US20170080077A1

US20170080077A1 - Heterologous expression of neisserial proteins

Info

Publication number: US20170080077A1
Application number: US15/368,435
Authority: US
Inventors: Maria Aricò; Maurizio Comanducci; Cesira Galeotti; Vega Masignani; Marzia Monica Giuliani; Mariagrazia Pizza
Original assignee: GlaxoSmithKline Biologicals SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 2000-02-28
Filing date: 2016-12-02
Publication date: 2017-03-23
Also published as: EP1947187A1; CN1800385B; ES2391153T3; JP2011101657A; US20040092711A1; CN1544462A; DK1790660T3; CN1201011C; DE60132978D1; CN100473663C; ES2360746T3; CA2875231A1; JP2013005816A; CN100334214C; DE60126249T2; CA2689666A1; RU2002125882A; AU2001239488B2; US9150898B2; CA2400562A1

Abstract

Alternative and improved approaches to the heterologous expression of the proteins of Neisseria meningitidis and Neisseria gonorrhoeae are disclosed. These approaches typically affect the level of expression, the ease of purification, the cellular localization, and/or the immunological properties of the expressed protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No. 14/244,806, filed Apr. 3, 2014; which is a Continuation of U.S. application Ser. No. 13/340,549, filed Dec. 29, 2011, now U.S. Pat. No. 8,703,914; which is a Divisional of U.S. application Ser. No. 12/825,210, filed Jun. 28, 2010, now U.S. Pat. No. 8,114,960; which is a Divisional of U.S. application Ser. No. 10/220,481, which claims an international filing date of Feb. 28, 2001, now U.S. Pat. No. 7,803,387; which is the National Phase of PCT Application No. PCT/IB2001/000452, filed Feb. 28, 2001; which claims the benefit of GB Application No. 0027675.8, filed Nov. 13, 2000, and GB Application No. 0004695.3, filed Feb. 28, 2000; all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of protein expression. In particular, it relates to the heterologous expression of proteins from Neisseria (e.g. N. gonorrhoeae or, preferably, N. meningitidis).

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 303822001_004 SegList.txt, date recorded: Nov. 29, 2016, size: 405 KB).

BACKGROUND

International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae. These proteins are typically described as being expressed in E. coli (i.e. heterologous expression) as either N-terminal GST-fusions or C-terminal His-tag fusions, although other expression systems, including expression in native Neisseria, are also disclosed.
It is an object of the present invention to provide alternative and improved approaches for the heterologous expression of these proteins. These approaches will typically affect the level of expression, the ease of purification, the cellular localisation of expression, and/or the immunological properties of the expressed protein.

DISCLOSURE

Nomenclature Herein

The 2166 protein sequences disclosed in WO99/24578, WO99/36544 and WO99/57280 are referred to herein by the following SEQ# numbers:


Application	Protein sequences	SEQ# herein

WO99/24578	Even SEQ IDs 2-892	SEQ#s 1-446
WO99/36544	Even SEQ IDs 2-90	SEQ#s 447-491
WO99/57280	Even SEQ IDs 2-3020	SEQ#s 492-2001
	Even SEQ IDs 3040-3114	SEQ#s 2002-2039
	SEQ IDs 3115-3241	SEQ#s 2040-2166

In addition to this SEQ# numbering, the naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used (e.g. ‘ORF4’, ‘ORIF40’, ‘ORF40-1’ etc. as used in WO99/24578 and WO99/36544; ‘m919’, ‘g919’ and ‘a919’ etc. as used in WO99/57280).
The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [Science (2000) 287:1809-1815] are referred to herein as SEQ4#s 21674326 [see also WO00/66791].
The term ‘protein of the invention’ as used herein refers to a protein comprising:

- (a) one of sequences SEQ#s 1-4326; or
- (b) a sequence having sequence identity to one of SEQ#s 1-4326; or
- (c) a fragment of one of SEQ#s 1-4326.

The degree of ‘sequence identity’ referred to in (b) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants [e.g. see WO00/66741]. Identity is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.
The ‘fragment’ referred to in (c) should comprise at least n consecutive amino acids from one of SEQ#s 1-4326 and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one of SEQ#s 14326. Preferred fragments are those disclosed in WO00/71574 and WO01/04316.
Preferred proteins of the invention are found in N. meningitidis serogroup B.
Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain 2996 or strain 394198 (a New Zealand strain). Unless otherwise stated, proteins mentioned herein are from N. meningitidis strain 2996. It will be appreciated, however, that the invention is not in general limited by strain. References to a particular protein (e.g. ‘287’, ‘919’ etc.) may be taken to include that protein from any strain.

Non-Fusion Expression

In a first approach to heterologous expression, no fusion partner is used, and the native leader peptide (if present) is used. This will typically prevent any ‘interference’ from fusion partners and may alter cellular localisation and/or post-translational modification and/or folding in the heterologous host.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) no fusion partner is used, and (b) the protein's native leader peptide (if present) is used.
The method will typically involve the step of preparing an vector for expressing a protein of the invention, such that the first expressed amino acid is the first amino acid (methionine) of said protein, and last expressed amino acid is the last amino acid of said protein (i.e. the codon preceding the native STOP codon).
This approach is preferably used for the expression, of the following proteins using the native leader peptide: 111, 149, 206, 2254, 235, 247-1, 274, 283, 286, 292, 401, 406, 502-1, 503, 519-1, 525-1, 552, 556, 557, 570, 576-1, 580, 583, 664, 759, 907, 913, 920-1, 936-1, 953, 961, 983, 989, Orf4, Orf7-1, Orf9-1, Orf23, Orf25, Orf37, Orf38, Orf40, Orf40.1, Orf40.2, Orf72-1, Orf76-1, Orf85-2, Orf91, Orf97-1, Orf119, Orf143.1, NMB0109 and NMB2050. The suffix ‘L’ used herein in the name of a protein indicates expression in this manner using the native leader peptide.
Proteins which are preferably expressed using this approach using no fusion partner and which have no native leader peptide include: 008, 105, 117-1, 121-1, 122-1, 128-1, 148, 216, 243, 308, 593, 652, 726, 926, 982, Orf83-1 and Orf143-1.
Advantageously, it is used for the expression of ORF25 or ORF40, resulting in a protein which induces better anti-bactericidal antibodies than GST- or His-fusions.
This approach is particularly suited for expressing lipoproteins.

Leader-Peptide Substitution

In a second approach to heterologous expression, the native leader peptide of a protein of the invention is replaced by that of a different protein. In addition, it is preferred that no fusion partner is used. Whilst using a protein's own leader peptide in heterologous hosts can often localise the protein to its ‘natural’ cellular location, in some cases the leader sequence is not efficiently recognised by the heterologous host. In such cases, a leader peptide known to drive protein targeting efficiently can be used instead.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's leader peptide is replaced by the leader peptide from a different protein and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove nucleotides that encode the protein's leader peptide and to introduce nucleotides that encode a different protein's leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The expressed protein will consist of the replacement leader peptide at the N-tertninus, followed by the protein of the invention minus its leader peptide.
The leader peptide is preferably from another protein of the invention (e.g. one of SEQ#s 1-4326), but may also be from an E. coli protein (e.g. the OmpA leader peptide) or an Erwinia carotovora protein (e.g. the PelB leader peptide), for instance.
A particularly useful replacement leader peptide is that of ORF4. This leader is able to direct lipidation in E. coli, improving cellular localisation, and is particularly useful for the expression of proteins 287, 919 and ΔG287. The leader peptide and N-terminal domains of 961 are also particularly useful.
Another useful replacement leader peptide is that of E. coli OmpA. This leader is able to direct membrane localisation of E. coli. It is particularly advantageous for the expression of ORF1, resulting in a protein which induces better anti-bactericidal antibodies than both fusions and protein expressed from its own leader peptide.
Another useful replacement leader peptide is MICKYLFSAA. This can direct secretion into culture medium, and is extremely short and active. The use of this leader peptide is not restricted to the expression of Neisserial proteins—it may be used to direct the expression of any protein (particularly bacterial proteins).

Leader-Peptide Deletion

In a third approach to heterologous expression, the native leader peptide of a protein of the invention is deleted. In addition, it is preferred that no fusion partner is used.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's leader peptide is deleted and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove nucleotides that encode the protein's leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The first amino acid of the expressed protein will be that of the mature native protein.
This method can increase the levels of expression. For protein 919, for example, expression levels in E. coli are much higher when the leader peptide is deleted. Increased expression may be due to altered localisation in the absence of the leader peptide.
The method is preferably used for the expression of 919, ORF46, 961, 050-1, 760 and 287.

Domain-Based Expression

In a fourth approach to heterologous expression, the protein is expressed as domains. This may be used in association with fusion systems (e.g. GST or His-tag fusions).
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) at least one domain in the protein is deleted and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove at least one domain from within the protein. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. Where no fusion partners are used, the first amino acid of the expressed protein will be that of a domain of the protein.
A protein is typically divided into notional domains by aligning it with known sequences in databases and then determining regions of the protein which show different alignment patterns from each other.
The method is preferably used for the expression of protein 287. This protein can be notionally split into three domains, referred to as A B & C (see FIG. 5). Domain B aligns strongly with IgA proteases, domain C aligns strongly with transferrin-binding proteins, and domain A shows no strong alignment with database sequences. An alignment of polymorphic forms of 287 is disclosed in WO00/66741.
Once a protein has been divided into domains, these can be (a) expressed singly (b) deleted from with the protein e.g. protein ABCD→ABD, ACD, BCD etc, or (c) rearranged e.g. protein ABC→ACB, CAB etc. These three strategies can be combined with fusion partners is desired.
ORF46 has also been notionally split into two domains a first domain (amino acids 1-433) which is well-conserved between species and serogroups, and a second domain (amino acids 433-608) which is not well-conserved. The second domain is preferably deleted. An alignment of polymorphic forms of ORF46 is disclosed in WO00/66741.
Protein 564 has also been split into domains (FIG. 8), as have protein 961 (FIG. 12) and protein 502 (amino acids 28-167 of the MC58 protein).

Hybrid Proteins

In a fifth approach to heterologous expression, two or more (e.g. 3, 4, 5, 5 or more) proteins of the invention are expressed as a single hybrid protein. It is preferred that no non-Neisserial fusion partner (e.g. GST or poly-His) is used.
This offers two advantages. Firstly, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Secondly, commercial manufacture is simplified only one expression and purification need be employed in order to produce two separately-useful proteins.
Thus the invention provides a method for the simultaneous heterologous expression of two or more proteins of the invention, in which said two or more proteins of the invention are fused (i.e. they are translated as a single polypeptide chain).
The method will typically involve the steps of obtaining a first nucleic acid encoding a first protein of the invention; obtaining a second nucleic acid encoding a second protein of the invention; ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Preferably, the constituent proteins in a hybrid protein according to the invention will be from the same strain.
The fused proteins in the hybrid may be joined directly, or may be joined via a linker peptide e.g. via a poly-glycine linker (i.e. G where n=3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence which facilitates cloning. It is evidently preferred not to join a ΔG protein to the C-terminus of a poly-glycine linker.
The fused proteins may lack native leader peptides or may include the leader peptide sequence of the N-terminal fusion partner.
The method is well suited to the expression of proteins orf1, orf4, orf25, orf40, Orf46/46.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.
The 42 hybrids indicated by ‘X’ in the following table of form NH₂-A-B-COOH are preferred:


	B

A	ORF46.1	287	741	919	953	961	983

ORF46.1		X	X	X	X	X	X
287	X		X	X	X	X	X
741	X	X		X	X	X	X
919	X	X	X		X	X	X
953	X	X	X	X		X	X
961	X	X	X	X	X		X
983	X	X	X	X	X	X

Preferred proteins to be expressed as hybrids are thus ORF46.1, 287, 741, 919, 953, 961 and 983. These may be used in their essentially full-length form, or poly-glycine deletions (ΔG) forms may be used (e.g. ΔG-287, ΔGTbp2, ΔG741, ΔG983 etc.), or truncated forms may be used (e.g. Δ1-287, Δ2-287 etc.), or domain-deleted versions may be used (e.g. 287B, 287C, 287BC, ORP46_1-433, ORF46_433-608, ORF46, 961c etc.).
Particularly preferred are: (a) a hybrid protein comprising 919 and 287; (b) a hybrid protein comprising 953 and 287; (c) a hybrid protein comprising 287 and ORF46.1; (d) a hybrid protein comprising ORF1 and ORF46.1; (e) a hybrid protein comprising 919 and ORF46.1; (1) a hybrid protein comprising ORF46.1 and 919; (g) a hybrid protein comprising ORF46.1, 287 and 919; (h) a hybrid protein comprising 919 and 519; and (1) a hybrid protein comprising ORF97 and 225. Further embodiments are shown in FIG. 14.
Where 287 is used, it is preferably at the C-terminal end of a hybrid; if it is to be used at the N-terminus, if is preferred to use a ΔG form of 287 is used (e.g. as the N-terminus of a hybrid with ORF46.1, 919, 953 or 961).
Where 287 is used, this is preferably from strain 2996 or from strain 394/98.
Where 961 is used, this is preferably at the N-terminus. Domain forms of 961 may be used.
Alignments of polymorphic forms of ORF46, 287, 919 and 953 are disclosed in WO00/66741, Any of these polymorphs can be used according to the present invention.

Temperature

In a sixth approach to heterologous expression, proteins of the invention are expressed at a low temperature.
Expressed Neisserial proteins (e.g. 919) may be toxic to E. coli, which can be avoided by expressing the toxic protein at a temperature at which its toxic activity is not manifested.
Thus the present invention provides a method for the heterologous expression of a protein of the invention, in which expression of a protein of the invention is carried out at a temperature at which a toxic activity of the protein is not manifested.
A preferred temperature is around 30° C. This is particularly suited to the expression of 919.

Mutations

As discussed above, expressed Neisserial proteins may be toxic to E. coli. This toxicity can be avoided by mutating the protein to reduce or eliminate the toxic activity. In particular, mutations to reduce or eliminate toxic enzymatic activity can be used preferably using site-directed mutagenesis.
In a seventh approach to heterologous expression, therefore, an expressed protein is mutated to reduce or eliminate toxic activity.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which protein is mutated to reduce or eliminate toxic activity.
The method is preferably used for the expression of protein 907, 919 or 922. A preferred mutation in 907 is at Glu-117 (e.g. Glu→Gly); preferred mutations in 919 are at Glu-255 (e.g. Glu→Gly) and/or Glu-323 (e.g. Glu→Gly); preferred mutations in 922 are at Glu-164 (e.g. Glu→Gly), Ser-213 (e.g. Ser→Gly) and/or Asn-348 (e.g. Asn→Gly).

Alternative Vectors

In a eighth approach to heterologous expression, an alternative vector used to express the protein. This may be to improve expression yields, for instance, or to utilise plasmids that are already approved for GMP use.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which an alternative vector is used. The alternative vector is preferably pSM214, with no fusion partners. Leader peptides may or may not be included.
This approach is particularly useful for protein 953. Expression and localisation of 953 with its native leader peptide expressed from pSM214 is much better than from the pET vector.
pSM214 may also be used with: ΔG287, Δ2-287, Δ3-287, Δ4-287, Orf46.1, 961L, 961, 961(MC58), 961c, 961c-L, 919, 953 and ΔG287-Orf46.1.
Another suitable vector is pET-24b (Novagen; uses kanamycin resistance), again using no fusion partners. pET-24b is preferred for use with: ΔG287K, Δ2-287K, Δ3-287K, Δ4-287K, Orf46.1-K, Orf46A-K, 961-K (MC58), 961a-K, 961b-K, 961c-K, 961c-L-K, 961d-K, ΔG287-919-K, ΔG287-Orf46.1-K and ΔG287-961-K.

Multimeric Form

In a ninth approach to heterologous expression, a protein is expressed or purified such that it adopts a particular multimeric form.
This approach is particularly suited to protein 953. Purification of one particular multimeric form of 953 (the monomeric form) gives a protein with greater bactericidal activity than other forms (the dimeric form).
Proteins 287 and 919 may be purified in dimeric forms.
Protein 961 may be purified in a 180 kDa oligomeric form (e.g. a tetramer).

Lipidation

In a tenth approach to heterologous expression, a protein is expressed as a lipidated protein.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which the protein is expressed as a lipidated protein.
This is particularly useful for the expression of 919, 287, ORF4, 406, 576-1, and ORF25. Polymorphic forms of 919, 287 and ORF4 are disclosed in WO00/66741.
The method will typically involve the use of an appropriate leader peptide without using an N-terminal fusion partner.

