingival plaque as detected using a  P. gingivalis  specific DNA probe. Sera was pooled from these patients and compared to a pool of sera from periodontally healthy patients. 
     Immunization and Murine Lesion Model Protocols 
     The mouse abscess model was used to assess the efficacy of immunising mice with recombinant  P. gingivalis  proteins in protecting mice from formation of a subcutaneous abscess. This model has been used by others as a predictor of potential vaccines against periodontal disease (Bird P S, et al. 1995 J. Periodontol. 66:351-362. BALB/c mice 6-8 weeks old were immunised by subcutaneously injecting them with 0.1 ml containing either 10 or 20 μg of recombinant  P. gingivalis  protein, 20 μg of  E. coli  lysate protein, 2×10 9  formalin killed cells of  P. gingivalis  strain 33277 emulsified in incomplete Freund&#39;s adjuvant (IFA; Sigma) on day 0. At day 21 mice were re-injected with the same dose and then bled 1 week later and evaluated for antibody levels. At day 35 mice all mice were challenged with approximately 2×10 9  cells of live  P. gingivalis  (ATCC 33277) by subcutaneous injection in the abdomen. Following challenge mice were monitored daily for weight loss and the size of the lesion measured for the next 10 days. Lesion sizes were measured by length and width and expressed as mm 2 . Groups were statistically analysed using a Kruskal-Wallis one-way ANOVA and were also individually examined using the unpaired t test or Mann-Whitney rank sum test using the Instat statistical package. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the results of one experiment at day 4 after challenge (lesions were at maximum size at this time point). Control mice immunised with  E. coli  lysate showed large lesions while mice immunised with killed cells of  P. gingivalis  strain 33277 were fully protected. This indicates that whole cells provide protection against  P. gingivalis  while  E. coli  protein immunised mice were not protected. Mice given the various PG recombinant proteins showed significant levels of protection for PG2, PG22, PG24 and PG29 (p&lt;0.05 unpaired t test) while PG8A was not quite significantly different (p=0.07) compared to the  E. coli  control group. 
         FIG. 2  shows the results of a separate experiment using combinations of recombinant proteins. Mice given PG1+PG2 showed a significant level of protection compared to control mice give  E. coli  lysate (p&lt;0.026 unpaired t test). 
     
    
    
