Abstract:
The invention relates to a nucleic acid encoding a signal peptide from Bordetella pertussis, a recombinant molecule comprising the signal peptide, and processes for optimizing protein expression in Gram-negative bacteria employing the nucleic acid or signal peptide.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of application Ser. No. 08/242,070, filed May 13, 1994, now abandoned, which is a continuation of application Ser. No. 08/108,170, filed Aug. 18, 1993, now abandoned, which is a continuation of application Ser. No. 07/871,107, filed Apr. 20, 1992, now abandoned, which is a divisional of application Ser. No. 07/467,551, filed Jan. 19, 1990, now U.S. Pat. No. 5,159,062. 
    
    
     BACKGROUND AND SUMMARY OF THE INVENTION 
     The invention relates to the signal peptide of a protein from Bordetella pertussis which is able to direct heterologous proteins into the periplasmic space between the inner and outer membranes of Gram-negative species of bacteria. The invention additionally relates to DNA sequences which code for this signal peptide, to plasmids which contain a gene structure of this type, and to host organisms with plasmids of this type. The invention furthermore relates to plasmid vectors with whose aid it is possible to determine and compare the efficiency of known and new signal sequences. It is possible as a consequence of such comparative study for particularly efficient signal sequences to be identified, cloned and used in all three possible translation reading frames for the expression of heterologous proteins. 
     It is possible in principle to distinguish between two different types of signal sequences: a &#34;hydrophobic&#34; type and a &#34;hydrophilic&#34; type. The &#34;hydrophobic&#34; group of signal sequences usually comprises about 13-30 amino acids, whereas the &#34;hydrophilic&#34; group comprises about 12-70 amino acids. The signal sequence of the &#34;hydrophobic&#34; type can be divided into three structural elements. It is composed of a relatively hydrophilic NH 2  terminus with one or two basic amino acids, of an apolar, mostly hydrophobic block of seven or eight amino acids, and of a relatively hydrophilic COOH terminus which is terminated by an amino acid with a small side-chain. Such &#34;hydrophobic&#34; signal sequences guide proteins through the membrane of the endoplasmic reticulum (ER) and through bacterial membranes. Although bacterial and ER signal sequences differ slightly from one another, they are functionally interchangeable. The structure of the &#34;hydrophilic&#34; type differs greatly from that of the abovementioned &#34;hydrophobic&#34; type: there are no lengthy uninterrupted sections of hydrophobic amino acids in the &#34;hydrophilic&#34; type, but there are usually many basic and hydroxylated amino acids and few or no acidic amino acids. The &#34;hydrophilic&#34; type of signal sequences guides proteins into mitochondria, chloroplasts and, possibly, into peroxisomes too. It has no significance for the present invention. 
     Although, as shown above, the &#34;hydrophobic&#34; type of signal sequences of prokaryotic and eukaryotic origin have common characteristics and may be functionally interchangeable, there are also observable differences: thus, most of the prokaryotic signal sequences hitherto known have, by comparison with the &#34;hydrophobic&#34; type (=ER type) of eukaryotic signal sequences, a lower hydrophobicity in the apolar section plus, usually, an additional basic amino acid in the NH 2  region. This is possibly the reason why the natural signal sequence of a heterologous protein is usually less efficiently recognized and processed in microorganisms than is a bacterial signal sequence preceding this protein. 
     The secretion of a heterologous protein in E. coli usually takes place as transport through the inner membrane into the periplasmic space; only a few exceptions in which heterologous proteins are secreted into the surrounding medium are known. The transport of a heterologous protein in to the periplasmic space in E. coli substantially corresponds functionally to the transport of a protein into the lumen of the endoplasmic reticulum of eukaryotic cells. It is possible as a consequence of this process for proteins to be correctly folded and for intramolecular disulfide bridges to be correctly produced in E. coli too. The signal sequence is eliminated by proteolysis by specific signal peptidases, and thus the mature, &#34;processed&#34; heterologous protein is synthesized in E. coli. 
     Some proteins are unstable after cytoplasmic expression in bacteria, for example Escherichia coli, and are very rapidly broken down again by proteases. This breakdown can be prevented by, inter alia, these proteins being, owing to a preceding, very efficient signal sequence, rapidly secreted into the periplasmic space. Hence the object was to isolate particularly efficient signal sequences and to design processes suitable for this. 
     Hoffman and Wright (Proc. Acad. Natl. Sci. USA; (1985) 82, 5107-5111) describe plasmids which code for the periplasmic alkaline phosphatase from E. coli (PhoA, EC 3.1.3.1) without the signal sequence belonging thereto. In in vitro fusions with fusion partners with their own signal sequence there is now secretion of active alkaline phosphatase in the form of a fusion protein, whereas when there is no fused-on signal sequence there is no detectable activity for the alkaline phosphatase released into the cytoplasm. Manoil and Beckwith (Proc. Natl. Acad. Sci. USA (1985) 82, 8129-8133) continued this work by placing the cDNA coding for PhoA without a signal sequence and 5 subsequent amino acids on the 3&#39; side in front of the transposon Tn5 (loc. cit.) and were thus able to show that fusions not only with secreted proteins but also with membrane proteins result in active PhoA. The said construct &#34;TnPhoA&#34; is consequently suitable for identifying signal sequences or structures resembling signal sequences. 
     S. Knapp and J. Mekalanos (J. Bacteriology (1988) 170, 5059-5066) have now generated, by means of TnPhoA mutagenesis, mutants in Bordetella pertussis which are influenced by modulation signals (in this case nicotinic acid and MgSO 4 ), with the majority of these mutants being repressed and some being activated, which suggests that there are at least two trans-acting regulatory genes. 
     We have found that the mutant SK6 mentioned therein contains a new and very efficient signal sequence. 
     This new signal sequence belongs to a secretory protein from Bordetella pertussis and has the following sequence (cf. Tab. 2 and 3) 
     