C-Terminal Deletions

In an eleventh approach to heterologous expression, the C-terminus of a protein of the invention is mutated. In addition, it is preferred that no fusion partner is used.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's C-terminus region is mutated and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to mutate nucleotides that encode the protein's C-terminus portion. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The first amino acid of the expressed protein will be that of the mature native protein.
The mutation may be a substitution, insertion or, preferably, a deletion.
This method can increase the levels of expression, particularly for proteins 730, ORF29 and ORF46. For protein 730, a C-terminus region of around 65 to around 214 amino acids may be deleted; for ORF46, the C-terminus region of around 175 amino acids may be deleted; for ORF29, the C-terminus may be deleted to leave around 230-370 N-terminal amino acids.

Leader Peptide Mutation

In a twelfth approach to heterologous expression, the leader peptide of the protein is mutated. This is particularly useful for the expression of protein 919.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which the protein's leader peptide is mutated.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; and manipulating said nucleic acid to mutate nucleotides within the leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.

Poly-Glycine Deletion

In a thirteenth approach to heterologous expression, poly-glycine stretches in wild-type sequences are mutated. This enhances protein expression.
The poly-glycine stretch has the sequence (Gly)_n, where n≧4 (e.g. 5, 6, 7, 8, 9 or more). This stretch is mutated to disrupt or remove the (Gly)_n. This may be by deletion (e.g. CGGGGS→CGGGS, COGS, CGS or CS), by substitution (e.g. CGCGGS→CGXGGS, CGCXGS, CGXGXS etc.), and/or by insertion (e.g. CGGGGS→CGGXGGS, CGXGOGS, etc.
This approach is not restricted to Neisserial proteins—it may be used for any protein (particularly bacterial proteins) to enhance heterologous expression. For Neisserial proteins, however, it is particularly suitable for expressing 287, 741, 983 and Thp2. An alignment of polymorphic forms of 287 is disclosed in WO00/66741.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) a poly-glycine stretch within the protein is mutated.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; and manipulating said nucleic acid to mutate nucleotides that encode a poly-glycine stretch within the protein sequence. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Conversely, the opposite approach (i.e. introduction of poly-glycine stretches) can be used to suppress or diminish expression of a given heterologous protein.

Heterologous Host

Whilst expression of the proteins of the invention may take place in the native host (i.e. the organism in which the protein is expressed in nature), the present invention utilises a heterologous host. The heterologous host may be prokaryotic or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonenna typhimurium, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactarnica, Neisseria cinerea, Mycobateria (e.g. M. tuberculosis), yeast etc.

Vectors etc.

As well as the methods described above, the invention provides (a) nucleic acid and vectors useful in these methods (b) host cells containing said vectors (c) proteins expressed or expressable by the methods (d) compositions comprising these proteins, which may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions (e) these compositions for use as medicaments (e.g. as vaccines) or as diagnostic reagents (f) the use of these compositions in the manufacture of (1) a medicament for treating or preventing infection due to Neisserial bacteria (2) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria, and/or (3) a reagent which can raise antibodies against Neisserial bacteria and (g) a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.

Sequences

The invention also provides a protein or a nucleic acid having any of the sequences set out in the following examples. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of ‘sequence identity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).
Furthermore, the invention provides nucleic acid which can hybridise to the nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).
The invention also provides nucleic acid encoding proteins according to the invention.
It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg, for antisense or probing purposes).
Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).
In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a construct used to express orf1 protein using a heterologous leader peptide.

FIG. 2 shows a construct used to express 287 protein using a heterologous leader peptide.

FIG. 3A-FIG. 3E show expression data for ORF1. FIG. 3A shows purification of ORF1.

FIG. 3B shows Western blot analysis. FIG. 3C shows the results of a bactericidal assay with ORF1. FIG. 3D shows FACS analysis. FIG. 3E shows the results of an ELISA assay.

FIG. 4A-FIG. 4E show expression data for protein 961. FIG. 4A shows purification of protein 961. FIG. 4B shows Western blot analysis. FIG. 4C shows the results of a bactericidal assay with protein 961. FIG. 4D shows FACS analysis. FIG. 4E shows the results of an ELISA assay.

FIG. 5 shows domains of protein 287.

FIG. 6 shows deletions within domain A of protein 287.

FIG. 7 shows specific deletions within domain A of protein 287.

FIG. 8 shows domains of protein 564.

FIG. 9 shows the PhoC reporter gene driven by the 919 leader peptide.

FIG. 10A-FIG. 10B show the results obtained using mutants of the 919 leader peptide driving the PhoC reporter. FIG. 10A shows results for control, phoC_wt, 9phoC, 9L1a, 9l1d, 9L1f, and 9S1e. FIG. 10B shows results for control, phoC_wt, 9phoC, 9S1b, 9S1c, and 9Sli.

FIG. 11A-FIG. 11B show insertion mutants of protein 730. FIG. 11A shows 730-C1.

FIG. 11B shows 730-C2.

FIG. 12 shows domains of protein 961.

FIG. 13 shows SDS-PAGE of ΔG proteins. Dots show the main recombinant product.

FIG. 14A-FIG. 14Z show 26 hybrid proteins according to the invention. FIG. 14A shows ΔG287-919. FIG. 14B shows ΔG287-953. FIG. 14C shows ΔG287-961. FIG. 14D shows ΔG287NZ-919, FIG. 14E shows ΔG287NZ-953. FIG. 14F shows ΔG287NZ-961. FIG. 14G shows ΔG983-ORF46.1. FIG. 14H shows ΔG983-741. FIG. 14I shows ΔG983-961. FIG. 14J shows ΔG983-961c. FIG. 14K shows ΔG741-961. FIG. 14I, shows ΔG741-961c. FIG. 14M shows ΔG741-983. FIG. 14N shows ΔG741-ORF46.1. FIG. 14O shows ORF46.1-741, FIG. 14P shows ORF46.1-961. FIG. 14Q shows ORF46.1-961c. FIG. 14R shows 961-ORF46.1. FIG. 14S shows 961-741. FIG. 14T shows 961-983. FIG. 14U shows 961c-ORF46.1. FIG. 14V shows 961c-741. FIG. 14W shows 961c-983. FIG. 14X shows 961cL-ORF46.1. FIG. 14Y shows 961cL-741. FIG. 14Z shows 961cL-983.

MODES FOR CARRYING OUT THE INVENTION

Example 1—919 and its Leader Peptide

Protein 919 from N. meningitidis (serogroup B, strain 2996) has the following sequence:

1	MKKYLFRAAL YGLAAAILAA CQSKSIQTFP QPDTSVINGP DRPVGIPDPA

51	GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV

101	CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR

151	RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT

201	HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA

251	EDPVELFEMH IGQSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL

301	KLGQTSMQGI KAYMRQNPQR LAEVLGQNPS YIFFRELAGS SNDGPVGALG

351	TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG

401	AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P*

The leader peptide is underlined.
The sequences of 919 from other strains can be found in FIGS. 7 and 18 of WO00/667411.
Example 2 of WO99/57280 discloses the expression of protein 919 as a His-fusion in E. coli. The protein is a good surface-exposed immunogen.
Three alternative expression strategies were used for 919:

- 1) 919 without its leader peptide (and without the mature N-terminal cysteine) and without any fusion partner (‘919^untagged’):

1	QSKSIQTFP QPDTSVINGP DRPVGIPDPA GTTVGGGGAV YTVVPHLSLP

50	HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV CAQATQFPVH SFQAKQFFER

100	YETPWQVAGN GSLAGTVTGY YEPVLKGDDR RTAQARFPIY GIPDDFISVP

150	LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT HTADLSRFPI TARTTAIKGR

200	FEGSRELPYH TRNQINGGAL DGKAPILGYA EDPVELFFMH IQGSGRLKTP

250	SGKYIRIGYA DKNEHPYVSI GRYMADKGYL KLGQTSMQGI KAYMRQNPQR

300	LAEVLGQNPS YIFFRELAGS SNDGPVGALG TPLMGEYAGA VDRHYITLGA

350	PLFVATAHPV TRKALNRLIM AQDTGSAIKG AVRVDYFWGY GDEAGELAGK

400	QKTTGYVWQL LPNGMKPEYR P*

- - The leader peptide and cysteine were omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.
- 2) 919 with its own leader peptide but without any fusion partner (‘919L’); and
- 3) 919 with the leader peptide (MKTFFKTLSAAALALILAA from ORF4 (‘919LOrf4’).

1	MKTFFKTLS AAALALILAA CQSKSIQTFP QPDTSVINGP DRPVGIPDPA

50	GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV

100	CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR

150	RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT

200	HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA

250	EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL

300	KLGQTSMQGI KSYMRQNPQR LAEVLGQNTS YIFFRELAGS SNDGPVGALG

350	TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG

400	AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P

- - To make this construct, the entire sequence encoding the ORF4 leader peptide was included in the 5′-primer as a tail (primer 919Lorf4 For). A NheI restriction site was generated by a double nucleotide change in the sequence coding for the ORF4 leader (no amino acid changes), to allow different genes to be fused to the ORF4 leader peptide sequence. A stop codon was included in all the T-end primer sequences.

All three forms of the protein were expressed and could be purified.
The ‘919’ and ‘919LOrf4’ expression products were both lipidated, as shown by the incorporation of [³H]-palmitate label. 919^untaggeddid not incorporate the ³H label and was located intracellularly.
919LOrf4 could be purified more easily than 919L. It was purified and used to immunise mice. The resulting sera gave excellent results in FACS and ELISA tests, and also in the bactericidal assay. The lipoprotein was shown to be localised in the outer membrane.
919^untaggedgave excellent ELISA titres and high serum bactericidal activity. FACS confirmed its cell surface location.

Example 2—919 and Expression Temperature

Growth of E. coli expressing the 919LOrf4 protein at 37° C. resulted in lysis of the bacteria. In order to overcome this problem, the recombinant bacteria were grown at 30° C. Lysis was prevented without preventing expression.

Example 3—Mutation of 907, 919 and 922

It was hypothesised that proteins 907, 919 and 922 are murein hydrolases, and more particularly lytic transglycosylases. Murein hydrolases are located on the outer membrane and participate in the degradation of peptidoglycan.
The purified proteins 919^untagged, 919Lorf4, 919-His (i.e. with a C-terminus His-tag) and 922-His were thus tested for murein hydrolase activity [Ursinus & Holtje (1994) J. Bact. 176:338-343]. Two different assays were used, one determining the degradation of insoluble murein sacculus into soluble muropeptides and the other measuring breakdown of poly(MurNAc-GlcNAc)_n>30glycan strands.
The first assay uses murein sacculi radiolabelled with meso-2,6-diamino-3,4,5-[³H]pimelic acid as substrate. Enzyme (3-10 μg total) was incubated for 45 minutes at 37° C. in a total volume of 100 μl comprising 10 mM Tris-maleate (pH 5.5), 10 mM MgCl₂, 0.2% v/v Triton X-100 and [³H]A₂pm labelled murein sacculi (about 10000 cpm). The assay mixture was placed on ice for 15 minutes with 100 μl of 1% w/v N-acetyl-N,N,N-trimethylammonium for 15 minutes and precipitated material pelleted by centrifugation at 10000 g for 15 minutes. The radioactivity in the supernatant was measured by liquid scintillation counting. E. coli soluble lytic transglycosylase Slt70 was used as a positive control for the assay; the negative control comprised the above assay solution without enzyme.
All proteins except 919-His gave positive results in the first assay.
The second assay monitors the hydrolysis of poly(MurNAc-GlcNAc)glycan strands. Purified strands, poly(MurNAc-GlcNAc)_n>30labelled with N-acetyl-D-1-[³H]glucosamine were incubated with 3 μg of 919L in 10 mM Tris-maleate (pH 5.5), 10 mM MgCl₂and 0.2% v/v Triton X-100 for 30 min at 37° C. The reaction was stopped by boiling for 5 minutes and the pH of the sample adjusted to about 3.5 by addition of 10 μl of 20% v/v phosphoric acid. Substrate and product were separated by reversed phase HPLC on a Nucleosil 300 C₁₈column as described by Harz et. al. [Anal. Biochem. (1990) 190:120-128]. The E. coli lytic transglycosylase Mlt A was used as a positive control in the assay. The negative control was performed in the absence of enzyme.
By this assay, the ability of 919LOrf4 to hydrolyse isolated glycan strands was demonstrated when anhydrodisaccharide subunits were separated from the oligosaccharide by HPLC.
Protein 919Lorf4 was chosen for kinetic analyses. The activity of 919Lorf4 was enhanced 3.7-fold by the addition of 0.2% v/v Triton X-100 in the assay buffer. The presence of Triton X-100 had no effect on the activity of 919. The effect of pH on enzyme activity was determined in Tris-Maleate buffer over a range of 5.0 to 8.0. The optimal pH for the reaction was determined to be 5.5, Over the temperature range 18° C. to 42° C., maximum activity was observed at 37° C. The effect of various ions on murein hydrolase activity was determined by performing the reaction in the presence of a variety of ions at a final concentration of 10 mM, Maximum activity was found with Mg²⁺, which stimulated activity 2.1-fold. Mn²⁺ and Ca²⁺ also stimulated enzyme activity to a similar extent while the addition Ni²⁺ and EDTA had no significant effect. In contrast, both Fe²⁺ and Zn²⁺ significantly inhibited enzyme activity. The structures of the reaction products resulting from the digestion of unlabelled E. coli murein sacculus were analysed by reversed-phase HPLC as described by Glauner [Anal. Biochem. (1988) 172:451-464]. Murein sacculi digested with the muramidase Cellosyl were used to calibrate and standardise the Hypersil ODS column. The major reaction products were 1,6 anhydrodisaccharide tetra and tri peptides, demonstrating the formation of 1,6 anhydronmraminic acid intramolecular bond.
These results demonstrate experimentally that 919 is a murein hydrolase and in particular a member of the lytic transglycosylase family of enzymes. Furthermore the ability of 922-His to hydrolyse murein sacculi suggests this protein is also a lytic transglycosylase.
This activity may help to explain the toxic effects of 919 when expressed in E. coli.
In order to eliminate the enzymatic activity, rational mutagenesis was used, 907, 919 and 922 show fairly low homology to three membrane-bound lipidated murein lytic transglycosylases from Exalt:

- 919 (441aa) is 27.3% identical over 440aa overlap to E. coli MLTA (P46885);
- 922 (369aa) is 38.7% identical over 310aa overlap to E. coli MLTB (P41052); and
- 901-2 (207aa) is 26.8% identical over 149aa overlap to E. coli MLTC (P52066).