     IMMUNOSCREENING 
     Cloned candidates were cultured in 15 ml of Terrific broth, induced with IPTG and sampled at 4 h post-induction. One ml of culture was removed, pelleted and the cells resuspended in a volume of PBS determined by dividing the OD A 600 nm  of the culture by 8. An aliquot of lysate (100 μl) was added to 100 μl of 2× sample reducing buffer (125 mM Tris pH 6.8, 20% glycerol, 4% SDS, 80 mM DTT, 0.03% bromophenol blue) and boiled for 10 min. SDS-PAGE was performed according to the method of Laemmli UK. 1970 (Nature 227:680-685) using 4-20% 1.0 mm Tris-Glycine gels (Novex) according to the manufacturers recommendations. Proteins were transferred onto Hybond-C Extra nitrocellulose membranes (Amersham) by transblotting and the membranes were then blocked for 2 h at room temperature (RT) in 5% skim milk in 20 mM Tris, 0.5M NaCl, 0.05% Tween-20, pH 7.5 (TTBS). 
     Immunoscreening was performed separately with the rabbit anti- P. gingivalis  whole cell serum, the rat protective serum, a pool of human periodontal patients serum, and in many cases an anti-T7-Tag antibody HRP conjugate (Novagen). Prior to use, the rabbit, rat and human sera were diluted 1/5000, 1/1000 and 1/500 respectively in 5% skim milk in TTBS and absorbed with 100 μl (for the rabbit serum) or 250 μl (for the rat and human sera)  E. coli  extract (20 mg/ml; Promega) for 6 h at RT. 
     Membranes were incubated overnight at RT with the absorbed antisera, or for 1 hr at RT with 1/5000 diluted anti-T7-Tag conjugate. Following 3×10 min washes with TTBS, HRP-conjugated anti-rabbit (Silenus), anti-mouse (Silenus) or anti-human (KPL) antibody, diluted 1/5000 in 5% skim milk in TTBS, was added for 1 h at RT. Membranes were washed as before, prior to addition of TMB membrane peroxidase substrate (KPL) for detection of immunoreactive proteins. Results of reactivity for the recombinant  P. gingivalis  proteins is shown in Table 7. 
     In addition some of the sera (pooled sera diluted 1/1000) from the mice immunised with  P. gingivalis  recombinant proteins (prior to challenge) were analysed for their reactivity against Western blots of whole native W50  P. gingivalis  proteins using similar techniques as those outlined above. PG2, PG8A, PG29 and PG3 all showed bands at a similar molecular weight to that of the recombinant PG protein in the native W50 blot. This indicates that PG proteins are expressed in the W50 strain and that the recombinant proteins have at least some identical immunogenicity to the native proteins. 
     m-RNA Analysis 
     Hot Phenol RNA Extraction 
       P. gingivalis  W50 cells (150ml culture) were grown anaerobically to mid log phase (OD A 600 =0.18) mixed with 50% glycerol and stored at −70° C. until RNA extraction. Cells were pelleted by centrifugation at 600 g, and resuspended in 8 ml ASE (20 mM NaOAc, 0.5% SDS, 1 mM EDTA). An equal volume of 20 mM NaOAc(pH 4.5)-saturated phenol was added and mixed by shaking for 30 seconds, incubated at 65° C. for 5 minutes, followed by a further 5 second shaking and repeated incubation. After cooling, 2 ml chloroform was added and mixed by shaking for 5 seconds, and the mixture spun at 10000 g for 10 minutes at 4° C. The top aqueous phase was transferred and re-extracted by repeating the phenol and chloroform steps. The aqueous phase was transferred again and 100 U RNase inhibitor (RNAsin; Promega) were added. RNA was precipitated with 3 volumes 100% ethanol at −20° C. overnight. The RNA precipitate was recovered by centrifugation at 10000 g at 4° C. for 15 minutes, then washed with 100% ethanol, dried and resuspended in 600 μl sterile, deionised, dH 2 O with 1 μl of fresh RNase inhibitor. RNA was aliquoted and stored at −70° C. The RNA concentration was determined spectrophotometrically. A formaldehyde RNA gel confirmed RNA integrity (Sambrook J. et al. 1989, Molecular Cloning. A laboratory manual. Cold Spring Laboratory Press, New York. 2nd Edition). 
     RT-PCR 
     The isolated RNA was used as a template for Reverse Transcription (RT) to produce cDNA. Varying RNA concentrations were used for the RT as each RNA transcript was potentially present at different levels. Subsequent amplification of the cDNA was performed using Polymerase Chain Reaction (PCR). RT-PCR was performed using GeneAmp® RNA PCR Kit (Perkin Elmer) according to the manufacturer&#39;s protocol with the following exception to the PCR; 35 cycles were performed as follows: Melt phase 95° C. for 30 seconds, Anneal phase varied between 50-60° C. for 30 seconds, Extension phase 72° C. for 1 minute. Amplification was performed in a PTC-100 Programable Thermal Controller (MJ Research Inc.). As a control to demonstrate that the amplified product did not arise from contaminating DNA, Reverse Transcriptase (RTase) was omitted from a parallel tube. The PCR products were examined against DNA markers (GIBCO 1 kB ladder) on a 1% agarose gel stained with ethidium bromide. 
     RT-PCR results are shown in Table 6 using the oligonucleotide primers as used in “Cloning, expression and purification of recombinant  P. gingivalis  genes” section described above,except for the following changes. For PG1 the 3′ reverse primer used was GCGCCTCGAGATTCATTTCCTTATAGAG, (SEQ ID NO: 714) for PG4 the 5′ forward primer was CTTCTTGTCGACTACAGCGGACATCATAAAATC (SEQ ID NO: 715) and the 3′ reverse primer was TTCCACCTCGAGTTAACGCAACTCTTCTTCGAT, (SEQ ID NO: 716) for PG6 the 5′ forward primer was TAAAGAATTCTGCCTCGAACCCATAATTGCTCCG, (SEQ ID NO: 717) for PG10 the 5′ forward primer was CGCGCATATGGATAAAGTGAGCTATGC (SEQ ID NO: 718) and the 3′ reverse primer was CGCGCTCGAGTTTGTTGATACTCAATAATTC, (SEQ ID NO: 719) for PG13 the 5′ forward primer was GCCCGGCGCCATGCGGACAAAAACTATCTTTTTTGCG (SEQ ID NO: 720) and the 3′ reverse primer was GCCCGGCGCCTTAGTTGTTGAATCGAATCGCTATTTGAGC (SEQ ID NO: 721). 
     Amplification of  P. gingivalis  transcripts is a likely indication that RNA for a specific candidate is present and that the protein is produced. However, where there is no amplification achieved this does not indicate that this gene is never transcribed and may be the result of the culture conditions or the state of the cells when harvested. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Expression of PG m-RNA with in vitro grown  P. gingivalis  W50. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Approx. 
                 Expected 
               