         MKKWFVAAGIGAAGLMLSSAA 
    
     Also described are PhoA-containing plasmids which, on the one hand, are very well suited as &#34;signal-sequence cloning vectors&#34; and, on the other hand, make it possible to compare quantitatively various signal sequences in terms of their &#34;secretion efficiency&#34;. Particularly useful for both purposes is the vector pTrc99C-PhoA (FIG. 1, Tab. 1 and Example 2). This vector has been constructed from pTrc99C (Amann et al. Gene 69 (1988) 301-315) and from a PhoA DNA which has been modified to that effect and has no signal peptide sequence, in such a way that the structural gene for PhoA is located in the correct reading frame with respect to the translation initiation codon of pTrc99C, and an NcoI cleavage site has been generated directly at the 5&#39; end of the PhoA structural gene (without signal sequence). 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1a and 1b: (Parts a and b) Construction of plasmid pTrc99C-PhoA is shown. N=NcoI; S=SacI; P=PstI; [N]=NcoI site which is not regenerated after ligation; &#39;pho=phoA structural gene lacking a signal sequence; and oligo=synthetic oligonucleotide sequence. The arrows indicate the direction of transcription or the NH 2  →COOH orientation of translated regions. 
     FIG. 2: The plasmid structure of pSEC-Bp1 is shown. 
     FIG. 3: The plasmid structure of pMAC5-8 is shown. F1-ORI=origin of replication of the phage f1; ORI=origin of replication of the ColE1 type CAT=coding region for chloramphenicol acetyltransferase; and AMP=coding region for β-lactamase. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Accodingly, the invention relates to: 
     a) the signal sequence 
     
         MKKWFVAAGIGAAGLMLSSAA 
    
     b) plasmids which carry a sequence of this type, 
     c) the use thereof for the secretion of proteins, and 
     d) plasmids which are particularly suitable for the closing and quantitative evaluation of signal sequences, due to the fact that a strong promoter which can be regulated, such as trc, is followed by the lacZ ribosome-binding site (RBS) and by a vector-encoded translation initiation codon at a distance from the lacZ RBS which is optimized for high expression, with an NcoI cleavage site being present directly at the 5&#39; end of the PhoA structural gene which has no signal sequence, but having been deleted from within the PhoA sequence by mutation, and with pTrc99C-PhoA being preferred. 
     Furthermore, the invention is further detailed in the examples and the patent claims. 
     EXAMPLE 1 
     Identification and Isolation of the Bordetella pertussis Signal Sequence 
     The transposon TnPhoA used hereinafter is a derivative of the transposon Tn5. PhoA carries in the left IS50 insertion element an E. coli PhoA structural gene derivative which has no signal sequence. The latter was constructed by Manoil and Beckwith (loc. cit.) in such a manner that when TnPhoA has been transposed into a chromosomal or plasmid-encoded gene the result is a PhoA-positive gene fusion only if the reading frames of the E. coli PhoA structural gene from TnPhoA and the signal sequence of the structural gene affected by the transposition coincide. It is easy to identify such PhoA positive colonies using the dyestuff indicator 5-bromo-4-chloro-indoxyl phosphate toluidine (XP). The described technique was used to carry out a TnPhoA mutagenesis in the Bordetella pertussis wild strain 18323 (Knapp and Mekalanos (1988) loc. cit.). This resulted, inter alia, in the generation of the PhoA-positive TnPhoA mutant SK6, whose TnPhoA gene fusion is called vrg6. The vrg6 gene fusion was cloned on a 20 kb BamHI fragment in the vector plasmid pBR322 as follows: genomic DNA of the mutant SK6 was cleaved with BamHI and ligated with pBR322 DNA cut with BamHI and was transformed into the E. coli strain CC118 (=PhoA negative). Clones which contain the genomic fragment with the TnPhoA gene fusion were selected on kanamycin/ampicillin agar plates (TnphoA codes like Tn5 for a kanamycin-resistance gene which is located between the 5&#39; phoA portion of TnphoA and the unique BamHI cleavage site within TnphoA). 
     A genomic BamHI fragment from a TnphoA mutant which has kanamycin resistance must therefore also carry the PhoA structural gene and the genomic B. pertussis DNA, located upstream, as far as the next genomic BamHI cleavage site. In the case of the BamHI fragment which is 20 kb in size and carries the vrg6 gene fusion, about 14 kb correspond to genomic B. pertussis DNA and about 6 kb correspond to TnphoA-encoding DNA. Transcriptional and translational regulation sequences of the vrg6 gene fusion were further localized. For this purpose, the BamHI fragment which is 20 kb in size was subjected to restriction analysis, and subfragments which carry the entire PhoA sequence from TnphoA but, compared to the 20 kb fragment, truncated B. pertussis DNA regions were cloned into pBR322 and pUC18. The deletion derivatives obtained in this way were recloned into the plasmid pLAFR2 which is able to replicate in B. pertussis (Friedmann et al. (1982), Gene 18, 289-196) and, after conjugative transfer into B. pertussis, examined for PhoA activity susceptible to modulation. In this way a PstI fragment which is about 3.2 kb in size was identified and subcloned into pUC18 (called pUC-PI hereinafter) which now contains only about 500 base-pairs of B. pertussis DNA upstream of the TnphoA insertion site of the vrg6 gene fusion and is PhoA positive in B. pertussis after induction. Since the PhoA activity of B. pertussis derivatives which contain the cloned BamHI fragment which is 20 kb in size or the PstI fragment which is 3.2 kb in size do not differ essentially in their phosphatase activity, the transcriptional and translational regulation sequences of the vrg6 gene fusion on the latter fragment must still be completely present. Starting from pUC-PI, deletions were introduced into the DNA region located 500 base-pairs upstream from the TnphoA insertion site using the enzymes exonuclease III and S1 nuclease by the method of Henikoff ((1984) Gene 28, 351-359). This resulted, inter alia, in the two pUC-PI derivatives vrg6-delta12 and vrg6-delta11. vrg6-delta12 still contains about 200 base-pairs B. pertussis-specific DNA upstream from the TnphoA insertion site and is likewise PhoA positive. DNA sequencing was used to determine the B. pertussis signal sequence on this recombinant plasmid. 
     The signal sequence is as follows: 
     