907-2 also shares homology with E. coli MLTD (P23931) and Slt70 (P03810), a soluble lytic transglycosylase that is located in the periplasmic space. No significant sequence homology can be detected among 919, 922 and 907-2, and the same is true among the corresponding MLTA, MLTB and MLTC proteins.
Crystal structures are available for Slt70 [1QTEA; 1QTEB; Thunnissen et al. (1995) Biochemistry 34:12729-12737] and for Slt35 [1LTM; 1QUS; 1QUT; van, Asselt et at (1999) Structure Fold Des 7:1167-80] which is a soluble form of the 40 kDa MLTB.
The catalytic residue (a glutamic acid) has been identified for both Slt70 and MLTB.
In the case of Slt70, mutagenesis studies have demonstrated that even a conservative substitution of the catalytic Glu505 with a glutamine (Gln) causes the complete loss of enzymatic activity. Although Slt35 has no obvious sequence similarity to Slt70, their catalytic domains shows a surprising similarity. The corresponding catalytic residue in MLTB is Glu162.
Another residue which is believed to play an important role in the correct folding of the enzymatic cleft, is a well-conserved glycine (Gly) downstream of the glutamic acid. Recently, Terrak et al. [Mol. Microbiol. (1999) 34:350-64] have suggested the presence of another important residue which is an aromatic amino acid located around 70-75 residues downstream of the catalytic glutamic acid.
Sequence alignment of Slt70 with 907-2 and of MLTB with 922 were performed in order to identify the corresponding catalytic residues in the MenB antigens.
The two alignments in the region of the catalytic domain are reported below:

907-2/Slt70:

	90 100 110 ▾120 130 140
907-2.pep	ERRRLLVNIQYESSRAG--LDTQIVLGLIEVESAFRQYAISGV G AR G LMQVMPFWKNYIG
	\|\| \| \| :: :\| : : :::: : \|\|\|: : \| \|\|\| \|\|\|\|:\|\| ::
slty_ecoli	ERFPLAYNDLFKRYTSGKEIPQSYAMAIARQESAWNPKVKSPVGASGLMQIMPGTATHTV
	480 490 500 ▴ 510 520 530
	GLU505

922/MLTB

	150 160 ▾ 170 180 190 200
922. pep	VAQKYGVPAELIVAVIGIETNY G KNT G SFRVADALATLGFDYPRRAGFFQKELVELLKLA
	: \| \|\|\|\| \|:\|\|::\|\|:\|\| :\|: \|: \|: \|\|\|\|\|\|:\|:\|\|\|\|\| :\|: \|\| :\| :\|
mltb_ecoli	AWQVYGVPPEIIVGIIGVETRWGRVMGKTRILDALATLSFNYPRRAEYFSGELETFLLMA
	150 160 ▴ 170 180 190 200
	GLU162

	210 220 230 240 250 260
922.pep	KEEGGDVFAFKGSYAGAMGMPQFMPSS Y RKWAVDYDGDGHRDIWGNVGDVAASVANYMKQ
	::\| \| : :\|\|\|:\|\|\|\|\|: \|\|\|\|\|\|\|:::\|\|\|::\|\|\|\| ::\| \| \|: :\|\|\|\|\|:\|
mltb_ecoli	RDEQDDPLNLKGSFAGAMGYGQFMPSSYKQYAVDFSGDGHINLWDPV-DAIGSVANYFKA
	210 220 230 240 250 260

From these alignments, it results that the corresponding catalytic glutamate in 907-2 is Glu117, whereas in 922 is Glu164. Both antigens also share downstream glycines that could have a structural role in the folding of the enzymatic cleft (in bold), and 922 has a conserved aromatic residue around 70aa downstream (in bold).
In the case of protein 919, no 3D structure is available for its E. coli homologue MLTA, and nothing is known about a possible catalytic residue. Nevertheless, three amino acids in 919 are predicted as catalytic residues by alignment with MLTA:

919/MLTA

	240 250 ▾ 260 270 280 290
919.pep	ALDGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYIRI-GYADKNEHPYVSIGRYMADK
	\|\|: \| \|\|:\|::: :: \|:\| :\|\|\|\| : :\|: : : :\|\| \|\| \| \| \|\|\|: : \|:
mlta_ecoli.p	ALSDKY-ILAYSNSLMDNFIMDVQGSGYIDFGDGSPLNFFSYAGKNGHAYRSIGKVLIDR
	170 180 190 200 210

	300 310 320 ▾ 330 340 ⋄350 ⋄
919.pep	GYLKLGQTSMQGIKSYMRQNPQ-RLAEVLGQNPSYIFFRELAGSSNDGPV-GALGTPLMG
	\| :\| : \|\|\|:\|: : : : : :: \|:\| \|\|\|\|::\|\|: : : \|\| \|\| ::\|\|:\|
mlta_ecoli.p	GEVKKEDMSMQAIRHWGETHSEAEVRELLEQNPSFVFFKPQSFA----PVKGASAVPLVG
	220 230 240 250 260 270

	360 ▾ ∘ 380 390 400 ⋄⋄410
919. pep	EYAGAVDRHYITLGAPLFVATAHPVTRKALN-----RLIMAQDTGSAIKGAVRVDYFWGY
	: : \| \|\| \| \|: \|:: : : :\| \|\|::\| \|:\|:\|\|\|\| : \| : \|
mlta_ecoli.p	RASVASDRSIIPPGTTLLAEVPLLDNNGKFNGQYELRLMVALDVGGAIKGQ-HFDIYQGI
	280 290 300 310 320 330

	420 ∘
919.pep	GDEAGELAGKQKTTGYVWQLLP
	\| \|\|\|: \|\| : \| \|\| \|
mlta_ecoli.p	GPEAGHRAGWYNHYGRVWVLKT
	340 350

The three possible catalytic residues are shown by the symbol ▾:

1) Glu255 (Asp in MLTA), followed by three conserved glycines (Gly263, Gly265 and Gly272) and three conserved aromatic residues located approximately 7577 residues downstream. These downstream residues are shown by □.
2) Glu323 (conserved in MLTA), followed by 2 conserved glycines Gly347 and Gly355) and two conserved aromatic residues located 84-85 residues downstream (Tyr406 or Phe407). These downstream residues are shown by 0.
3) Asp362 (instead of the expected Glu), followed by one glycine (Gly 369) and a conserved aromatic residue (Trp428). These downstream residues are shown by ∘.

Alignments of polymorphic forms of 919 are disclosed in WO00/66741.
Based on the prediction of catalytic residues, three mutants of the 919 and one mutant of 907, containing each a single Amino acid substitution, have been generated. The glutamic acids in position 255 and 323 and the aspartic acids in position 362 of the 919 protein and the glutamic acid in position 117 of the 907 protein, were replaced with glycine residues using PCR-based SDM. To do this, internal primers containing a codon change from Glu or Asp to Gly were designed:


		Codon
Primers	Sequences	change

919-E255 for	CGAAGACCCCGTCGgtCTTTTTTTTATG	GAA → Ggt
919-E255 rev	GTGCATAAAAAAAAGacCGACGGGGTCT

919-E323 for	AACGCCTCGCCGgtGTTTTGGGTCA	GAA → Ggt
919-E323 rev	TTTGACCCAAAACacCGGCGAGGCG

919-D362 for	TGCCGGCGCAGTCGgtCGGCACTACA	GAC → Ggt
919-D362 rev	TAATGTAGTGCCGacCGACTGCGCCG

907-E117 for	TGATTGAGGTGGgtAGCGCGTTCCG	GAA → Ggt
907-E117 rev	GGCGGAACGCGCTacCCACCTCAAT

Underlined nucleotides code for glycine; the mutated nucleotides are in lower case.

To generate the 919-E255, 919-E323 and 919-E362 mutants, PCR was performed using 20 ng of the pET 919-LOrf4 DNA as template, and the following primer pairs:

- 1) Orf4L for/919-E255 rev
- 2) 919-E255 for/919L rev
- 3) Orf4L far 919-E323 rev
- 4) 919-E323 for/919L rev
- 5) Orf4L for/919-D362 rev
- 6) 919-D362 for/919L rev

The second round of PCR was performed using the product of PCR 1-2, 3-4 or 5-6 as template, and as forward and reverse primers the “Orf4L for” and “919L rev” respectively.
For the mutant 907-E117, PCR have been performed using 200 ng of chromosomal DNA of the 2996 strain as template and the following primer pairs:

- 7) 907L for/907-E117 rev
- 8) 907-E117 for/907L rev

The second round of PCR was performed using the products of PCR 7 and 8 as templates and the oligos “907L for” and “907L rev” as primers.
The PCR fragments containing each mutation were processed following the standard procedure, digested with NdeI and XhoI restriction enzymes and cloned into pET-21b+ vector. The presence of each mutation was confirmed by sequence analysis.
Mutation of Glu117 to Gly in 907 is carried out similarly, as is mutation of residues Glu164, Ser213 and Asn348 in 922.
The E255G mutant of 919 shows a 50% reduction in activity; the E3230 mutant shows a 70% reduction in activity; the E362G mutant shows no reduction in activity.

Example 4—Multimeric Form

287-GST, 919^untaggedand 953-His were subjected to gel filtration for analysis of quaternary structure or preparative purposes. The molecular weight of the native proteins was estimated using either FPLC Superose 12 (H/R 10/30) or Superdex 75 gel filtration columns (Pharmacia). The buffers used for chromatography for 287, 919 and 953 were 50 mM Tris-HO (pH 8.0), 20 mM Bicine (pH 8.5) and 50 mM Bicine (pH 8.0), respectively.
Additionally each buffer contained 150-200 mM NaCl and 10% v/v glycerol. Proteins were dialysed against the appropriate buffer and applied in a volume of 204.1. Gel filtration was performed with a flow rate of 0.5-2.0 ml/min and the eluate monitored at 280 nm. Fractions were collected and analysed by SDS-PAGE. Blue dextran 2000 and the molecular weight standards ribonuclease A, chymotrypsin A ovalbumin, albumin (Pharmacia) were used to calibrate the column. The molecular weight of the sample was estimated from a calibration curve of K_avlog M_rof the standards. Before gel filtration, 287-GST was digested with thrombin to cleave the GST moiety.
The estimated molecular weights for 287, 919 and 953-His were 73 kDa, 47 kDa and 43 kDa respectively. These results suggest 919 is monomeric while both 287 and 953 are principally dimeric in their nature. In the case of 953-His, two peaks were observed during gel filtration. The major peak (80%) represented a dimeric conformation of 953 while the minor peak (20%) had the expected size of a monomer. The monomeric form of 953 was found to have greater bactericidal activity than the dimer.

Example 5—pSM214 and pET-24b Vectors

953 protein with its native leader peptide and no fusion partners was expressed from the pET vector and also from pSM214 [Velati Bellini et al. (1991) J. Biotechnol. 18, 177-192].
The 953 sequence was cloned as a full-length gene into pSM214 using the E. coli MM1294-1 strain as a host. To do this, the entire DNA sequence of the 953 gene (from ATG to the STOP codon) was amplified by PCR using the following primers:
953L for/2

CCGGAATTCTTATGAAAAAAATCATCTTCGCCGC Eco RI

953L rev/2

GCCCAAGCTTTTATTGTTTGGCTGCCTCGATT Hind III

which contain EcoRI and HindIII restriction sites, respectively. The amplified fragment was digested with EcoRI and HindIII and ligated with the pSM214 vector digested with the same two enzymes. The ligated plasmid was transformed into E. coli MM294-1 cells (by incubation in ice for 65 minutes at 37° C.) and bacterial cells plated on LB agar containing 20 μg/ml of chloramphenicol.
Recombinant colonies were grown over-night at 37° C. in 4 ml of LB broth containing 20 μg/ml of chloramphenicol; bacterial cells were centrifuged and plasmid DNA extracted as and analysed by restriction with EcoRI and HindIII. To analyse the ability of the recombinant colonies to express the protein, they were inoculated in LB broth containing 20 μg/ml of chloramphenicol and let to grown for 16 hours at 37° C. Bacterial cells were centrifuged and resuspended in PBS. Expression of the protein was analysed by SDS-PAGE and Coomassie Blue staining.
Expression levels were unexpectedly high from the pSM214 plasmid.
Oligos used to clone sequences into pSM-214 vectors were as follows:

ΔG287	Fwd	CCGGAATTCTTATG-TCGCCCGATGTTAAATCGGCGGA	EcoRI
(pSM-214)	Rev	GCCCAAGCTT- TCA ATCCTGCTTTTTTGCCG	HindIII

Δ2
287	Fwd	CCGGAATTCTTATG-AGCCAAGATATGGCGGCAGT	EcoRI
(pSM-214)	Rev	GCCCAAGCTT- TCA ATCCTGCTCTTTTTTGCCG	HindIII

Δ3
287	Fwd	CCGGAATTCTTATG-TCCGCCGAATCCGCAAATCA	EcoRI
(pSM-214	Rev	GCCCAAGCTT- TCA ATCCTGCTCTTTTTTGCCG	HindIII

Δ4
287	Fwd	CCGGAATTCTTATG-GGAGGGTTGATTTGGCTAATG	EcoRI
(pSM-214)	Rev	GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG	HindIII

Orf46.1	Fwd	CCGGAATTCTTATG-TCAGATTTGGCAAACGATTCTT	EcoRI
(pSM-214)	Rev	GCCCAAGCTT-TTACGTATCATATTTCACGTGCTTC	HindIII

ΔG287-Orf46.1	Fwd	CCGGAATTCTTATG-TCGCCCGATGTTAAATCGGCGGA	EcoRI
(pSM-214)	Rev	GCCCAAGCTT- TTA CGTATCATATTTCACGTGCTTC	HindIII

919	Fwd	CCGGAATTCTTATG-CAAAGCAAGAGCATCCAAACCT	EcoRI
(pSM-214)	Rev	GCCCAAGCTT-TTACGGGCGGTATTCGGGCT	HindIII

961L	Fwd	CCGGAATTCATATG-AAACACTTTCCATCC	EcoRI
(pSM-214)	Rev	GCCCAAGCTT- TTA CCACTCGTAATTGAC	HindIII

961	Fwd	CCGGAATTCATATG-GCCACAAGCGACGAC	EcoRI
(pSM-214)	Rev	GCCCAAGCTT- TTA CCACTCGTAATTGAC	HindIII

961c L	Fwd	CCGGAATTCTTATG-AAACACATTTCCATCC	EcoRI
pSM-214	Rev	GCCCAAGCTT- TCA ACCCACGTTGTAAGGTTG	HindIII

961c	Fwd	CCGGAATTCTTATG-GCCACAAACGACGACG	EcoRI
pSM-214	Rev	GCCCAAGCTT- TCA ACCCACGTTGTAAGGTTG	HindIII

953	Fwd	CCGGAATTCTTATG-GCCACCTACAAAGTGGACGA	EcoRI
(pSM-214)	Rev	GCCCAAGCTT-TTATTGTTTGGCTGCCTCGATT	HindIII

These sequences were manipulated, cloned and expressed as described for 953L.
For the pET-24 vector, sequences were cloned and the proteins expressed in pET-24 as described below for pET21. pET2 has the same sequence as pET-21, but with the kanamycin resistance cassette instead of ampicillin cassette.
Oligonucleotides used to clone sequences into pET-24b vector were:

ΔG 287 K	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC §	NheI
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC *	XhoI

Δ2 287 K	Fwd	CGCGGATCCGCTAGC-CAAGATATGGCGGCAGT§	NheI

Δ3 287 K	Fwd	CGCGGATCCGCTAGC-GCCGAATCCGCAAATCA §	NheI

Δ4 287 K	Fwd	CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG§	NheI

Orf46.1 K	Fwd	GGGAATTCCATATG-GGCATTTCCCGCAAAATATC	NdeI
	Rev	CCCGCTCGAG-TTACGTATCATATTTCACGTGC	XhoI

Orf46A K	Fwd	GGGAATTCCATATG-GGCATTTCCCGCAAAATATC	NdeI
	Rev	CCCGCTCGAG-TTATTCTATGCCTTGTGCGGCAT	XhoI

961 K	Fwd	CGCGGATCCCATATG-GCCACAAGCGACGACGA	NdeI
(MC58)	Rev	CCCGCTCGAG- TTA CCACTCGTAATTGAC	XhoI

961a K	Fwd	CGCGGATCCCATATG-GCCACAAACGACG	NdeI
	Rev	CCCGCTCGAG- TCA TTTAGCAATATTATCTTTGTTC	XhoI

961b K	Fwd	CGCGGATCCCATATG-AAAGCAAACAGTGCCGAC	NdeI
	Rev	CCCGCTCGAG- TTA CCACTCGTAATTGAC	XhoI

961c K	Fwd	CGCGGATCCCATATG-GCCACAAACGACG	NdeI
	Rev	CCCGCTCGAG-TTAACCCACGTTGTAAGGT	XhoI

961cL K	Fwd	CGCGGATCCCATATG-ATGAAACACTTTCCATCC	NdeI
	Rev	CCCGCTCGAG- TTA ACCCACGTTGTAAGGT	XhoI

961d K	Fwd	CGCGGATCCCATATG-GCCACAAACGACG	NdeI
	Rev	CCCGCTCGAG-TCAGTCTGACACTGTTTTATCC	XhoI

ΔG 287-	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
919 K	Rev	CCCGCTCGAG-TTACGGGCGGTATTCGG	XhoI

ΔG 287-	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
Orf46.1 K	Rev	CCCGCTCGAG-TTACGTATCATATTTCACGTGC	XhoI

ΔG 287-	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
961 K	Rev	CCCGCTCGAG-TTACCACTCGTAATTGAC	XhoI

*This primer was used as a Reverse primer for all the 287 forms.
§Forward primers used in combination with the ΔG278 K reverse primer.