               
                   
                 RNA 
                 Annealing 
                 RT- 
                   
                 fragment 
                 fragment 
               
               
                 PG # 
                 μg 
                 temp. ° C. 
                 PCR 
                 PCR (−RT) 
                 size bp 
                 size bp 
               
               
                   
               
             
          
           
               
                  1 
                 0.15 
                 55 
                 + 
                 − 
                 1300 
                 1362 
               
               
                  2 
                 1.0 
                 50 
                 + 
                 − 
                 3200 
                 3051 
               
               
                  3 
                 0.15 
                 60 
                 + 
                 − 
                 720 
                 690 
               
               
                  4 
                 2.9 
                 55 
                 − 
                 − 
                 N.D. 
                 2000 
               
               
                  5 
                 0.02 
                 50 
                 + 
                 − 
                 1000 
                 947 
               
               
                  6 
                 1.0 
                 55 
                 + 
                 − 
                 1000 
                 972 
               
               
                   8A 
                 0.15 
                 50 
                 + 
                 − 
                 1200 
                 1278 
               
               
                 10 
                 0.15 
                 55 
                 + 
                 − 
                 590 
                 585 
               
               
                 11 
                 0.10 
                 60 
                 + 
                 − 
                 960 
                 942 
               
               
                 12 
                 0.02 
                 60 
                 + 
                 − 
                 880 
                 831 
               
               
                 13 
                 1.0 
                 50 
                 + 
                 − 
                 2150 
                 2274 
               
               
                 14 
                 0.15 
                 60 
                 + 
                 − 
                 1050 
                 996 
               
               
                 22 
                 1.0 
                 60 
                 − 
                 − 
                 N.D. 
                 228 
               
               
                 24 
                 1.0 
                 55 
                 + 
                 + 
                 1150 
                 1194 
               
               
                 29 
                 0.15 
                 60 
                 + 
                 − 
                 880 
                 885 
               
               
                   
               
               
                 The symbols are + band visible on agarose gel, − no band present on agarose gel, ND not detected. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Immunoblot results of proteins expressed in  E. coli  against rabbit, 
               
               
                 rat and human antisera. Deduced MW was calculated from amino 
               
               
                 acid sequence of the  P. gingivalis  proteins, some of which 
               
               
                 had their N-terminal signal sequences removed. Apparent MW was 
               
               
                 determined from SDS-PAGE gels. The N- and C- terminal tags add 
               
               
                 approximately 2.5 KDa to the deduced MW of the recombinant proteins. 
               
             
          
           
               
                 Protein 
                 Deduced 
                 Apparent 
                 Antisera reactivity 
               
             
          
           
               
                 number 
                 MW (KDa) 
                 MW (KDa) 
                 T7 
                 Rabbit 
                 Rat 
                 Human 
               
               
                   
               
             
          
           
               
                 PG1 
                 47.5 
                 63 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG2 
                 112.4 
                 125.7 
                 ND 
                 + 
                 − 
                 − 
               
               
                 PG3 
                 22.6 
                 18.3 
                 ND 
                 − a   
                 − 
                 − 
               
               
                 PG4 
                 75 
                 90.6 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG5 
                 34.9 
                 43.8 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG6 
                 36.7 
                 47.1 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG8 
                 67.5 
                 63.1 
                 ND 
                 − b   
                 − 
                 − 
               
               
                 PG8A 
                 47.7 
                 90.6 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG10 
                 21.3 
                 25.5 
                 ND 
                 + 
                 − 
                 + 
               
               
                 PG11 
                 36.2 
                 42.4 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG12 
                 30.7 
                 30.6 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG13 
                 84.5 
                 101 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG14 
                 36 
                 42.4 
                 ND 
                 − 
                 + 
                 + 
               
               
                 PG22 
                 8.6 
                 11.1 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG24A 
                 47 
                 63.1 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG29 
                 31.1 
                 40.9 
                 ND 
                 + 
                 + 
                 + 
               
               
                 PG30 
                 35.1 
                 46.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG31 
                 16.7 
                 — 
                 − 
                 − 
                 − 
                 − 
               
               
                 PG32 
                 41.2 
                 59.5 
                 + 
                 + 
                 + 
                 − 
               
               
                 PG33 
                 39.9 
                 52.7 
                 + 
                 + 
                 + 
                 − 
               