         MKKWFVAAGIGAAGLMLSSAA 
    
     (cf. also Tab. 2) The B. pertussis signal sequence characterized in this way comprises 21 amino acids and was subsequently prepared and cloned as described in Example 3 and is suitable for the secretion of heterologous proteins. 
     vrg6-delta11 contains only four B. pertussis-specific nucleotides upstream from the TnphoA insertion site, followed by a pUC18-specified SacI cleavage site (Tab. 1). PstI/SacI cleavage of the vrg6-delta11 DNA results in the complete PhoA structural gene from TnphoA, which has no signal sequence and is on a fragment which is about 2.6 kb in size and which serves as a source of the phoA structural gene which has no signal sequence in Example 2. 
     EXAMPLE 2 
     Construction of a Vector Plasmid (pTrc99C-phoA) for the Cloning and Comparative Efficiency Measurement of Signal Sequences 
     The construction of the vector plasmid pTrc99C-phoA is described hereinafter. This vector plasmid carries as essential element the phoA structural gene which has already been described above, has no signal sequence and was isolated from TnphoA. The phoA structural gene carries an internal NcoI cleavage site. This cleavage site was eliminated by the method of site-directed mutagenesis while retaining the amino acid sequence. 
     For this purpose, initially the recombinant PhoA-negative plasmid pvrg6-delta11 (see Example 1) was cleaved with EcoRI, and the fragment which is 330 base-pairs in size from the internal region of the phoA structural gene was isolated. This fragment, which contains the NcoI cleavage site which is to be mutated, was ligated into the EcoRI site of the mutagenesis vector pMa5-8 (FIG. 3). The resulting plasmid pMa5-8-EcoRI330 was isolated and used to prepare a single strand. The single strand with the cloned EcoRI fragment obtained in this way was then isolated by known methods and subjected to the published gapped-duplex mutagenesis protocol (Kramer et al. (1984) Nucl. Acids Res. 12, 9441-9456), using the following oligodeoxynucleotide: 
     
         5&#39; ATCGATATTGCCGTGGTACGTTGCTTTC 3&#39; 
    