Example 6—ORF1 and its Leader Peptide

ORF1 from N. meningitidis (serogroup B, strain MC58) is predicted to be an outer membrane or secreted protein. It has the following sequence:

1	MKTTDKRTTE THRKAPKTGR IRFSPAYLAI CLSFGILPQA WAGHTYFGIN

51	YQYYRDFAEN KGRFAVGAKD IEVYNKKGEL VGKSMTKAPM IDFSVVSRNG

101	VAALVGDQYI VSVAHNGGYN NVDFGAEGRN PDQHRFTYKI VKRNNYKAGT

151	KGHPYGGDYH MPRLHKFVTD AEPVEMTSYM DGRKYIDQNN YPDRVRIGAG

201	RQYWRSDEDE PNNRESSYHI ASAYSWLVGG NTFAQNGSGG GTVNLGSEKI

251	KHSPYGFLPT GGSFGDSGSP MFIYDAQKQK WLINGVLQTG NPYIGKSNGF

301	QLVRKDWFYD EIFAGDTHSV FYEPRQNGKY SFNDDNNGTG KINAKHEHNS

351	LPNRLKTRTV QLFNVSLSET AREPVYHAAG GVNSYRPRLN NGENISFIDE

401	GKGELILTSN INQGAGGLYF QGDFTVSPEN NETWQGAGVH ISEDSTVTWK

451	VNGVANDRLS KIGKGTLHVQ AKGENQGSIS VGDGTVILDQ QADDKGKKQA

501	FSEIGLVSGR GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH SLSPHRIQNT

551	DEGAMIVNHN QDKESTVTIT GNKDIATTGN NNSLDSKKEI AYNGWFGEKD

601	TTKTNGRLNL VYQPAAEDRT LLLSGGTNLN GNITQTNGKL FFSGRPTPHA

651	YNELNDHWSQ KEGIPRGEIV WDNDWINRTF KAENFQIKGG QAVVSRNVAK

701	VKGDWHLSNH AQAVFGVAPH QSHTICTRSD WTGLTNCVEK TITDDKVIAS

751	LTKTDISGNV DLADHAHLNL TGLATLNGNL SANGDTRYTV SHNANQNGNL

801	SLVGNAQATF NQATLNGNTS ASGNASFNLS DHAVQNGSLT LSGNAKANVS

851	HSALNGNVSL ADKAVFHFES SRFTGQISGG KDTALHLKDS EWTLPSGTEL

901	GNLNLDNATI TLNSAYRHDA AGAQTGSATD APRRRSRRSR RSLLSVTPPT

951	SVESRFNTLT VNGKLNGQGT FRFMSELFGY RSDKLKLAES SEGTYTLAVN

1001	NTGNEPASLE QLTVVEGKDN KPLSENLNFT LQNEHVDAGA WRYQLIRKDG

1051	EFRLHNPVKE QELSDKLGKA EAKKQAEKDN AQSLDALIAA GRDAVEKTES

1101	VAEPARQAGG ENVGIMQAEE EKKRVQADKD TALAKQREAE TRPATTAFPR

1151	ARRARRDLPQ LQPQPQPQPQ RDLISRYANS GLSEFSATLN SVFAVQDELD

1201	RVPAEDRRNA VWTSGIRDTK HYRSQDFRAY RQQTDLRQIG MQKNLGSGRV

1251	GILFSHNRTE NTFDDGIGNS ARLAHGAVFG QYGIDRFYIG ISAGAGFSSG

1301	SLSDGIGGKI RRRVLHYGIQ ARYRAGFGGF GIEPHIGATR YFVQKADYRY

1351	ENVNIATPGL AFNRYRAGIK ADYSFKPAQH ISITPYLSLS YTDAASGKVR

1401	TRVNTAVLAQ DFGKTRSAEW GVNAEIKGFT LSLHAAAAKG PQLEAQHSAG

1451	IKLGYRW*

The leader peptide is underlined.
A polymorphic form of ORF1 is disclosed in WO99/55873.
Three expression strategies have been used for ORF1:

- 1) ORF1 using a His tag, following WO99/24578 (ORF1-His);
- 2) ORF1 with its own leader peptide but without any fusion partner (‘ORF1L’); and
- 3) ORF1 with the leader peptide (MKKTAIAIAVALAGFATVAQA) from E. coli OmpA (‘Orf1LOmpA’):

MKKTAIAIAVALAGFATVAQAASAGHTYFGINYQYYRDFAENKGKFAVGA

KDIEVYNKKGELVGKSMTKAPMIDFSVVSRNGVAALVGDQYIVSVAHNGG

YNNVDFGAEGRNPDQHRFTYKIVKRNNYKAGTKGHPYGGDYHMPRLHKFV

TDAEPVEMTSYMDGRKYIDQNNYPDRVRIGAGRQYWRSDEDEPNNRESSY

HIASAYSWLVGGNTFAQNGSGGGTVNLGSEKIKHSPYGFLPTGGSFGDSG

SPMFIYDAQKQKWLINGVLQTGNPYIGKSNGFQLVRKDWFYDEIFAGDTH

SVFYEPRQNGKYSFNDDNNGTGKINAKHEHNSLPNRLKTRTVQLFNVSLS

ETAREPVYHAAGGVNSYRPRLNNGENISFIDEGKGELILTSNINQGAGGL

YFQGDFTVSPENNETWQGAGVHISEDSTVTWKVNGVANDRLSKIGKGTLH

VQAKGENQGSISVGDGTVILDQQADDKGKKQAFSEIGLVSGRGTVQLNAD

NQFNPDKLYFGFRGGRLDLNGHSLSFHRIQNTDEGAMIVNHNQDKESTVT

ITGNKDIATTGNNNSLDSKKEIAYNGWFGEKDTTKTNGRLNLVYQPAAED

RTLLLSGGTNLNGNITQTNGKLFFSGRPTPHAYNHLNDHWSQKEGIPRGE

IVWDNDWINRTPKAENFQIKGGQAVVSRNVAKVKGDWHLSNHAQAVFGVA

PHQSHTICTRSDWTGLTNCVEKTITDDKVIASLTKTDISGNVDLADHAHL

NLTGLATLNGNLSANGDTRYTVSHNATQNGNLSLVGNAQATFNQATLNGN

TSQSGNASFNLSDHAVQNGSLTLSGNAKANVSHSALNGNVSLADKAVFHF

ESSRFTGQISGGKDTALHLKDSEWTLPSGTELGNLNLDNATITLNSAYRH

DAAGAQTGSATDAPRRRSRRSRRSLLSVTPPTSVESRFNTLTVNGKLNGQ

GTFRFMSELFGYRSDKLKLAESSEGTYTLAVNNTGNEPASLEQLTVVEGK

DNKPLSENLNFTLQNEHVDAGAWRYQLIRKDGEFRLHNPVKEQELSDKLG

KAEAKKQAEKDNAQSLDALIAAGRDAVEKTESVAEPARQAGGENVGIMQA

EEEKKRVQADKDTALAKQREAETRPATTAFPRARRARRDLPQLQPQPQPQ

PQRDLISRYANSGLSEFSATLNSVFAVQDELDRVFAEDRRNAVWTSGIRD

TKHYRSQDFRAYRQQTDLRQIGMQKNLGSGRVGILFSHNRTENTFDDGIG

NSARLAHGAVFGQYGIDRFYIGISAGAGFSSGSLSDGIGGKIRRRVLHYG

IQARYRAGFGGFGIEPHIGATRYFVQKADYRYENVNIATPGLAFNRYRAG

IKADYSFKPAQHISITPYLSLSYTDAASGKVRTRVNTAVLAQDFGKTRSA

EWGVNAEIKGFTLSLHAAAAKGPQLEAQHSAGIKLGYRW*

- - To make this construct, the clone pET911LOmpA (see below) was digested with the NheI and XhoI restriction enzymes and the fragment corresponding to the vector carrying the OmpA leader sequence was purified (pETLOmpA). The ORF1 gene coding for the mature protein was amplified using the oligonucleotides ORF1-For and ORF1-Rev (including the NheI and XhoI restriction sites, respectively), digested with NheI and XhoI and ligated to the purified pETOmpA fragment (see FIG. 1). An additional AS dipeptide was introduced the NheI site.

All three forms of the protein were expressed. The His-tagged protein could be purified and was confirmed as surface exposed, and possibly secreted (see FIG. 3). The protein was used to immunise mice, and the resulting sera gave excellent results in the bactericidal assay.
ORF1LOmpA was purified as total membranes, and was localised in both the inner and outer membranes. Unexpectedly, sera raised against. ORF1LOmpA show even better ELISA and anti-bactericidal properties than those raised against the His-tagged protein.
ORF1L was purified as outer membranes, where it is localised.

Example 7—Protein 911 and its Leader Peptide

Protein 911 from N. meningitidis (serogroup B, strain MC58) has the following sequence:

1	MKKNILEFWV GLFVLIGAAA VAFLAFRVAG GAAFGGSDKT

	YAVYADFGDI

51	GGLKVNAPVK SAGVLVGRVG AIGLDPKSYQ ARVRLDLDGK

	YQFSSDVSAQ

101	ILTSGLLGEQ YIGLQQGGDT ENLAAGDTIS VTSSAMVLEN

	LIGKFMTSFA

151	EKAMDGGNAE KAAE*

The leader peptide is underlined.
Three expression strategies have been used for 911:

- 1) 911 with its own leader peptide but without any fusion partner (‘911L’);
- 2) 911 with the leader peptide from Emil OmpA (‘911LOmpA’).
  - To make this construct, the entire sequence encoding the OmpA leader peptide was included in the 5′-primer as a tail (primer 911LOmpA Forward). A NheI restriction site was inserted between the sequence coding for the OmpA leader peptide and the 911 gene encoding the predicted mature protein (insertion of one amino acid, a serine), to allow the use of this construct to clone different genes downstream the OmpA leader peptide sequence.
- 3) 911 with the leader peptide (MKYLLPTAAAGLLLAAQPAMA) from Erwinia carotovora PelB (‘911LpelB’).
  - To make this construct, the 5′-end PCR primer was designed downstream from the leader sequence and included the NcoI restriction site in order to have the 911 fused directly to the PelB leader sequence; the 3′-end primer included the STOP codon. The expression vector used was pET22b+(Novagen), which carries the coding sequence for the PelB leader peptide. The NcoI site introduces an additional methionine after the PelB sequence.

All three forms of the protein were expressed. ELISA titres were highest using 911L, with 919LOmpA also giving good results.

Example 8—ORF46

The complete ORF46 protein from N. meningitidis (serogroup B, strain 2996) has the following sequence:

1	LGISRKISLI LSILAVCLPM HAHASDLAND SFIRQVLDRQ

	HFEPDKYHL

51	FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY

	IVRFSDHGHE

101	VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD

	GYDGPQGGGY

151	PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH

	NAGSMLTQGV

201	GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE

	IVGAGDAVQG

251	ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA

	AIRDWAVQNP

301	NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK

	RSQGAIALP

351	KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI

	TSSTVPPSNG

401	KNVKLADQRH PRTGVPFDGK GFPNFEKHVK YDTKLDIQEL

	SGGGIPKAKP

451	VSDAKPRWEV DRKLNKLTTR EQVEKNVQEI RNGNKNSNFS

	QHAQLEREIN

501	KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN

	PVVEYVEING

551	KAYIVRGNNR VFAAEYLGRI HELKFKKVDF PVPNTSWKNP

	TDVLNESGNV

601	KRPRYRSK*

The leader peptide is underlined.
The sequences of ORF46 from other strains can be found in WO00/66741.
Three expression strategies have been used for ORF46:

- 1) ORF46 with its own leader peptide but without any fusion partner (‘ORF46-2L’);
- 2) ORF46 without its leader peptide and without any fusion partner (‘ORF46-2’), with the leader peptide omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence:

1	SDLANDSFIR QVLDRQHFEP DGKYHLFGSR GELAERSGHI

	GLGKIQSHQL

51	GNLMIQQAAI KGNIGYIVRF SDHGHEVHSP FDNHASHSDS

	DEAGSPVDGF

101	SLYRIHWDGY EHHPADGYDG PQGGGYPAPK GARDIYSYDI

	KGVAQNIRLN

151	LTDNRSTGQR LADRFHNAGS MLTQGVGDGF KRATRYSPEL

	DRSGNAAEAF

201	NGTADIVKNI IGAAGEIVGA GDAVQGISEG SNIAVMHGLG

	LLSTENKMAR

251	INDLADMAQL KDYAAAAIRD WAVQNPNAAQ GIEAVSNIFM

	AAIPIKGIGA

301	VRGKYGLGGI TAHPIKRSQM GAIALPKGKS AVSDNFADAA

	YAKYPSPYHS

351	RNIRSNLEQR YGKENITSST VPPSNGKNVK LADQRHPKTG

	VPFDGKGFPN

401	FEKHVKYDTK LDIQELSGGG IPKAKPVSDA KPRWEVDRKL

	NKLTTREQVE

451	KNVQEIRNGN KNSNFSQHAQ LEREINKLKS ADEINFADGM

	GKFTDSMNDK

501	AFSRLVKSVK ENGFTNPVVE YVEINGKAYI VRGNNRVFAA

	EYLGRIHELK

551	FKKVDFPVPN TSWKNPTDVL NESGNVKRPR YRSK*

- 3) ORF46 as a truncated protein, consisting of the first 433 amino acids (‘ORF46.1L’), constructed by designing PCR primers to amplify a partial sequence corresponding to as 1-433.
  - A STOP codon was included in the 3′-end primer sequences.

ORF46-2L is expressed at a very low level to E. coli. Removal of its leader peptide (ORF46-2) does not solve this problem. The truncated ORF46.1L form (first 423 amino acids, which are well conserved between serogroups and species), however, is well-expressed and gives excellent results in ELISA test and in the bactericidal assay.
ORF46.1 has also been used as the basis of hybrid proteins. It has been fused with 287, 919, and ORF1. The hybrid proteins were generally insoluble, but gave some good ELISA and bactericidal results (against the homologous 2996 strain):


Protein	ELISA	Bactericidal Ab

Orf1-Orf46.1-His	850	256
919-Or146.1-His	12900	512
919-287-Orf46-His	n.d.	n.d.
Orf46.1-287His	150	8192
Orf46.1-919His	2800	2048
Orf46.1-287-919His	3200	16384

For comparison, ‘triple’ hybrids of ORF46.1, 287 (either as a GST fusion, or in ΔG287 form) and 919 were constructed and tested against various strains (including the homologous 2996 strain) versus a simple mixture of the three antigens. FCA was used as adjuvant


2996	BZ232	MC58	NGH38	F6124	BZ133

Mixture	8192	256	512	1024	>2048	>2048
ORF46.1-287-	16384	256	4096	8192	8192	8192
919his
ΔG287-919-	8192	64	4096	8192	8192	16384
ORF46.1his
ΔG287-	4096	128	256	8192	512	1024
ORF46.1-
919his

Again, the hybrids show equivalent or superior immunological activity.
Hybrids of two proteins (strain 2996) were compared to the individual proteins against various heterologous strains:


1000	MC58	F6124 (MenA)

ORF46.1-His	<4	4096	<4
ORF1-His	8	256	128
ORF1-ORF46.1-His	1024	512	1024

Again, the hybrid shows equivalent or superior immunological activity.

Example 9—Protein 961

The complete 961 protein from N. meningitidis (serogroup B, strain MC58) has the following sequence:

1	MSMKHFPAKV LTTAILATFC SGALAATSDD DVKKAATVAI

	VAAYNNGQEI

51	NGFKAGETIY DIGEDGTITQ KDATAADVEA DDFKGLGLKK

	VVTNLTKTVN


103	ENKQNVDAKV KAAESEIEKL TTKLADTDAA LADTDAALDE

	TTNALNKLGE

151	NITTFAEETK TNIVKIDEKL SAVADTVDKH AEAFNDIADS

	LDETNTKADE

201	AVKTANEAKQ TAEETKQNVD AKVKAAETAA GKAEAAAGTA

	NTAADKAEAV

251	AAKVTDIKAD LATNKADIAK NSARIDSLDK NVANLRKETR

	QGLAEQAALS

301	GLFQPYNVGR FNVTAAVGGY KSESAVAIGT GFRFTENFAA

	KAGVAVGTSS

351	GSSAAYHVGV NYEW*

The leader peptide is underlined.
Three approaches to 961 expression were used:

- 1) 961 using a GST fusion, following WO99/57280 (‘GST961’);
- 2) 961 with its own leader peptide but without any fusion partner (‘961L’); and
- 3) 961 without its leader peptide and without any fusion partner (‘961^untagged’), leader peptide omitted by designing the 5′-end PCR primer downstream from the predicted leader sequence.

All three forms of the protein were expressed. The GST-fusion protein could be purified and antibodies against it confirmed that 961 is surface exposed (FIG. 4). The protein was used to immunise mice, and the resulting sera gave excellent results in the bactericidal assay. 961L could also be purified and gave very high ELISA titres.
Protein 961 appears to be phase variable. Furthermore, it is not found in all strains of N. meningitidis.