               
                 PG35 
                 92.6 
                 116.6 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG36 
                 98.9 
                 120.2 
                 − 
                 − 
                 − 
                 − 
               
               
                 PG37 
                 18.8 
                 23.1 
                 + 
                 + 
                 − 
                 − 
               
               
                 PG38 
                 16.1 
                 22.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG39 
                 87.9 
                 116.6 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG40 
                 76.6 
                 103.1 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG41 
                 48.3 
                 81.1 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG42 
                 59.3 
                 73.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG43 
                 27.1 
                 50.3 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG44 
                 28.6 
                 32.3 
                 + 
                 − 
                 + 
                 − 
               
               
                 PG45 
                 84 
                 100.6 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG46 
                 83 
                 97.7 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG47 
                 93.7 
                 42.5 
                 + 
                 + 
                 − 
                 + 
               
               
                 PG48 
                 45.2 
                 37.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG49 
                 33.3 
                 64.1 
                 + 
                 − 
                 + 
                 − 
               
               
                 PG50 
                 91.9 
                 113.2 
                 + 
                 + 
                 − 
                 − 
               
               
                 PG51 
                 19.6 
                 27.2 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG52 
                 50.4 
                 64.4 
                 + 
                 + 
                 − 
                 + 
               
               
                 PG53 
                 47.4 
                 45.4 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG54 
                 101.4 
                 46.7 
                 + 
                 + 
                 − 
                 − 
               
               
                 PG55 
                 70.4 
                 68.4 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG56 
                 142.3 
                 — 
                 − 
                 − 
                 − 
                 − 
               
               
                 PG57 
                 100 
                 134.5 
                 + 
                 + 
                 + 
                 + 
               
               
                 PG58 
                 63 
                 82.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG59 
                 33.3 
                 43.6 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG60 
                 55.6 
                 77.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG61 
                 81.5 
                 107.3 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG62 
                 51.9 
                 58.4 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG63 
                 29.6 
                 43.6 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG64 
                 18.5 
                 26.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG65 
                 25.9 
                 28.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG66 
                 22.2 
                 25.1 
                 + 
                 + 
                 − 
                 − 
               
               
                 PG67 
                 103.7 
                 105 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG68 
                 133.3 
                 30.7 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG69 
                 44.4 
                 50.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG70 
                 25.9 
                 30.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG71 
                 88.9 
                 105.5 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG72 
                 40.7 
                 49.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG73 
                 40.7 
                 29 
                 +/− 
                 − 
                 − 
                 − 
               
               
                 PG74 
                 22.2 
                 32.5 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG75 
                 40.7 
                 46.7 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG76 
                 48.1 
                 55.6 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG77 
                 29.6 
                 36.9 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG78 
                 33.3 
                 35.4 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG79 
                 33.3 
                 — 
                 − 
                 − 
                 − 
                 − 
               
               
                 PG80 
                 25.9 
                 20.5 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG81 
                 23 
                 25.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG82 
                 44.8 
                 48.5 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG84 
                 41.7 
                 52.4 
                 + 
                 − 
                 − 
                 +/− 
               
               
                 PG85 
                 62.7 
                 72.4 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG86 
                 21.7 
                 27.4 
                 + 
                 − 
                 − 
                 +/− 
               
               
                 PG87 
                 83 
                 91.3 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG88 
                 27 
                 40.1 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG89 
                 26.2 
                 29.4 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG90 
                 23 
                 28.4 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG91 
                 57.2 
                 85.7 
                 + 
                 + 
                 + 
                 + 
               
               
                 PG92 
                 83.6 
                 110.4 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG93 
                 83.4 
                 110.4 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG96 
                 59.3 
                 70.3 
                 + 
                 + 
                 + 
                 + 
               
               
                 PG97 
                 44.4 
                 57.5 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG98 
                 33.3 
                 36 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG99 
                 40.7 
                 55.6 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG100 
                 29.6 
                 10.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG101 
                 14.8 
                 19.7, 14.1 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG102 
                 59.3 
                 70.3 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG104 
                 40.7 
                 57.5 
                 + 
                 − 
                 − 
                 + 
               
               
                   
               
               
                 The symbols are + positive, − negative, +/− weak positive, ND not done. 
               
               
                   a Positive reaction detected with the rabbit antiserum to sarkosyl insoluble  P. gingivalis  antigen. 
               
               
                   b Purified protien demonstrated weak positive reaction with the rabbit antiserum to whole  P. gingivalis . 
               
             
          
         
       
     
     It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. 
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