     A plasmid which had the desired NcoI mutation was identified by appropriate restriction analysis, and the relevant region was sequenced and confirmed as correct. Subsequently the EcoRI fragment which is 330 base-pairs in size was reisolated from this plasmid and sited in place of the corresponding fragment of the plasmid pvrg-6-delta11. For this purpose, pvrg-6-delta11 was partially digested with EcoRI, and a fragment which was shorter by 330 base-pairs than the starting plasmid pvrg-delta11 (about 6700 bp), which had been linearized by partial EcoRI digestion, was isolated. The EcoRI fragment of this size (about 6400 bp) was treated with alkaline phosphatase and ligated to the mutated EcoRI fragment which was 330 base-pairs in size, and the ligation mixture was transformed into E. coli. Recombinant plasmids which contain a restored phoA structural gene with the correctly inserted 330 base-pair EcoRI fragment were identified by restriction analysis and DNA sequencing. A recombinant plasmid of this type, pvrg6-delta11-deltaNcoI, was replicated and used to construct the hybrid plasmid pTrc99C-phoA. For this purpose, a SacI-ScaI fragment which was about 2600 base-pairs in size was isolated from pvrg6-delta11-deltaNcoI. In the next step the SacI-ScaI fragment which is about 900 base-pairs in size from pTrc99C (Amann et al. (1988) Gene 69, 301-315) was replaced by this SacI-ScaI fragment which is about 2600 base-pairs in size. The resulting recombinant plasmid pTrc99C-phoA now carries, as a result of the above manipulations, a unique NcoI cleavage site directly at the 5&#39; end of the phoA structural gene which has no signal sequence, and it can be used, as shown in the following example, for cloning any desired synthetic or natural signal sequences. pTrc99C-phoA carries the structural gene of phoA in the correct reading frame with respect to the translation initiation codon of the expression vector pTrc99C but is unable, because of the absence of the phoA signal sequence, to bring about in transformed Escherichia coli cells the synthesis of an enzymatically active alkaline phosphatase and is therefore suitable as a &#34;signal-sequence cloning vector&#34;. In addition, pTrc99C-phoA carries, upstream from the hybrid trc promoter (Amann and Brosius (1985) Gene 40, 183-190), the lacZ ribosome-binding site (RBS) and a translation initiation codon at a distance from the lacZ RBS which is optimized for high expression. E. coli cells which contain the recombinant plasmid pTrc99C-phoA do not produce any plasmid-encoded biologically active alkaline phosphatase activity because the phoA structural gene of this plasmid lacks the signal sequence. PhoA-positive colonies can now be generated by placing a DNA fragment coding for a signal sequence in front of the phoA structural gene in the correct reading frame. This can take place by cutting pTrc99C-phoA with NcoI and inserting synthetic DNA fragments which code for signal sequences into this vector DNA. Bacterial colonies which carry hybrid plasmids of this manipulation can now easily be identified by means of their new PhoA-positive phenotype using the dyestuff indicator XP which has already been described above. The principle which has been presented is explained hereinafter in the form of exemplary embodiments. Cloning of signal sequences of various secretory proteins into the pTrc99C-phoA vector results in isogenic recombinant plasmids which differ only in the signal sequence. For this reason, the phoA activity of the E. coli cells which contain such constructs provides a measure of the efficiency of the relevant cloned signal sequences. 
     Another possible use of the vector pTrc99C-phoA comprises the cloning of the synthetic DNA fragments which do not code for an unambiguously defined signal sequence but are degenerate in such a way that a plurality of amino acids is possible for each position of the signal sequence. This is to a certain extent a shotgun cloning, and the phoA activity measurement which is now possible due to the vector represents a measure of the efficiency of the artificial signal sequence. It is possible to use this method to prepare and evaluate new signal sequences which can be used for the heterologous expression of cloned genes. 
     The principle of the construction of pTrc99C-phoA is illustrated in FIG. 1. The abbreviations means: N=NcoI, S=SacI, P=PstI, [N]=NcoI site is not regenerated after ligation, &#39;phoA=phoA structural gene which has no signal sequence, arrows indicate the direction of transcription or the NH 2  →COOH orientation of translated regions. Oligo means=synthetic oligonucleotide sequence. Tab. 1 shows the relevant cloning and translation initiation region of pTrc99C-phoA. 
     EXAMPLE 3 
     DNA synthesis and cloning of the Bordetella pertussis signal sequence and of five other naturally occurring microbial signal sequences of secretory proteins. 
     The vector pTrc99C-phoA was used to clone six different signal sequences whose amino acid sequences are depicted in Tab. 2. Five other signal sequences, besides the new Bordetella pertussis signal sequence, were selected on the basis of the following criteria: 
     a) Signal sequence of a periplasmic protein 
     Alkaline phosphatase (phoA) from E. coli (Kikuchi et al. (1981) Nucleic Acid Res. 9, 5671-5678) 
     b) Signal sequence of an outer membrane protein 
     Outer membrane protein (ompA) from E. coli (Movva et al. (1980) J. Biol. Chem. 255, 27-29) 
     c) Signal sequences of three proteins secreted into the medium 
     Heat stable toxin I (STI) from E. coli (So and McCarthy (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4011-4015) 
     Heat stable toxin II (STII) from E. coli (Lee et al. (1983) Infect. Immun. 42, 264-268) 
     Amylase from Bacillus subtilis (Yang et al (1983) Nucleic Acids Res. 11, 237-249). 
     The following simplified nomenclature has been used for the synthesis and cloning of these signal sequences: 
     
         ______________________________________Bordetella portussis vrg-6            signal sequence = Seq 1PhoA             signal sequence = Seq 2OmpA             signal sequence = Seq 3STI              signal sequence = Seq 4STII             signal sequence = Seq 5Bacillus subtilis amylase            signal sequence = Seq 6______________________________________ 
    
     All six signal sequences mentioned were prepared by DNA synthesis. The DNA fragments synthesized for this purpose (depicted in Tab. 3) were cloned and identified in the test vector pTrc99C-phoA using the selection for alkaline phosphatase described in Example 2. The synthetic DNA fragments encoding the signal sequence were designed in such a way that, after insertion in the correct orientation in the vector pTrc99C-phoA, only one NcoI site is regenerated, specifically downstream from the region encoding the signal sequence (cf. Also FIG. 1, Tab. 3 and Tab. 4). It is thus possible for this NcoI site to be used further, as further detailed in Example 4, as cloning site for the insertion of heterologous genes into the pSEC vectors (pSEC=secretion). 
     The twelve DNA fragments shown in Tab. 3 were synthesized by known methods (Sinha et al. (1984) Nucl. Acids Res. 12, 4539-4557) using β-cyanoethylamidites. The syntheses were carried out by the phosphite triester method (Letsinger (1975) J. Amer. Chem. Soc. 97, 3278; Letsinger (1976) J. Amer. Chem. Soc. 98, 3655) using a Biosearch synthesizer. After cleavage off the carrier (CPG) with concentrated ammonia at room temperature for 5-8 h, and after the protective groups on the bases had been cleaved off in the same solution at 55° C. for about 12 h, the oligodeoxynucleotides were purified by gel electrophoresis or reverse-phase HPLC. The oligodeoxynucleotides were taken up in annealing buffer (100 mM NaCl, 10 mM TRIS-Cl (pH 7.8), 0.1 mM EDTA), molar amounts of each strand mixed, incubated at 95° C. for 5 min and slowly cooled to room temperature. The double-stranded DNA fragments have at the 5&#39; ends single-stranded regions which are four bases long and are complementary to an NcoI recognition site. The test vector pTrc99C-phoA was linearized with NcoI and ligated in various mixtures together with hybridized DNA fragments. Competent E. coli cells were transformed with the ligation mixtures by known methods, plated out on LB/amp agar plates and incubated at 37° C. overnight. The colonies were transferred by the replica-plating method to LB/Amp/XP/IPTG indicator plates and again incubated at 37° C. PhoA-positive colonies have a blue color on this indicator plate. Plasmid DNA of these colonies was isolated and sequenced, and it was possible to confirm the correct orientation of the synthetic DNA fragments as well as the expected correct signal sequence for the six abovementioned examples. The plasmids which were obtained in this way and had the particular signal sequence confirmed as correct by sequencing were called, in accordance with the above table, pTrc99C-phoA-Seq-1, -2, -3, -4, -5 and -6. It is now possible under standardized conditions to compare and evaluate, on the basis of the extinction (measurement of the liberated dyestuff), these signal sequences, those found from B. pertussis being among the relatively strongest. 
     EXAMPLE 4 
     Construction of the Secretion Vectors pSEC-Bp-1, pSEC-Bp-2 and pSEC-Bp-3 
     Plasmid DNA of the clone pTrc99C-phoA-Seq-1 was digested with SacI and ScaI, and the fragment which is about 3.1 kb in size was isolated. This fragment carries only pTrc99C-specific sequences in addition to the B. pertussis signal sequence (see also FIG. 1). This fragment was ligated, in each of three separate mixtures, with one of the approximately 0.9 kb SacI/ScaI fragments of the plasmids pTrc97A, pTrc97B and pTrc97C (Amann et al. loc. cit.), and the resulting plasmids were called pSEC-Bp-1, pSEC-Bp-2 and pSEC-Bp-3. This manipulation made use of the long polylinker region of the plasmids pTrc97A, pTRC97B and pTrc97C in order to make available in all three reading frames a plurality of restriction sites downstream from the region encoding the Bordetella pertussis signal sequence (Tab. 4). It is possible in analogy to these constructions to prepare similar secretion vectors for the expression and secretion of heterologous proteins by use of the plasmids pTrc99C-phoA-Seq-2, -3, -4, -5 and -6. The secretion vectors prepared in this way differ in their relative efficiency and in the cellular location of the expressed products in accordance with the origin of the signal sequence used in each case. As an example, FIG. 2 shows the plasmid structure of pSEC-BP1, and Tab. 5 shows the complete DNA sequence of pSEC-BP1, where xxx stands for a start or stop codon. 
     Legend to FIG. 1 
     Map of the plasmids pMAC5-8 (=pMA5-8 and pMC5-8). 
     F1-ORI: Origin of replication of the phage f1; 
     ORI: Origin of replication of the ColE1 type; 
     CAT: Coding region for chloramphenicol acetyltransferase; 
     AMP: Coding region for β-lactamase. 
     pMA5-8 carries an amber mutation in CAT (A at position 3409) and pMC5-8 carries an amber mutation in AMP (C at position 2238). 
     
                                           TABLE 1__________________________________________________________________________pTrc99C-phoA__________________________________________________________________________ ##STR1##__________________________________________________________________________ 
    
     
                                           TABLE 2__________________________________________________________________________Amino Acids__________________________________________________________________________ ##STR2##__________________________________________________________________________ 
    
     
         TABLE 3  - Bordetella pectussis signal sequence  5&#39;                                                                      TT  CATG AAA AAG TGG TTCGTTGCTGCCGGCATCGGCGCTGCCGGA CTCATG CTCTCCAGCGCCGCCCA AG CAA CGA CGG CCG TAG CCG CGA CGG CCTGAG TACGAG AGG TCG CGG CGG TAC5&#39;    E. coli phoA signal sequence  5&#39; CATG AAA CAA AGCACTATTGCA CTG GCA CTCTTA CCG TTA CTG TTTACCCCTGTG ACA AAA GCTTTGTTTCG TGA TAA CGTGACCGTGAG AATGGCAATGACAAA TGG GGA         TC CACTGTTTG TAC5&#39;  E. coli ompA signal sequence  5&#39; CATG AAA AAG ACA GCTATCGCG ATTGCA GTG GCA CTG GCTGGTTTCGCTACCGTA GCG CAG GCTTTTTCTGTCGA TAG CGCTAA CGTCACCGTGCA CGA CCA AAG CGA TGG CATCGCGTCCG G TAC5&#39;  E. coli heat-stable toxin I signal sequence  5&#39;  CATG AAA AAG CTA ATG TTG GCA ATTTTTATTTCTGTA TTA TCTTTCCCCTCTTTTAGTCAG  CA T CCTTTTTCGATTACAACCGTTAA AAA TAA AGA CATAATAGA AAG GGG AGA AAA TCA GTCAGTGGG TAC5&#39;  E. coli heat-stable toxin II signal sequence  5&#39; CATG AAA AAG AATATCGCA TTTCTTCTTGCA TCTATG TTCGTTTTTTCTATTGCTACA AATGCCTATGCTTTTTCTTA TAG CGTAAA GCCGAA CGTAGA TACAAG CAA AAA AGA TAA CGA TGTTTA CGG ATA CGG TAC5&#39;  Bacillus subtilis Amylase signal sequence  5&#39; CATG TTTGCA AAA CGA TTCAAA ACCTCTTTA CTG CCG TTA TTCGCTGGA TTTTTA TTG CTG TTTCATTTG GTTAAA CGTTTTGCTAAG TTTTGG AGA AATGACGGCAATAAG CGA CCTAAA AATAACGACAAA GTA AACCAA  CTG GCA GGA CCG GCG GCTGCG AGTCC  GACCGTCCTGGCCGCCGA CGCTCA GGG TAC5&#39; 
    
     
         TABLE 4  -  ##STR3##  ##STR4##  ##STR5## 
    
     
                                           TABLE 5__________________________________________________________________________1  GTTTGACAGC       TTATCATCGA                CTGCACGGTG                         CACCAATGCT                                  TCTGGCGTCA51 GGCAGCCATC       GGAAGCTGTG                GTATGGCTGT                         GCAGGTCGTA                                  AATCACTGCA101   TAATTCGTGT       CGCTCAAGGC                GCACTCCCGT                         TCTGGATAAT                                  GTTTTTTGCG                                  -35151   CCCACATCAT       AACCGTTCTC                GCAAATATTC                         TGAAATGAGC                                  TGTTGACAAT   trcP     -10201   TAATCATCCG       GCTCGTATAA                TGTGTGGAAT                         TGTGAGCGGA                                  TAACAATTTC       M        KKWF     VAA      GIG251   ACACAGGAAA       CAGACCATGA                AAAAGTGGTT                         CGTTGCTGCC                                  GGCATCGGCG       ***   AAGL     MLS      SAA301   CTGCCGGACT       CATGCTCTCC                AGCGCCGCCA                         TGGAATTCGA                                  GCTCGGTACCNcoIEcoRISstIKpnI351   CGGGGATCCT       CTAGAGTCGA                CCTGCAGGCA                         TGCAAGCTTG                                  GCTGTTTTGGSmaIBamHIXbaISalIPstISphIHindIII401   CGGATGAGAG       AAGATTTTCA                GCCTGATACA                         GATTAAATCA                                  GAACGCAGAA   ***               ***      ***451   GCGGTCTGAT       AAAACAGAAT                TTGCCTGGCG                         GCAGTAGCGC                                  GGTGGTCCCA501   CCTGACCCCA       TGCCGAACTC                AGAAGTGAAA                         CGCCGTAGCG                                  CCGATGGTAG551   TGTGGGGTCT       CCCCATGCGA                GAGTAGGGAA                         CTGCCAGGCA                                  TCAAATAAAA601   CGAAAGGCTC       AGTCGAAAGA                CTGGGCCTTT                         CGTTTTATCT                                  GTTGTTTGTC651   GGTGAACGCT       CTCCTGAGTA                GGACAAATCC                         GCCGGGAGCG                                  GATTTGAACG701   TTGCGAAGCA       ACGGCCCGGA                GGGTGGCGGG                         CAGGACGCCC                                  GCCATAAACT751   GCCAGGCATC       AAATTAAGCA                GAAGGCCATC                         CTGACGGATG                                  GCCTTTTTGC801   GTTTCTACAA       ACTCTTTTTG                TTTATTTTTC                         TAAATACATT                                  CAAATATGTA851   TCCGCTCATG       AGACAATAAC                CCTGATAAAT                         GCTTCAATAA                                  TATTGAAAAA901   GGAAGAGTAT       GAGTATTCAA                CATTTCCGTG                         TCGCCCTTAT                                  TCCCTTTTTT951   GCGGCATTTT       GCCTTCCTGT                TTTTGCTCAC                         CCAGAAACGC                                  TGGTGAAAGT1001   AAAAGATGCT       GAAGATCAGT                TGGGTGCACG                         AGTGGGTTAC                                  ATCGAACTGG1051   ATCTCAACAG       CGGTAAGATC                CTTGAGAGTT                         TTCGCCCCGA                                  AGAACGTTTT1101   CCAATGATGA       GCACTTTTAA                AGTTCTGCTA                         TGTGGCGCGG                                  TATTATCCCG1151   TGTTGACGCC       GGGCAAGAGC                AACTCGGTCG                         CCCCATACAC                                  TATTCTCAGA1201   ATGACTTGGT       TGAGTACTCA                CCAGTCACAG                         AAAAGCATCT                                  TACGGATGGC1251   ATGACAGTAA       GAGAATTATG                CAGTGCTGCC                         ATAACCATGA                                  GTGATAACAC1301   TGCGGCCAAC       TTACTTCTGA                CAACGATCGG                         AGGACCGAAG                                  GAGCTAACCG1351   CTTTTTTGCA       CAACATGGGG                GATCATGTAA                         CTCCCCTTGA                                  TCGTTGGGAA1401   CCGGAGCTGA       ATGAAGCCAT                ACCAAACGAC                         GAGCGTGACA                                  CCACGATGCC1451   TACAGCAATG       GCAACAACGT                TGCGCAAACT                         ATTAACTGGC                                  GAACTACTTA1501   CTCTAGCTTC       CCGGCAACAA                TTAATAGACT                         GGATGGAGGC                                  GGATAAAGTT1551   GCAGGACCAC       TTCTGCGCTC                GGCCCTTCCG                         GCTGGCTGGT                                  TTATTGCTCA1601   TAAATCTGGA       GCCGGTGAGC                GTGGGTCTCG                         CGGTATCATT                                  GCAGCACTGG1651   GGCCAGATGG       TAAGCCCTCC                CGTATCGTAG                         TTATCTACAC                                  GACGGGGAGT1701   CAGGCAACTA       TGGATGAACG                AAATAGACAG                         ATCGCTGAGA                                  TAGGTGCCTC1751   ACTGATTAAG       CATTGGTAAC                TGTCAGACCA                         AGTTTACTCA                                  TATATACTTT1801   AGATTGATTT       AAAACTTCAT                TTTTAATTTA                         AAAGGATCTA                                  GGTGAAGATC1851   CTTTTTGATA       ATCTCATGAC                CAAAATCCCT                         TAACGTGAGT                                  TTTCGTTCCA1901   CTGAGCGTCA       GACCCCGTAG                AAAAGATCAA                         AGGATCTTCT                                  TGAGATCCTT1951   TTTTTCTGCG       CGTAATCTGC                TGCTTGCAAA                         CAAAAAAACC                                  ACCGCTACCA2001   GCGGTGGTTT       GTTTGCCGGA                TCAAGAGCTA                         CCAACTCTTT                                  TTCCGAAGGT2051   AACTGGCTTC       AGCAGAGCGC                AGATACCAAA                         TACTGTCCTT                                  CTAGTGTAGC2101   CGTAGTTAGG       CCACCACTTC                AAGAACTCTG                         TAGCACCGCC                                  TACATACCTC2151   GCTCTGCTAA       TCCTGTTACC                AGTGGCTGCT                         GCCAGTGGCG                                  ATAAGTCGTG2201   TCTTACCGCG       TTGGACTCAA                GACGATAGTT                         ACCGGATAAG                                  GCGCAGCGGT2251   CGGGCTGAAC       GGGGGGTTCG                TGCACACAGC                         CCAGCTTGGA                                  GCGAACGACC2301   TACACCGAAC       TGAGATACCT                ACAGCGTGAG                         CTATGAGAAA                                  GCGCCACGCT2351   TCCCGAAGGG       AGAAAGGCGG                ACAGGTATCC                         GGTAAGCGGC                                  AGGGTCGGAA2401   CAGGAGAGCG       CACGAGGGAG                CTTCCAGGGG                         GAAACGCCTG                                  GTATCTTTAT2451   AGTCCTGTCG       GGTTTCGCCA                CCTCTGACTT                         GAGCGTCGAT                                  TTTTGTGATC2501   CTCGTCAGGG       GGGCGGAGCC                TATGGAAAAA                         CGCCAGCAAC                                  GCGGCCTTTT2551   TACGGTTCCT       GGCCTTTTGC                TGGCCTTTTG                         CTCACATGTT                                  CTTTCCTGCG2601   TTATCCCCTG       ATTCTGTGGA                TAACCGTATT                         ACCGCCTTTG                                  AGTGAGCTGA2651   TACCCCTCGC       CGCAGCCGAA                CGACCGAGCG                         CAGCGAGTCA                                  GTGAGCGAGG2701   AAGCGGAAGA       GCGCCTGATC                CGGTATTTTC                         TCCTTACGCA                                  TCTGTGCGGT2751   ATTTCACACC       GCATATGGTG                CACTCTCAGT                         ACAATCTGCT                                  CTGATGCCCC2801   ATAGTTAAGC       CAGTATACAC                TCCGCTATCG                         CTACGTGACT                                  GGGTCATGGC2851   TGCGCCCCGA       CACCCCCCAA                CACCCGCTGA                         CGCGCCCTGA                                  CGGGCTTSTC2901   TGCTCCCGGC       ATCCGCTTAC                AGACAAGCTG                         TGACCGTCTC                                  CGGGAGCTGC2951   ATGTGTCAGA       GGTTTTCACC                GTCATCACCG                         AAACGCGCGA                                  GGCAGCAGAT3001   CAATTCGCGC       GCGAAGGCGA                AGCGGCATGC                         ATTTACGTTG                                  ACACCATCGA3051   ATGGTGCAAA       ACCTTTCGCG                GTATGGCATG                         ATAGCGCCCG                                  GAAGAGAGTC3101   AATTCAGGGT       GGTGAATGTG                AAACCAGTAA                         CGTTATACGA                                  TGTCGCAGAG3151   TATGCCGGTG       TCTCTTATCA                GACCGTTTCC                         CGCGTGGTGA                                  ACCAGGCCAG3201   CCACGTTTCT       GCGAAAACGC                GGGAAAAAGT                         GGAAGCGGCG                                  ATGGCGGAGC3251   TGAATTACAT       TCCCAACCGC                GTGGCACAAC                         AACTGGCGGG                                  CAAACAGTCG3301   TTGCTGATTG       GCGTTGCCAC                CTCCAGTCTG                         GCCCTGCACG                                  CGCCGTCGCA3351   AATTGTCGCG       GCGATTAAAT                CTCGCGCCGA                         TCAACTGGGT                                  GCCAGCGTGG3401   TGGTGTCGAT       GGTAGAACGA                AGCGGCGTCG                         AAGCCTGTAA                                  AGCGGCGGTG3451   CACAATCTTC       TCGCGCAACG                CGTCAGTGGG                         CTGATCATTA                                  ACTATCCGCT3501   GGATGACCAG       GATGCCATTG                CTGTGGAAGC                         TGCCTGCACT                                  AATGTTCCGG3551   CGTTATTTCT       TGATGTCTCT                GACCAGACAC                         CCATCAACAG                                  TATTATTTTC3601   TCCCATGAAG       ACGGTACGCG                ACTGGGCGTG                         GAGCATCTGG                                  TCGCATTGGG3651   TCACCAGCAA       ATCGCGCTGT                TAGCGGGCCC                         ATTAAGTTCT                                  GTCTCGGCGC3701   GTCTGCGTCT       GGCTGGCTGG                CATAAATATC                         TCACTCGCAA                                  TCAAATTCAG3751   CCGATAGCGG       AACGGGAAGG                CGACTGGAGT                         GCCATGTCCG                                  GTTTTCAACA3801   AACCATGCAA       ATGCTGAATG                AGGGCATCGT                         TCCCACTGCG                                  ATGCTGGTTG3851   CCAACGATCA       GATGGCGCTG                GGCGCAATGC                         GCGCCATTAC                                  CGAGTCCGGG3901   CTGCGCGTTG       GTGCGGATAT                CTCGGTAGTG                         GGATACGACG                                  ATACCGAAGA3951   CAGCTCATGT       TATATCCCGC                CGTCAACCAC                         CATCAAACAG                                  GATTTTCGCC4001   TGCTGGGGCA       AACCAGCGTG                GACCGCTTGC                         TGCAACTCTC                                  TCAGGGCCAG4051   GCGGTGAAGG       GCAATCAGCT                GTTGCCCGTC                         TCACTGGTGA                                  AAAGAAAAAC4101   CACCCTGGCG       CCCAATACGC                AAACCGCCTC                         TCCCCGCGCG                                  TTGGCCGATT4151   CATTAATGCA       GCTGGCACGA                CAGGTTTCCC                         GACTGGAAAG                                  CGGGCAGTGA4201   GCGCAACGCA       ATTAATGTGA                GTTAGCGCGA                         ATTGATCTG__________________________________________________________________________