Example 10—Protein 287

Protein 287 from N. meningitidis (serogroup B, strain 2996) has the following sequence;

1	MFERSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP

	VVAEKETEVK

51	EDAPQAGSQG QGAPSTQGSQ DMAAVSAENT GNGGAATTDK

	PKNEDEGPQN

101	DMPQNSAESA NQTGNNQPAD SSDSAPASNP APANGGSNFG

	RVDLANGVLI

151	DGPSQNITLT HCKGDSCNGD NLLDEEAPSK SEFENLNESE

	RIEKYKKDGK

201	SDKFTNLVAT AVQANGTNKY VIIYKDKSAS SSSARFRRSA

	RSRRSLPAEM

251	PLIPVNQADT LIVDGEAVSL TGHSGNIFAP EGNYRYLTYG

	AEKLPGGSYA

301	LRVQGEPAKG EMLAGTAVYN GEVLHFHTEN GRPYPTRGRF

	AAKVDFGSKS

351	VDGIIDSGDD LHMGTQKFKA AIDGNGFKGT WTENGGGDVS

	GRFYGPAGEE

401	VAGKYSYRPT DAEKGGFGVF AGKKEQD*

The leader peptide is shown underlined.
The sequences of 287 from other strains can be found in FIGS. 5 and 15 of WO00/66741.
Example 9 of WO99/57280 discloses the expression of 287 as a GST-fusion in E. coli.
A number of further approaches to expressing 287 in E. coli have been used, including:

- 1) 287 as a His-tagged fusion (287-His′);
- 2) 287 with its own leader peptide but without any fusion partner (‘287L’);
- 3) 287 with the ORF4 leader peptide and without any fusion partner (‘287LOrf4’); and
- 4) 287 without its leader peptide and without any fusion partner (‘287^untagged’):

1	CGGGGGGSPD VKSADTLSKP AAPVVAEKET EVKEDAPQAG

	SQGQGAPSTQ

51	GSQDMAAVSA ENTGNGGAAT TDKPKNEDEG PQNDMPQNSA

	ESANQTGNNQ

101	PADSSDSAPA SNPAPANGGS NFGRVDLANG VLIDGPSQNI

	TLTHCKGDSC

151	NGDNLLDEEA PSKSEFENLN ESERIEKYKK DGKSDKFTNL

	VATAVQANGT


20	NKYVIIYKDK SASSSSARFR RSARSRRSLP AEMPLIPVNQ

	ADTLIVDGEA

251	VSLTGHSGNI PAPEGNYRYL TYGAEKLPGG SYALRVQGEP

	AKGEMLAGTA

301	VYNGEVLHFH TENGRPYPTR GRFAAKVDFG SKSVDGIIDS

	GDDLHMGTQK

351	FKAAIDGNGF KGTWTENGGG DVSGRFYGPA GEEVAGKYSY

	RPTDAEKGGF

401	GVFAGKKEQD *

All these proteins could be expressed and purified.
‘287L’ and ‘287 LOrf4’ were confirmed as lipoproteins.
As shown in FIG. 2, ‘287LOrf4’ was constructed by digesting 919LOrf4 with NheI and XhoI. The entire ORF4 leader peptide was restored by the addition of a DNA sequence coding for the missing amino acids, as a tail, in the 5′-end primer (287LOrf4 for), fused to 287 coding sequence. The 287 gene coding for the mature protein was amplified using the oligonucleotides 287LOrf4 For and Rev (including the NheI and XhoI sites, respectively), digested with NheI and XhoI and ligated to the purified pETOrf4 fragment.

Example 11—Further Non-Fusion Proteins with/without Native Leader Peptides

A similar approach was adopted for E. coli expression of further proteins from WO99/24578, WO99/36544 and WO99/57280.
The following were expressed without a fusion partner: 008, 105, 117-1, 121-1, 122-1, 128-1, 148, 216, 243, 308, 593, 652, 726, 982, and Orf143-1. Protein 117-1 was confirmed as surface-exposed by FACS and gave high ELISA titres.
The following were expressed with the native leader peptide but without a fusion partner: 111, 149, 206, 225-1, 235, 247-1, 274, 283, 286, 292, 401, 406, 502-1, 503, 519-1, 525-1, 552, 556, 557, 570, 576-1, 580, 583, 664, 759, 907, 913, 920-1, 926, 936-1, 953, 961, 983, 989, Orf4, Orf7-1, Orf9-1, Orf23, Orf25, Orf37, Orf38, Orf40, Orf40.1, Orf40.2, Orf72-1, Orf76-1, Orf85-2, OrF91, Orf97-1, Orf119, Orf143.1. These proteins are given the suffix ‘L’.
His-tagged protein 760 was expressed with and without its leader peptide. The deletion of the signal peptide greatly increased expression levels. The protein could be purified most easily using 2M urea for solubilisation.
His-tagged protein 264 was well-expressed using its own signal peptide, and the 30 kDa protein gave positive Western blot results.
All proteins were successfully expressed.
The localisation of 593, 121-1, 128-1, 593, 726, and 982 in the cytoplasm was confirmed.
The localisation of 920-1L, 953L, ORF9-1L, ORF85-2L, ORF97-1L, 570L, 580L and 664L in the periplasm was confirmed.
The localisation of ORF40L in the outer membrane, and 008 and 519-1L in the inner membrane was confirmed. ORF25L, ORF4L, 406L, 576-1L were all confirmed as being localised in the membrane.
Protein 206 was found not to be a lipoprotein.
ORF25 and ORF40 expressed with their native leader peptides but without fusion partners, and protein 593 expressed without its native leader peptide and without a fusion partner, raised good anti-bactericidal sera. Surprisingly, the forms of ORF25 and ORF40 expressed without fusion partners and using their own leader peptides (i.e. ‘ORF25L’ and ‘ORF40L’) give better results in the bactericidal assay than the fusion proteins.
Proteins 920L and 953L were subjected to N-terminal sequencing, giving HRVWVETAH and ATYKVDEYHANARFAF, respectively. This sequencing confirms that the predicted leader peptides were cleaved and, when combined with the periplasmic location, confirms that the proteins are correctly processed and localised by E. coli when expressed from their native leader peptides.
The N-terminal sequence of protein 519.1L localised in the inner membrane was MEFFIILLA, indicating that the leader sequence is not cleaved. It may therefore function as both an uncleaved leader sequence and a transmembrane anchor in a manner similar to the leader peptide of PBP1 from N. gonorrhoeae [Ropp & Nicholas (1997) J. Bact. 179:2783-2787.]. Indeed the N-terminal region exhibits strong hydrophobic character and is predicted by the Tmpred. program to be transmembrane.

Example 12—Lipoproteins

The incorporation of palmitate in recombinant lipoproteins was demonstrated by the method of Kraft et. al. [J. Bact. (1998) 180:3441-3447.]. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. The culture was diluted to an OD₅₅₀of 0.1 in 5.0 ml of fresh medium LB/Amp medium containing 5 μC/ml [³H] palmitate (Amersham). When the OD₅₅₀of the culture reached 0.4-0.8, recombinant lipoprotein was induced for 1 hour with IPTG (final concentration 1.0 mM). Bacteria were harvested by centrifugation in a bench top centrifuge at 2700 g for 15 min and washed twice with 1.0 ml cold PBS. Cells were resuspended in 120 μl of 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1.0% w/v SDS and lysed by boiling for 10 min. After centrifugation at 13000 g for 10 min the supernatant was collected and proteins precipitated by the addition of 1.2 ml cold acetone and left for 1 hour at −20° C. Protein was pelleted by centrifugation at 13000 g for 10 min and resuspended in 20-50 μl (calculated to standardise loading with respect to the final 0.1) of the culture) of 1.0% w/v SDS. An aliquot of 15 μl was boiled with 5 μl of SDS-PAGE sample buffer and analysed by SDS-PAGE. After electrophoresis gels were fixed for 1 hour in 10% v/v acetic acid and soaked for 30 minutes in Amplify solution (Amersham). The gel was vacuum-dried under heat and exposed to Hyperfilm (Kodak) overnight −80° C.
Incorporation of the [³H] palmitate label, confirming lipidation, was found for the following proteins: Orf4L, Orf25L, 287L, 287LOrf4, 406.L, 576L, 926L, 919L and 9191, Orf4.

Example 13—Domains in 287

Based on homology of different regions of 287 to proteins that belong to different functional classes, it was split into three ‘domains’, as shown in FIG. 5. The second domain shows homology to IgA proteases, and the third domain shows homology to transferrin-binding proteins.
Each of the three ‘domains’ shows a different degree of sequence conservation between N. meningitidis strains domain C is 98% identical, domain A is 83% identical, whilst domain B is only 71% identical. Note that protein 287 in strain MC58 is 61 amino acids longer than that of strain 2996. An alignment of the two sequences is shown in FIG. 7, and alignments for various strains are disclosed in WO00/66741 (see FIGS. 5 and 15 therein).
The three domains were expressed individually as C-terminal His-tagged proteins. This was done for the MC58 and 2996 strains, using the following constructs:

- 287a-MC58 (aa 1-202), 287b-MC58 (aa 203-288), 287c-MC58 (an 311-488).
- 287a-2996 (aa 1-139), 287b-2996 (aa 140-225), 287c-2996 (aa 250-427).

To make these constructs, the stop codon sequence was omitted in the 3′-end primer sequence. The 5′ primers included the NheI restriction site, and the 3′ primers included a Kiwi as a tail, in order to direct the cloning of each amplified fragment into the expression vector pRT21b4 rising NdeI-XhoI, NheI-XhoI or NdeI-HindIII restriction sites.
All six constructs could be expressed, but 287b-MC8 required denaturation and refolding for solubilisation.
Deletion of domain A is described below (‘Δ4 287-His’).
Immunological data (serum bactericidal assay) were also obtained using the various domains from strain 2996, against the homologous and heterologous MenB strains, as well as MenA (F6124 strain) and MenC (BZ133 strain):


2996	BZ232	MC58	NGH38	394/98	MenA	MenC

287-His	32000	16	4096	4096	512	8000	16000
287(B)-His	256	—	—	—	—	16	—
287(C)-His	256	—	32	512	32	2048	>2048
287(B-C)-His	64000	128	4096	64000	1024	64000	32000

Using the domains of strain MC58, the following results were obtained:


MC58	2996	BZ232	NGH38	394/98	MenA	MenC

287-His	4096	32000	16	4096	512	8000	16000
287(B)-His	128	128	—	—	—	—	128
287(C)-His	—	16	—	1024	—	512	—
287(B-C)-His	16000	64000	128	64000	512	64000	>8000

Example 14—Deletions in 287

As well as expressing individual domains, 287 was also expressed (as a C-terminal His-tagged protein) by making progressive deletions within the first domain. These Four deletion mutants of protein 287 from strain 2996 were used (FIG. 6):

- 1) ‘287-His’, consisting of amino acids 18-427 (i.e. leader peptide deleted);
- 2) ‘Δ1 287-His’, consisting of amino acids 26-427;
- 3) ‘Δ2 287-His’, consisting of amino acids 70-427;
- 4) ‘Δ3 287-His’, consisting of amino acids 107-427; and
- 5) ‘Δ4 287-His’, consisting of amino acids 140427 (=287-bc).

The ‘Δ4’ protein was also made for strain MC58 (‘Δ4 287MC58-His’; an 203-488).
The constructs were made in the same way as 287a/b/c, as described above.
All six constructs could be expressed and protein could be purified. Expression of 287-His was, however, quite poor.
Expression was also high when the C-terminal His-tags were omitted.
Immunological data (serum bactericidal assay) were also obtained using the deletion mutants, against the homologous (2996) and heterologous MenB strains, as well as MenA (F6124 strain) and MenC (BZ133 strain):


2996	BZ232	MC58	NGH38	394/98	MenA	MenC

287-his	32000	16	4096	4096	512	8000	16000
Δ1 287-His	16000	128	4096	4096	1024	8000	16000
Δ2 287-His	16000	128	4096	>2048	512	16000	>8000
Δ3 287-His	16000	128	4096	>2048	512	16000	>8000
Δ4 287-His	64000	128	4096	64000	1024	64000	32000

The same high activity for the Δ4 deletion was seen using the sequence from strain MC58.
As well as showing superior expression characteristics, therefore, the mutants are immunologically equivalent or superior.

Example 15—Poly-Glycine Deletions

The ‘Δ1 287-His’ construct of the previous example differs from 287-His and from ‘287^untagged’ only by a short N-terminal deletion (GGGGGGS). Using an expression vector which replaces the deleted serine with a codon present in the Nhe cloning site, however, this amounts to a deletion only of (Gly)₆. Thus, the deletion of this (Gly)₆sequence has been shown to have a dramatic effect on protein expression.
The protein lacking the N-terminal amino acids up to GGGGGG is called ‘ΔG287’. In strain MC58, its sequence (leader peptide underlined) is:

	ΔG287
1	MFKRSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP

	VVSEKETEAK

51	EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN

	PKNEDEVAQN

101	DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP

	ANQPDMANAA

151	DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA

	SNPAPANGGS

201	NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV

	QLKSEFEKLS

251	DADKISNYKK DGKNDKFVGL VADSVQMKGI NQYIIFYKPK

	PTSFARFRRS

301	ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS LTGHSGNIFA

	PEGNYRYLTY

351	GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY NGEVLHFHTE

	NGRPYPTRGR

401	FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKG

	TWTENGSGDV

451	SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD*

ΔG287, with or without His-tag (‘ΔG287-His’ and ‘ΔG287K’, respectively), are expressed at very good levels in comparison with the ‘287-His’ or ‘287^untagged’.
On the basis of gene variability data, variants of ΔG287-His were expressed in E. coli from a number of MenB strains, in particular from strains 2996, MC58, 1000, and BZ232. The results were also good.
It was hypothesised that poly-Gly deletion might be a general strategy to improve expression. Other MenB lipoproteins containing similar (Gly)_nmotifs (near the N-terminus, downstream of a cysteine) were therefore identified, namely Tbp2 (N/v1130460), 741 (NMB 1870) and 983 (NMB1969):

TBP2	ΔGTbp2
1	MNNPLVNQAA MVLPVFLLSA CLGGGGSFDL DSVDTEAPRP APKYQDVFSE

51	KPQAQKDQGG YGFAMRLKRR NWYPQAKEDE VKLDESDWEA TGLPDEPKEL

101	PKRQKSVIEK VETDSDNNIY SSPYLKPSNH QNGNTGNGIN QPKNQAKDYE

151	NFKYVYSGWF YKHAKREFNL KVEPKSAKNG DDGYIFYHGK EPSRQLPASG

201	KITYKGVWHF ATDTKKGQKF REIIQPSKSQ GDRYSGFSGD DGEEYSNKNK

251	STLTDGQEGY GFTSNLEVDF HNKKLTGKLI RNNANTDNNQ ATTTQYYSLE

301	AQVTGNRFNG KATATDKPQQ NSETKEHPFV SDSSSLSGGF FGPQGEELGF

351	RFLSDDQKVA VVGSAKTKDK PANGNTAAAS GGTDAAASNG AAGTSSENGK

401	LTTVLDAVEL KLGDKEVQKL DNFSNAAQLV VDGIMIPLLP EASESGNNQA

451	NQGTNGGTAF TRKFDHTPES DKKDAQAGTQ TNGAQTASNT AGDTNGKTKT

501	YEVEVCCSNL NYLKYGMLTR KNSKSAMQAG ESSSQADAKT EQVEQSMFLQ

551	GERTDEKEIP SEQNIVYRGS WYGYIANDKS TSWSGNASNA TSGNRAEFTV

601	NFADKKITGT LTADNRQEAT FTIDGNIKDN GFEGTAKTAE SGFDLDQSNT

651	TRTPKAYITD AKVQGGFYGP KAEELGGWFA YPGDKQTKNA TNASGNSSAT

701	VVFGAKRQQP VR*

741	ΔG741
1	VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL

51	QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ

101	IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI

151	GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI

201	EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA

251	QEVAGSAEVK TVNGIRHIGL AAKQ*

983	ΔG983
1	MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS

51	NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN

101	LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE

151	LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT

201	DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM

251	MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL

301	DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIFSTGN DAQAQPNTYA

351	LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA

401	MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN

451	LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT

501	SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGRTIIE GGSLVLYGNN

551	KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ

601	LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS

651	AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART

701	ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT

751	DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG

801	RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA

851	KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL

901	FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE

951	GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL

1001	FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE

1051	FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*

Thp2 and 741 genes were from strain MC58; 983 and 287 genes were from strain 2996. These were cloned in pET vector and expressed in E. coli without the sequence coding for their leader peptides or as “ΔG forms”, both fused to a C-terminal His-tag. In each case, the same effect was seen expression was good in the clones carrying the deletion of the poly-glycine stretch, and poor or absent if the glycines were present in the expressed protein:


ORF	Express.	Purification	Bact. Activity

287-His(2996)	+/−	+	+
‘287^untagged’(2996)	+/−	nd	nd
ΔG287-His(2996)	+	+	+
ΔG287K(2996)	+	+	+
ΔG287-His(MC58)	+	+	+
ΔG287-His(1000)	+	+	+
ΔG287-His(BZ232)	+	+	+
Tbp2-His(MC58)	+/−	nd	nd
ΔGTbp2-His(MC58)	+	+
741-His(MC58)	+/−	nd	nd
ΔG741-His(MC58)	+	+
983-His(2996)
ΔG983-His(2996)	+	+

SDS-PAGE of the proteins is shown in FIG. 13.
ΔG287 and Hybrids
ΔG287 proteins were made and purified for strains MC58, 1000 and BZ232. Each of these gave high ELISA titres and also serum bactericidal titres of >8192. ΔG287K, expressed from pET-24b, gave excellent titres in ELISA and the serum bactericidal assay. ΔG287-ORF46.1K may also be expressed in pET-24b.
ΔG287 was also fused directly in-frame upstream of 919, 953, 961 (sequences shown below) and ORF46.1:

ΔG287-919

1	ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC

51	TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG

101	CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCNG CCAAGATATG

151	GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA

201	CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT

251	CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA

301	GATTCCGCCC CCGCGTCAAA CCCTCCACCT GCGANAGGCG GTAGCAATTT

351	TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA

401	ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTAA

451	TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC

501	TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA

551	ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC

601	ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC

651	TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA

701	ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG

751	CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA

801	CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG

851	AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA

901	GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG

951	GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG

1001	ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC

1051	GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT

1101	TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA

1151	GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC

1201	AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGATGCCAAA GCAAGAGCAT

1251	CCAAACCTTT CCGCAACCCG ACACATCCGT CATCAACGCC CCGGACCGGC

1301	CGGTCGGCAT CCCCGACCCC GCCGGAACGA CGGTCGGCGG CGGCGGGGCC

1351	GTCTATACCG TTGTACCGCA CCTGTCCCTG CCCCACTGGG CGGCGCAGGA

1401	TTTCGCCAAA AGCCTGCAAT CCTTCCGCCT CGGCTGCGCC AATTTGAAAA

1451	ACCGCCAAGG CTGGCAGGAT GTGTGCGCCC AAGCCTTTCA AACCCCCGTC

1501	CATTCCTTTC AGGCAAAACA GTTTTITGAA CGCTATTTCA CGCCGTGGCA

1551	GGTTGCAGGC AACGGAAGCC TTGCCGGTAC GGTTACCGGC TATTACGAGC

1601	CGGTGCTGAA GGGCGACGAC AGGCGGACGG CACAAGCCCG CTTCCCGATT

1651	TACGGTATTC CCGACGATTT TATCTCCGTC CCCCTGCCTG CCGGTTTGCG

1701	GAGCGGAAAA GCCCTTGTCC GCATCAGGCA GACGGGAAAA AACAGCGGCA

1751	CAATCGACAA TACCGGCGGC ACACATACCG CCGACCTCTC CCGATTCCCC

1801	ATCACCGCGC GCACAACGGC AATCAAAGGC AGGTTTGAAG GAAGCCGCTT

1851	CCTCCCCTAC CACACGCGCA ACCAAATCAA CGGCGGCGCG CTTGACGGCA

1901	AAGCCCCGAT ACTCGGTTAC GCCGAAGACC CCGTCGAACT TTTTTTTATG

1951	CACATCCAAG GCTCGGGCCG TCTGAAAACC CCGTCCGGCA AATACATCCG

2001	CATCGGCTAT GCCGACAAAA ACGAACATCC CTACGTTTCC ATCGGACGCT

2051	ATATGGCGGA CAAAGGCTAC CTCAAGCTCG GGCAGACCTC GATGCAGGGC

2101	ATCAAAGCCT ATATGCGGCA AAATCCGCAA CGCCTCGCCG AAGTTTTGGG

2151	TCAAAACCCC AGCTATATCT TTTTCCGCGA GCTTGCCGGA AGCAGCAATG

2201	ACGGTCCCGT CGGCGCACTG GGCACGCCGT TGATGGGGGA ATATGCCGGC

2251	GCAGTCGACC GGCACTACAT TACCTTGGGC GCGCCCTTAT TTGTCGCCAC

2301	CGCCCATCCG GTTACCCGCA AAGCCCTCAA CCGCCTGATT ATGGCGCAGG

2351	ATACCCGCAG CGCGATTAAA GGCGCGGTGC GCGTGGATTA CCACGGGTTA

2401	TACGGCGACG AAGCCGGCGA ACTTGCCGGC AAACAGAAAA CCACGGGTTA

2451	CGTCTGGCAG CTCCTACCCA ACGGTATGAA GCCCGAATAC CGCCCGTAAC

2501	TCGAG

1	MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM

51	AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS

101	DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL

151	LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI

201	IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG

251	HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE

301	VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI

351	DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG

401	KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA

451	VYTVVPHLSL PHWAAQDFAK SLQSPRLGCA NLKNRQGWQD VCAQAFQTPV

501	HSFQAKQFFE RYFTPMQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARFPI

551	YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP

601	ITARTTAIKG RFEGSRPLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM

651	HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LKLGQTSMQG

701	IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG

751	AVDRHYITLG APLFVATAHP VTRKALNRLI MAQDTGSAIK GAVRVDYFWG

901	YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP*

ΔG287-953

1	ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC

51	TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG

101	CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG

151	GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA

201	CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT

251	CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA

301	GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT

351	TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA

401	ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA

451	TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC

501	TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA

551	ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC

601	ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC

651	TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA

701	ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG

751	CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA

801	CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG

851	AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAAGGGCGAA

901	GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG

951	GTTTGCCGCA AAAGTCGATT TCGGCAGGAA ATCTGTGGAC GGCATTATCG

1001	ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC

1051	GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT

1101	TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA

1151	GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC

1201	AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGAGCCACCT ACAAAGTGGA

1251	CGAATATCAC GCCAACGCCC GTTTCGCCAT CGACCATTTC AACACCAGCA

1301	CCAACGTCGG CGGTTTTTAC GGTCTGACCG GTTCCGTCGA GTTCGACCAA

1351	GCAAAACGCG ACGGTAAAAT CGACATCACC ATCCCCGTTG CCAACCTGCA

1401	AAGCGGTTCG CAACACTTTA CCGACCACCT GAAATCAGCC GACATCTTCG

1451	ATGCCGCCCA ATATCCGGAC ATCCGCTTTG TTTCCACCAA ATTCAACTTC

1501	AACGGCAAAA AACTGGTTTC CGTTGACGGC AACCTGACCA TGCACGGCAA

1551	AACCGCCCCC GTCAAACTCA AAGCCGAAAA ATTCAACTGC TACCAAAGCC

1601	CGATGGCGAA AACCGAAGTT TGCGGGGGCG ACTTCAGCAC CACCATCGAC

1651	CGCACCAAAT GGGGCGTGGA CTACCTCGTT AACGTTGGTA TGACCAAAAG

1701	CGTCCGCATC GACATCCAAA TCGAGGCAGC CAAACAATAA CTCGAG

1	MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM

51	AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS

101	DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL

151	LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI

201	IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG

251	HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE

301	VLHFHTENGR PYPTKGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI

351	DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG

401	KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ

451	AKRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF

501	NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID

551	RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ*

ΔG287-961

1	ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC

51	TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG

101	CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG

151	GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA

201	CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT

251	CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA

301	GATTCCGCCC CCGCGTCAAA CGCTGCACCT GCGAATGGCG GTAGCAATTT

351	TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA

401	ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA

451	TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC

501	TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA

551	ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC

601	ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC

651	TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA

701	ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG

751	CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA

801	CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG

851	AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA

901	GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG

951	GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG

1001	ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC

1051	GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT

1101	TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA

1151	GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC

1201	AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGAGCCACAA ACGACGACGA

1251	TGTTAAAAAA GCTGCCACTG TGGCCATTGC TGCTGCCTAC AACAATGGCC

1301	AAGAAATCAA CGGTTTCAAA GCTGGAGAGA CCATCTACGA CATTGATGAA

1351	GACGGCACAA TTACCAAAAA AGACGCAACT GCAGCCGATG TTGAAGCCGA

1401	CGACTTTAAA GGTCTGGGTC TGAAAAAAGT CGTGACTAAC CTGACCAAAA

1451	CCGTCAATGA AAACAAACAA AACGTCGATG CCAAAGTAAA AGCTGCAGAA

1501	TCTGAAATAG AAAAGTTAAC AACCAAGTTA GCAGACACTG ATGCCGCTTT

1551	AGCAGATACT GATGCCGCTC TGGATGCAAC CACCAACGCC TTGAATAAAT

1601	TGGGAGAAAA TATAACGACA TTTGCTGAAG AGACTAAGAC AAATATCGTA

1651	AAAATTGATG AAAAATTAGA AGCCGTGGCT GATACCGTCG ACAAGCATGC

1701	CGAAGCATTC AACGATATCG CCGATTCATT GGATGAAACC AACACTAAGG

1751	CAGACGAAGC CGTCAAAACC GCCAATGAAG CCAAACAGAC GGCCGAAGAA

1801	ACCAAACAAA ACGTCGATGC CAAAGTAAAA GCTGCAGAAA CTGCAGCAGG

1851	CAAAGCCGAA GCTGCCGCTG GCACAGCTAA TACTGCAGCC GACAAGGCCG

1901	AAGCTGTCGC TGCAAAAGTT ACCGACATCA AAGCTGATAT CGCTACGAAC

1951	AAAGATAATA TTGCTAAAAA AGCAAACAGT GCCGACGTGT ACACCAGAGA

2001	AGAGTCTGAC AGCAAATTTG TCAGAATTGA TGGTCTGAAC GCTACTACCG

2051	AAAAATTGGA CACACGCTTG GCTTCTGCTG AAAAATCCAT TGCCGATCAC

2101	GATACTCGCC TGAACGGTTT GGATAAAACA GTGTCAGACC TGCGCAAAGA

2151	AACCCGCCAA GGCCTTGCAG AACAAGCCGC GCTCTCCGGT CTGTTCCAAC

2201	CTTACAACGT GGGTCGGTTC AATGTAACGG CTGCAGTCGG CGGCTACAAA

2251	TCCGAATCGG CAGTCGCCAT CGGTACCGGC TTCCGCTTTA CCGAAAACTT

2301	TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC TTCGTCCGGT TCTTCCGCAG

2351	CCTACCATGT CGGCGTCAAT TACGAGTGGT AACTCGAG

1	MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM

51	AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS

101	DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQINTLTHC KGDSCNGDNL

151	LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI

201	IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG

251	HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE

301	VLSFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI

351	DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGEGVFAG

401	KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE

451	DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE

501	SEIEKLTTKL ADTDAALADT DAALDAFTNA LNKLGENITT FAEETKTNIV

551	KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE

601	TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN

651	KDNIAKKANS ADVYTREESD SKFVRIDGLN ATTERLDTRL ASAEKSIADH

701	DTRLNGLDKT VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK

751	SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*


		ELISA	Bactericidal

ΔG287-953-His	3834	65536
ΔG287-961-His	108627	65536

The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens (using 287-GST) for 919 and ORF46.1:


		Mixture with 287	Hybrid with ΔG287

919	32000	128000
ORF46.1	128	16000

Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained:


		919	ORF46.1

	Strain	Mixture	Hybrid	Mixture	Hybrid

NGH38	1024	32000	—	16384
MC58	512	8192	—	512
BZ232	512	512	—	—
MenA (F6124)	512	32000	—	8192
MenC (C11)	>2048	>2048	—	—
MenC (BZ133)	>4096	164000	—	8192

The hybrid proteins with ΔG287 at the N-terminus are therefore immunologically superior to simple mixtures, with ΔG287-ORF46.1 being particularly effective, even against heterologous strains: ΔG287-ORF46.1K may be expressed in pET-24b.
The same hybrid proteins were made using New Zealand strain 394198 rather than 2996:

ΔG287NZ-919

1	ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC

51	CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG

101	CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG

151	GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA

201	CAAACCCAAA AATGAAGACG AGGGGGCCCA AAATGATATG CCGCAAAATG

251	CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG

301	CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA

351	GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG

401	ACGATCCGTC GGGAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT

451	ACAAATCAAG CCGAAAACAA TGAAACCGCC GGTTCTCAAA ATCCTGCCTC

501	TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA

551	ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG

601	ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA

651	AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA

701	GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC

751	GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT

801	CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC

851	OGTCGAGGCG GTCGCTTCCG GCCGAGATCC CGCTGATTCC CGTCAATCAG

901	GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC

951	CGOCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG

1001	CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT

1051	TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT

1101	GGATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG

1151	CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC

1201	GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG

1251	AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG

1301	GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT

1351	CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA

1401	AGAGCAGGAT GGATCCGGAG GAGGAGGATG CCAAAGCAAG AGCATCCAAA

1451	CCTTTCCGCA ACCCGACACA TCCGTCATCA ACGGCCCGGA CCGGCCGGTC

1501	GGCATCCCCG ACCCCGCCGG AACGACGGTC GGCGGCGGCG GGGCCGTCTA

1551	TACCGTTGTA CCGCACCTGT CCCTGCCCCA CTGGGCGGCG CAGGATTTCG

1601	CCAAAAGCCT GCAATCCTTC CGCCTCGGCT GCGCCAATTT GAAAAACCGC

1651	CAAGGCTGGC AGGATGTGTG CGCCCAAGCC TTTCAAACCC CCGTCCATTC

1701	CTTTCAGGCA AAACAGTTTT TTGAACGCTA TTTCACGCCG TGGCAGGTTG

1751	CAGGCAACGG AAGCCTTGCC GGTACGGTTA CCGGCTATTA CGAGCCGGTG

1801	CTGAAGGGCG ACGACAGGCG GACGGCACAA GCCCGCTTCC CGATTTACGG

1851	TATTCCCGAC GATTTTATCT CCGTCCCCCT GCCTGCCGGT TTGCGGAGCG

1901	GAAAAGCCCT TGTCCGCATC AGGCAGACGG GAAAAAACAG CGGCACAATC

1951	GACAATACCG GCGGCACACA TACCGCCGAC CTCTCCCGAT TCCCCATCAC

2001	CGCGCGCACA ACGOCAATCA AAGGCAGGTT TGAAGGAAGC CGCTTCCTCC

2051	CCTACCACAC GGGCAACCAA ATCAAGGGCG GCGCGCTTGA CGGCAAAGCC

2101	CCGATACTCG GTTACGCCGA AGACCCCGTC GAACTTTTTT TTATGCACAT

2151	CCAAGGCTCG GGCCGTCTGA AAACCCCGTC CGGCAAATAC ATCCGCATCG

2201	GCTATGCCGA CAAAAAGGAA CATCCCTACG TTTCCATCGG ACGCTATATG

2251	GCGGACAAAG GCTACCTCAA GCTCGGGCAG ACCTCGATGC AGGGCATCAA

2301	AGCCTATATG CGOCAAAATC COCAACGCCT CGCCGAAGTT TTOGGTCAAA

2351	ACCCCAGCTA TATCTTTTTC CGCGAGCTTG CCGGAAGCAG CAATGACGGT

2401	CCCGTCGGCG CACTGGGCAC GCCGTTGATG GGGGAATATG CCGGCGCAGT

2451	CGACCGGCAC TACATTACCT TGGGCGCGCC CTTATTTGTC GCCACCGCCC

2501	ATCCGGTTAC CCGCAAAGCC CTCAACCGCC TGATTATGGC GCAGGATACC

2551	GGCAGCGCGA TTAAAGGCGC GGTGCGCGTG GATTATTTTT GGGGATACGG

2601	CGACGAAGCC GGCGAACTTG CCGGCAAACA GAAAACCACG GGTTACGTCT

2651	GGCAGCTCCT ACCCAACGGT ATCAAGCCCG AATACCGCCC GTAAAAGCTT

1	MASPDVKSAD TLSKPAAPVV SEKETAAKED APQAGSQGQG APSAQGGQDM

51	AAVSERNTGN GGAAATDKPK NEDRGAQNDM PQNAADTDSL TPNHTPASNM

101	PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG

151	TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL

201	THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV

251	GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ

301	ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP

351	SKGEMLAGTA VYNGEVLHPH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS

401	GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY

451	RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV

501	GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR

551	QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV

601	LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI

651	DNTGGTHTAD LSRFPITART TAIKGRFRGS RFLPYHTRNQ INGGALDGKA

701	PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNE HPYVSIGRYM

751	ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG

801	PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTRKA LNRLIMAQDT

851	GSAIKGAVRV DYFWGYGDEA GKLAGKQKTT GYVWQLLPNG MKPEYRP*

ΔG287NZ-953

1	ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC

51	CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG

101	CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG

151	GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA

201	CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG

251	CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG

301	CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA

351	GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG

401	ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT

451	ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC

501	TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA

551	ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG

601	ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA

651	AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA

701	GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC

751	GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT

801	CTTTTATAAA CCTAAACCCA CTTCAATTGC GCGATTTAGG CGTTCTGCAC

851	GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG

901	GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC

951	CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG

1001	CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT

1051	TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT

1101	GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG

1151	CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC

1201	GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG

1251	AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG

1301	GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT

1351	CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA

1401	AGAGCAGGAT GGATCCGGAG GAGGAGGAGC CACCTACAAA GTGGACGAAT

1451	ATCACGCCAA CGCCCGTTTC GCCATCGACC ATTTCAACAC CAGCACCAAC

1501	GTCGGCGGTT TTTACGGTCT GACCGGTTCC GTCGAGTTCG ACCAAGCAAA

1551	ACGCGACGGT AAAATCGACA TCACCATCCC CGTTGCCAAC CTGCAAAGCG

1601	GTTC CAACA CTTTACCGAC CACCTGAAAT CAGCCGACAT CTTCGATGCC

1651	GCCCAATATC CGGACATCCG CTTTGTTTCC ACCAANTTCA ACTTCAACGG

1701	CAAAAAACTG GTTTCCGTTG ACGGCAACCT GACCATGCAC GGCAAAACCG

1751	CCCCCGTCAA ACTCAAAGCC GAAAAATTCA ACTGCTACCA AAGCCCGATG

1801	GCGAAAACCG AAGTTTGCGG CGGCGACTTC AGCACCACCA TCGACCGCAC

1651	CAAATGGGGC GTGGACTACC TCGTTAACGT TGGTATGACC AAAAGCGTCC

1901	GCATCGACAT CCAAATCGAG GCAGCCAAAC AATAAAAGCT T

1	MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM

51	AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM

101	PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG

151	TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL

201	THCKGDSCSG NNFLDEEVQL KSEPEKLSDA DKISNYKKDG KNDGKNDKFV

251	GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ

301	ADTLIVDGEA VSLTGHSGNI FAPEGNYKYL TYGAEKLPGG SYALRVQGEP

351	SKGEMLAGTA VYNGEVLEFH TDNGRPSPSR GRFAAKVDFG SKSVDGIIES

401	GDGLENGTQK FKAAIDGNGF KGTWTENGGG CUSGKEYGPA GEEVAGKYSY

451	APTDAEKGGF GVFACKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN

501	VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQEFTD HLKSADIFDA

551	AQYPDIRFVS TKENFNGALL VSVDGNLTMH GKTAPVKLKA ERFNCYOSPM

601	AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ*

ΔG287N2-961

1	ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC

51	CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG

101	CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG

151	GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA

201	CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG

251	CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG

301	CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGcCOGGG AATCGGAGCA

351	GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG

401	ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT

451	ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC

501	TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA

551	ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG

601	ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA

651	AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA

701	GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC

751	GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT

801	CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC

851	GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG

901	GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC

951	CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG

1001	CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT

1051	TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT

1101	GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG

1151	CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC

1201	GGCGAIGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG

1251	AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG

1301	GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT

1351	CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA

1401	AGAGCAGGAT GGATCCGGAC GAGGAGGAGC CACAAACGAC GACGATGTTA

1451	AAAAAGCTGC GACTGTGGCC ATTGCTGCTG CCTACAACAA TGGCCAAGAA

1501	ATCAACGGTT TCAAAGCTGG AGAGACCATC TACGACATTG ATGAAGACGG

1551	CACAATTACC AAAAAAGACG CAACTGCAGC CGATGTTGAA GCCGACGACT

1601	TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA CTAACCTGAC CAAAACCGTC

1651	AATGAAAACA AACAAAACGT CGATGCCAAA GTAAAAGCTG CAGAATCTGA

1701	AATAGAAAAG TTAACAACCA AGTTAGCAGA CACTGATGCC GCTTTAGCAG

1752	ATACTGATGC CGCTCTGGAT GCAACCACCA ACGCCTTGAA TAAATTGGGA

1801	GAAAATATAA CGACATTTGC TGAAGAGACT AAGACAAATA TCGTAAAAAT

1851	TGATGAAAAA TTAGAAGCCG TGGCTGATAC CGTCGACAAG CATGCCGAAG

1901	CATTCAACGA TATCGCCGAT TCATTGGATG AAACCAACAC TAAGGCAGAC

1951	GAAGCCGTCA AAACCGCCAA TGAAGCCAAA CAGACGGCCG AAGAAACCAA

2001	ACAAAACGTC GATGCCAAAG TAAAAGCTGC AGAAACTGCA GCAGGCAAAG

2051	CCGAAGCTGC CGCTGGCACA GCTAATACTG CAGCCGACAA GGCCGAAGCT

2101	GTCGCTGCAA AAGTTACCGA CATCAAAGCT GATATCGCTA CGAACAAAGA

2151	TAATATTGCT AAAAAAGCAA ACAGTGCCGA CGTGTACACC AGAGAAGAGT

2201	CTGACAGCAA ATTTGTCAGA ATTGATGGTC TGAACGCTAC TACCGAAAAA

2251	TTGGACACAC GCTTGGCTTC TGCTGAAAAA TCCATTGCCG ATCACGATAC

2301	TCGCCTGAAC GGTTTGGATA AAACAGTGTC AGACCTGCGC AAAGAAACCC

2351	GCCAAGGCCT TGCAGAACAA GCCGCGCTCT CCGGTCTGTT CCAATCTTAC

2401	AACGTGGOTC GGTTCAATGT AACGGCTGCA GTCGGCGGCT ACAAATCCGA

2451	ATCGGCAGTC GCCATCGGTC CCGGCTTCCG CTTTACCGAA AACTTTGCCG

2501	CCAAAGCAGG CGTGGCAGTC GGCACTTCGT CCGGTTCTTC CGCAGCCTAC

2551	CATGTCGGCG TCAATTACGA GTGGTAAAAG CTT

1	MAWPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM

51	AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM

101	PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG

151	TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL

201	THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV

251	GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ

301	ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP

351	SKGEMLAGTA SYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS

401	GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GKKVAGKYSY

451	RPTDAEKGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE

501	INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV

551	NENKONVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG

601	ENITTFAEET KTNIVKIDER LEAVADTVDK HAEAFNDIAD SLDETNTKAD

651	EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKADAAAGT ANTAADKAEA

701	VAAKVTDZKA DIATNKDNIA KKANSADVYT REESDSKEVR IDGLNATTEK

751	LDTRLASAEK SIADHDTELN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY

801	NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY

851	HVGVNYEW*

ΔG983 and Hybrids

Bactericidal titres generated in response to ΔG983 (His-fusion) were measured against various strains, including the homologous 2996 strain:


	2996	NGH38	BZ133

ΔG983	512	128	128

ΔG983 was also expressed as a hybrid, with ORF46.1, 741, 961 or 961c at its C-terminus:

ΔG983-ORF46.1

1	ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA

51	CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA

101	AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC

151	GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT

201	GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA

251	ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA

301	GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT

351	GTATGGCAGA AAAOAACACG GCTATAACGA AAATTACAAA AACTATACGG

401	CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA

451	GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA

501	TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA

551	TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT

601	GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT

651	GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC

701	GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC

751	CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA

801	CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA

851	GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC

901	ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT

951	ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG

1001	GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG

1051	GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT

1101	GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA

1151	ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC

1201	ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT

1251	GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG

1301	ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA

1351	CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC

1401	CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA

1451	TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG

1501	GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA

1551	ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG

1601	CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC

1651	GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT

1701	GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG

1751	ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG

1801	GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC

1851	CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG

1901	GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA

1951	GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC

2001	TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC

2051	AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAAGTGGA TGCCTCCGAA

2101	TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA

2151	TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG

2201	TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC

2251	GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG

2301	CCGCCTGAAA GoCGTATCGO ACGGGTTGGA CCACAACGGC ACGGGTCTGC

2351	GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT

2401	GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA

2451	AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA

2501	CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT

2551	GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT

2601	CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG

2651	AACATGCGGA AGGCAGCGTC AAGGGCACGC TGATGCAGCT GGGCGCACTG

2701	GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG

2751	CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA

2801	GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA

2851	CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT

2901	TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA

2951	CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC

3001	AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT

3651	CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC

3101	AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAC

3151	GGTGGCGGAG GCACTGGATC CTCAGATTTG GCAAACGATT CTTTTATCCG

3201	GCAGGTTCTC GACCGTCAGC ATTTCGAACC CGACGGGAAA TACCACCTAT

3251	TCGGCAGCAG GGGGGAACTT GCCGAGCGCA GCGGCCATAT CGGATTGGGA

3301	AAAATACAAA GCCATCAGTT GGGCAACCTG ATGATTCAAC AGGCGGCCAT

3351	TAAAGGAAAT ATCGGCTACA TTGTCCGCTT TTCCGATCAC GGGCACGAAG

3401	TCCATTCCCC CTTCGACAAC CATGCCTCAC ATTCCGATTC TGATGAAGCC

3451	GGTAGTCCCG TTGACGGATT TAGCCTTTAC CGCATCCATT GGGACGGATA

3501	CGAACACCAT CCCGCCGACG GCTATGACGG GCCACAGGGC GGCGGCTATC

3551	CCGCTCCCAA AGGCGCGAGG GATATATACA GCTACGACAT AAAAGGCGTT

3601	GCCCAAAATA TCCGCCTCAA CCTGACCGAC AACCGCAGCA CCGGACAACG

3651	GCTTGCCGAC CGTTTCCACA ATGCCGGTAG TATGCTGACG CAAGGAGTAG

3701	GCGACGGATT CAAACGCGCC ACCCGATACA GCCCCGAGCT GGACAGATCG

3751	GGCAATGCCG CCGAAGCCTT CAACGGCACT GCAGATATCG TTAAAAACAT

3801	CATCGGCGCG GCAGGAGAAA TTGTCGGCGC AGGCGATGCC GTGCAGGGCA

3851	TAAGCGAAGG CTCAAACATT GCTGTCATGC ACGGCTTGGG TCTGCTTTCC

3901	ACCGAAAACA AGATGGCGCG CATCAACGAT TTGGCAGATA TGGCGCAACT

3951	CAAAGACTAT GCCGCAGCAG CCATCCGCGA TTGGGCAGTC CAAAACCCCA

4001	ATGCCGCACA AGGCATAGAA GCCGTCAGCA ATATCTTTAT GGCAGCCATC

4051	CCCATCAAAG GGATTGGAGC TGTTCGGGGA AAATACGGCT TGGGCGGCAT

4101	CACGGCACAT CCTATCAAGC GGTCGCAGAT GGGCGCGATC GCATTGCCGA

4151	AAGGGAAATC CGCCGTCAGC GACAATTTTG CCGATGCGGC ATACGCCAAA

4201	TACCCGTCCC CTTACCATTC CCGAAATATC CGTTCAAACT TGGAGCAGCG

4251	TTACGGCAAA GAAAACATCA CCTCCTCAAC CGTGCCGCCG TCAAACGGCA

4301	AAAATGTCAA ACTGGCAGAC CAACGCCACC CGAAGACAGG CGTACCGTTT

4351	GACGGTAAAG GGTTTCCGAA TTTTGAGAAG CACGTGAAAT ATGATACGCT

4401	CGAGCACCAC CACCACCACC ACTGA

1	MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD

51	VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLIULKPAIE AGYTGRGVEV

101	GIVDTGESVG SISPPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE

151	ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD

201	ATLHINNTND ETKNDMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD

251	LFQIANSEEQ YRQALIPYSG GDKTDEGIRL MINSDYGNLS YHIRNKNMLF

301	IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP

351	GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG

401	TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG

451	PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT

501	GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT

551	DQSGANETVE IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK

601	GAGYLNSTGR RVPFLSAAKI GQDYSFETNI ETDGGLLASL DSVEKTAGSE

651	GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE

701	SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIENSLA

751	ATVYADSTAA HADMQGRRLK AVSGGLOHNG TGLRVIAQTQ QDGGTWEQGG

801	VEGKMRGSTQ TVGIAAKTGE NTTAAATIGM GRSTWSENSA NAKTDSISLF

851	AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL

901	GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG

951	LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGRTGAR

1001	NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD

1051	GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG

1101	KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA

1151	GSPVDGESLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DTYSYDIKGV

1201	AQMIRLNLTD NRSTGQRLAD REHMAGSMLT QGVGDGFKRA TRYSPELDRS

1251	GNAAFAENGT ADIVKNIIGA AORTVGAGDA VQGISEGSNI AVMHGLGLLS

1301	TENKMARDID LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI

1351	PIKGIGAVRG KYGLGGITAH PIKRSQHGAI ALPKGKSAVS DATADAAYAK

1401	YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRSPKTGVPF

1451	DGKGFPNFEK HVKYDTLEHH HHHH*

ΔG983-741

1	ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA

51	CASCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA

101	AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC

151	GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT

201	GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA

251	ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA

301	GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT

351	GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG

401	CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGAGATTGAA

451	GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAACCAA AGCCGACGGA

501	TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA

551	TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT

601	GGGACGCTAC AGATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT

651	GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC

701	GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC

751	CTTTTCCAAh TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA

801	CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA

851	GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC

901	ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT

951	ATTGCCATTT TATGAAAAAG ACGCTUAAAA AGGCATTATC ACAGTCGCAG

1001	GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG

1051	GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT

1101	GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA

1151	ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC

1201	ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT

1251	GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG

1301	ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA

1351	CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC

1401	CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA

1451	TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG

1501	GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA

1551	ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG

1601	CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC

1651	GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT

1701	GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG

1751	ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG

1801	GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGAGC

1851	CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG

1901	GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA

1951	GGCGACACGC TGTCCTATTA TGTCCCTCGC GGCAATGCGG CACGGACTGC

2001	TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGANC

2051	AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA

2101	TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA

2151	TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG

2201	TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC

2251	GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG

2301	CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC

2351	GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT

2401	GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA

2451	AACCGGCGAA AATACGACAG CAGCCGCCAC ACTCGGCATG GGACGCAGCA

2501	CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT

2551	GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT

2601	CTCCTACGGA CGcTACAAAA AGAGCATCAG CCGCAGCACC GGTGCGGACG

2651	AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG

2701	GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG

2751	CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA

2801	GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA

2851	CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT

2901	TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA

2951	CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC

3001	AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT

3051	cGGCAACGGC TGGAACGGCT TGGCACCTTA CAGCTACGCC GGTTCCAAAC

3101	AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG

3151	GGATCCGGAG GGGGTGGTGT CGCCGCCGAC ATCGGTGCGG GGCTTGccGA

3201	TGCACTAACC GCACCGCTCG ACCATAAAGA CAAAGGTTTG CAGTCTTTGA

3251	CGCTGGATCA GTCCGTCAGG AAAAACGAGA AACTGAAGCT GGCGGCACAA

3301	GGTGCGGAAA AAACTTATGG AAACGGTGAC AGCCTCAATA CGGGCAAATT

3351	GAAGAACGAC AAGGTCAGCC GTTTCGACTT TATCCGCCAA ATCGAAGTGG

3401	ACGGGCAGCT CATTACCTTG GAGAGTGGAG AGTTCCAAGT ATACAAACAA

3451	AGCCATTCCG CCTTAACCGC CTTTCAGACC GAGCAAATAC AAGATTCGGA

3501	GCATTCCGGG AAGATGGTTG CGAAACGCCA GTTCAGAATC GGCGACATAG

3551	CGGGCGAACA TACATCTTTT GACAAGCTTC CCGAAGGCGG CAGGGCGACA

3601	TATCGCGGGA COGCGTTCGG TTCAGACGAT GcCGGCGGAA AACTGAcCTA

3651	CACCATAGAT TTCGCCGCCA AGCAGGGAAA CGGCAAAATC GAACATTTGA

3701	AATCGcCAGA ACTCAATGTC GACCTGGCCO CCGCCGATAT CAAGCCGGAT

3751	GGAAAACGCC ATGCCGTCAT CAGCGGTTCC GTCCTTTACA ACCAAGCCGA

3801	GAAAGGCAGT TACTCCCTCG GTATCTTTGG CGGAAAACC CAGGAAGTTG

3851	CCGGCAGCGC GGAAGTGAAA ACCGTAAACG GCATACGCCA TATCGGCCTT

3901	GCCGCCAAGC AACTCGAGCA CCACCACCAC CACCACTGA

1	MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD

51	VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPATE AGYTGRGVEV

101	GIVDTGESVG SISFPELYGR KEHGYNENYK NTTAYMRKEA PEDGGGKDIE

151	ASFDDEAVIE TRARPTDIRM VREIGHIDLV SHIIGGRSVD GRPAGGIAPD

201	ATLHINETED ETKNEMMVAA IRNAWVKLGE RGVRIVENSF GTTSRAGTAD

251	LFQIANSBEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF

301	IFSTGEDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KYKEDIYGEP

351	GTEPLEYGSN MCGITAMWCL SAFYEASVRF TRTNPIQIAG TSFSAPIVTG

401	TAALLLQKYP WESNDNARTT LLTTAQDIGA VGVDSKFGWG LLDAGRAMNG

451	pASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLARRGGSQ LQLHGENTYT

501	GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT

551	DQSGANETVH IKGSLQLDGK GTLYTRDGKL LKVDGTAIIG GKLYMSARGK

601	GAGYLNSTGR RVFASSAAKI GODYSFFTET ETDGGLLASL DSVEKTAGSE

651	GDTLSYYVRR GNAARTASAA ANSAPAGLKH AVEQGGSNLE NLMVELDASE

701	SRATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFESLA

751	ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG

801	VEGEMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF

851	AGIRHDAGDI GYLKGLPSTO RYKNSISRST GADEHAEGSV NGTLMQLGAL

901	GGVEVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG

951	LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR

1001	NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNMSG RVGVGYRFLE

1051	GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ

1101	GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ

1151	SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT

1201	YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD

1251	GKRHAVISGS VLYNQARKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL

1301	AAKQLEHBEH HH*

ΔG983-961

1	ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA

51	CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA

101	AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC

151	GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT

201	GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA

251	ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA

301	GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT

351	GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG

401	CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA

451	GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA

501	TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA

551	TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT

601	GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT

651	GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC

701	GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG GACTGCCGAC

751	CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA

801	CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA

851	GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC

901	ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT

951	ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG

1001	GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG

1051	GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT

1101	GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA

1151	ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC

1201	ACGGCGGCTC TGCTGCTOCA GAAATACCCG TGGATGAGCA ACGACAACCT

1251	GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG

1301	ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA

1351	CCCGCGTCGT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC

1401	CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA

1451	TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG

1501	GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA

1551	ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG

1601	CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC

1651	GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT

1701	GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG

1751	ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGG ACGCGGCAAG

1801	GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC

1851	CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG

1901	GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA

1951	GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC

2001	TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC

2051	AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTOGA TGCCTCCGAA

2101	TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA

2151	TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG

2201	TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC

2251	GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG

2301	CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC

2351	GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCOGT

2401	GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA

2451	AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA

2501	CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT

2551	GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT

2601	CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG

2651	AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG

2701	GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG

2751	CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA

2801	GTGCTTTGGG CTGGAGCGGC AACAGCCTCA GTGAAGGCAC GCTGGTCGGA

2851	CTCGCGGGTC TGAAGCTGTG GCAACCCTTG AGCGATAAAG CCGTCCTGTT

2901	TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA

2951	CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC

3001	AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT

3051	CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC

3101	AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG

3151	OGTGGCGGAG GCACTGGATC CGCCACAAAC GACGACGATG TTAAAAAAGC

3201	TGCCACTGTG GCCATTGCTG CTGCCTACAA CAATGGCCAA GAAATCAACG

3251	GTTTCAAAGC TGGAGAGACC ATCTACGACA TTGATGAAGA CGGCACAATT

3301	ACCAAAAAAG ACGCAACTGC AGCCGATGTT GAAGCCGACG ACTTTAAAGG

3351	TCTGGGTCTG AAAAAAGTCG TGACTAACCT GACCAAAACC GTCAATGAAA

3401	ACAAACAAAA CGTCGATGCC AAAGTAAAAG CTGCAGAATC TGAAATAGAA

3451	AAGTTAACAA CCAAGTTAGC AGACACTGAT GCCGCTTTAG CAGATACTGA

3501	TGCCGCTCTG GATGCAACCA CCAACGCCTT GAATAAATTG GGAGAAAATA

3551	TAACGACATT TGCTGAAGAG ACTAAGACAA ATATCGTAAA AATTGATGAA

3601	AAATTAGAAG CCGTGGCTGA TACCGTCGAC AAGCATGCCG AAGCATTCAA

3651	CGATATCGCC GATTCATTGG ATGAAACCAA CACTAAGGCA GACGAAGCCG

3701	TCAAAACCGC CAATGAAGCC AAACAGACGG COGAAGAAAC CAAACAAAAC

3751	GTCGATGCCA AAGTAAAAGC TGCAGAAACT GCAGCAGGCA AAGCCGAAGC

3801	TGCCGCTGGC ACAGCTAATA CTGCAGCCGA CAAGGCCGAA GCTGTCGCTG

3851	CAAAAGTTAC CGACATCAAA GCTGATATCG CTACGAACAA AGATAATATT

3901	GCTAAAAAAG CAAACAGTGC CGACGTGTAC ACCAGAGAAG AGTCTGACAG

3951	CAAATTTGTC AGAATTGATG GTCTGAACGC TACTACCGAA AAATTGGACA

4001	CACGCTTGGC TTCTGCTGAA AAATCCATTG CCGATCACGA TACTCGCCTG

4051	AACGGTTTGG ATAAAACAGT GTCAGACCTG CGCAAAGAAA CCCGCCAAGG

4101	CCTTGCAGAA CAAGCCGCGC TCTCCGGTCT GTTCCAACCT TACAACGTGG

4151	GTCGGTTCAA TGTAACGGCT GCAGTCGGCG GCTACAAATC CGAATCGGCA

4201	GTCGCCATCG GTACCGGCTT CCGCTTTACC GAAAACTTTG CCGCCAAAGC

4251	AGGCGTGGCA GTCGGCACTT CGTCCGGTTC TTCCGCAGCC TACCATGTCG

4301	GCGTCAATTA CGAGTGGCTC GAGCACCACC ACCACCACCA CTGA

1	MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD

51	VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLEPAIE AGYTGRGVEV

101	GIVDTGESVG SISPPELYGR KEHGYNENYK NYTAYMPEZA PEDGCCKDIE

151	ASFDDEAVIE TEAKPTDIRH VREIGHIDLV SHIIGGRSVD GRPAGGIAPD

201	ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD

251	LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNENMLF

301	IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP

351	GTEPLEYGSN MCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG

401	TAALLLQKYP WNSNDNIATT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG

451	PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLBGENTYT

501	GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT

551	DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYEEARGK

601	GAGYLNSTGR RVPFLSAAKX GQDYSFFTNI ETDGGLLASL DSVERTAGSE

651	GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLEVELDASE

701	SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIPNSLA

751	ATVYADSTAA EADMQGRELK AVSDGLDHNG TGLEVIAQTQ QDGGTWEQGG

801	VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF

851	AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL

901	GGVNVPFAAT GDLTVEGGLR YDLLKOGAFA EKGSALGWSG NSLTEGTING

951	LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVIGGFTGA TAATGKTGAR

1001	NNEHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE

1051	GGGGTGSATN DMINKKAATV AIAAAYNNGQ EINGFRAGET IYDIDEDGTI

1101	TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KQTAEETKQN

1151	KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE

1201	KLEAVADTVD KHAEAFNDIA DSLDETNTKA DENVETANBA KQTAEETKQN

1251	VrAFVEAAET AAGKASAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI

1301	AKKANSADVY TREESDSKFV RIDGLNATTE KLDTPIASAE KSIADHDTRL

1351	NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNtTG1NVTA AVGGYKSESA

1401	VAIGTGFRFT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH*

ΔG983-961c

1	ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA

51	CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA

101	AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC

151	GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT

201	GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA

251	ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA

301	GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT

351	GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG

401	CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA

451	GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA

501	TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA

551	TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT

601	GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT

651	GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC

701	GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC

751	CTTTTCCAAA TAGCCAATTC GGAGGAGCAC TACCGCCAAG CGTTGCTCGA

801	CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA

851	GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC

901	ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT

951	ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG

1001	GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG

1051	GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT

1101	GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA

1151	ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC

1201	ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT

1251	GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGGGTGG

1301	ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA

1351	CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC

1401	CGATATTGCC TACTCcTTCc GTAACGACAT TTCAGGCACG GGCGGCCTGA

1451	TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG

1501	GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA

1551	ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG

1601	CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC

1651	GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT

1701	GGACGGCAAA GGTACGCTGT ACACACGTTT CTGAAAGTGG CTGAAAGTGG

1751	ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGcAAG

1801	GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC

1851	CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGAcG

1901	GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA

1951	GGCGACACGc TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC

2001	TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC

2051	AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA

2101	TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA

2151	TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG

2201	TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC

2251	GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG

2301	CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC

2351	GCGTCATCGC GCAAACCCAA CAGGACGOTG GAACGTGGGA ACAGGGCGGT

2401	GITGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA

2451	AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA

2501	CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT

2551	GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT

2601	CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG

2651	AACATGCGGA ACGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG

2701	GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG

2751	CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA

2801	GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA

2851	CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT

2901	TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA

2951	CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC

3001	ATTATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT

3051	CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC

3101	AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG

3151	GGTGGCGGAG GCACTGGATC CGCCACAAAC GACGACGATG TTAAAAAAGC

3201	TGCCACTGTG GCCATTGCTG CTGCCTACAA CAATGGCCAA GAAATCAACG

3251	GTTTCAAAGC TGGAGAGACC ATCTACGACA TTGATGAAGA CGGCACAATT

3301	ACCAAAAAAG ACGCAACTGC AGCCGATGTT GAAGCCGACG ACTTTAAAGG

3351	TCTGGGTCTG AAAAAAGTCG TGACTAACCT GACCAAAACC GTCAATGAAA

3401	ACAAACAAAA CGTCGATGCC AAAGTAAAAG CTGCAGAATC TGAAATAGAA

3451	AAGTTAACAA CCAAGTTAGC AGACACTGAT GCCGCTTTAG CAGATACTGA

3501	TGCCGCTCTG GATGCAACCA CCAACGCCTT GAATAAATTG GOAGAAAATA

3551	TAACGACATT TGCTGAAGAG ACTAAGACAA ATATCGTAAA AATTGATGAA

3601	AAATTAGAAG CCGTGGCTGA TACCGTCGAC AAGCATGCCG AAGCATTCAA

3651	CGATATCGCC GATTCATTGG ATGAAACCAA CACTAAGGCA GACGAAGCCG

3701	TCAAAACCGC CAATGAAGCC AAACAGACGG CCGAAGAAAC CAAACAAAAC

3751	GTCGATGCCA AAGTAAAAGC TGCAGAAACT GCAGCAGGCA AAGCCGAAGC

3801	TGCCGCTGGC ACAGCTAATA CTGCAGCCGA CAAGGCCGAA GCTGTCGCTG

3851	CAAAAGTTAC CGACATCAAA GCTGATATCG CTACGAACAA AGATAATATT

3901	GCTAAAAAAG CAAACAGTGC CGACGTGTAC ACCAGAGAAG AGTCTGACAG

3951	CAAATTTGTC AGAATTGATG GTCTGAACGC TACTACCGAA AAATTGGACA

4001	CACGCTTGGC TTCTGCTGAA AAATCCATTG CCGATCACGA TACTCGCCTG

4051	AACGGTTTGG ATAAAACAGT GTCAGACCTG CGCAAAGAAA CCCGCCAAGG

4101	CCTTGCAGAA CAAGCCGCGC TCTCCGGTCT GTTCCAACCT TACAACGTGG

4151	GTCTCGAGCA CCACCACCAC CACCACTGA

1	MTSAPDFNAG GTGIGSNSRA TTARSAAVSY AGIKNEMCKD RSMLCAGRDD

51	VAVTDRDAKI NAPPPNLMTG DFPNPNDAYK NLINLEPAIR AGYTGRGVEV

101	GIVDTGESVG SISFPELYGR KERGYNENYK NYTAYMRKEA PEDGGGKDIE

151	ASFDDEAVIE TEARPTDIRM VRE/GHIDLV SHIIGGRSVD GRPAGGIAPD

201	ATLHIMNTND ETRNEMMVAA IENAWVKLGR RGVRIVNNSF GTTSRAGTAD

251	LFQIANSKEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNRNMLF

301	IFSTGNDAQA QPNTYALLPF YERDAQRGII TVAGVDRSGE KFKREMYGEP

351	GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG

401	TAALLDQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGRAMNG

451	PASFPFGDFT ADTRGTSDLA YSERNDISGT GGLIKKGGSQ LQLHGNNTYT

501	GKTIIEGGSL VLYGNNKSDM RVETRGALIY NGAASGGSLN SDGIVYLADT

551	DQSGANETVH IKGSLQLDGK GTLYTRLGRL LKVDGTAIIG GRLYMSARGR

601	GAGYLNSTGR RVPFLSAAKI GODYSFETNI ETDGGLLASL DSVERTAGSE

651	GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE

701	SSATFETVET AAADRTDMPG IRPYGATFRA AAAVQMANAA DGVRIENSLA

751	ATVYADSTAA HADMQGRRLK AVSDGMOHNG TGLRVIAQTQ QDGGTWEQGG

801	VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF

851	AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NOTLMQLGAL

901	GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG

951	LAGLKLSOPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGRTGAR

1001	NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE

1051	GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI

1101	TKKDATAADV EADDFKGLGL KKVVTNATET VNENKQNVDA KVKAAESEIE

1151	RLTTKLADTD AALADTDAAL DATTNALNKL GENITTFARE TKTNIVRIDE

1201	RLRAVADTVD KHAEAFNDIA DSLDETNTKA DEAVRTANRA KQTAEETKQN

1251	VDAKVRAAET AAGRAEAAAG TANTAADKAE AVAAKVTDIK ADIATNK1N1

1301	AKKANSADVY TREESDSKEV RIDGLNATTE KIDTRIASAE KSIADHDTRL

1351	NGLDKTVSDL RKETRQGLAE QAALSGLFQP INVGLEHHHH HH*

ΔG741 and Hybrids

Bactericidal litres generated in response to ΔG741 (His-fusion) were measured against various strains, including the homologous 2996 strain:


	2996	MC58	NGH38	F6124	BZ133

ΔG741	512	131072	>2048	16384	>2048

As can be seen, the ΔG741-induced anti-bactericidal titre is particularly high against heterologous strain MC58.
ΔG741 was also fused directly in-frame upstream of proteins 961, 961c, 983 and ORF46.1:

ΔG741-961

1	ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC

51	GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG

101	TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT

151	TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT

201	CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA

251	CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA

301	ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT

351	GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CANAGCGGGC GAACATACAT

401	CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG

451	TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC

501	CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA

551	ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC

601	GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC

651	CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG

701	TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC

751	GAGGGTGGCG GAGGCACTGG ATCCGCCACA AACGACGACG ATGTTAAAAA

801	AGCTGCCACT GTGGCCATTG CTGCTGCCTA CAACAATGGC CAAGAAATCA

851	ACGGTTTCAA AGCTGGAGAG ACCATCTACG ACATTGATGA AGACGGCACA

901	ATTACCAAAA AAGACGCAAC TGCAGCCGAT GTTGAAGCCG ACGACTTTAA

951	AGGTCTGGGT CTGAAAAAAG TCGTGACTAA CCTGACCAAA ACCGTCAATG

1001	AAAACAAACA AAACGTCGAT GCCAAAGTAA AAGCTGCAGA ATCTGAAATA

1051	GAAAAGTTAA CAACCAAGTT AGCAGACACT GATGCCGCTT TAGCAGATAC

1101	TGATGCCGCT CTGGATGCAA CCACCAACGC CTTGAATAAA TTGGGAGAAA

1151	ATATAACGAC ATTTGCTGAA GAGACTAAGA CAAATATCGT AAAAATTGAT

1201	GAAAAATTAG AAGCCGTGGC TGATACCGTC GACAAGCATG CCGAAGCATT

1251	CAACGATATC GCCGATTCAT TGGATGAAAC CAACACTAAG GCAGACGAAG

1301	CCGTCAAAAC CGCCAATGAA GCCAAACAGA CGGCCGAAGA AACCAAACAA

1351	AACGTCGATG CCAAAGTAAA AGCTGCAGAA ACTGCAGCAG GCAAAGCCGA

1401	AGCTGCCGCT GGCACAGCTA ATACTGCAGC CGACAAGGCC GAAGCTGTCG

1451	CTGCAAAAGT TACCGACATC AAAGCTGATA TCGCTACGAA CAAAGATAAT

1501	ATTGCTAAAA AAGCAAACAG TGCCGACGTG TACACCAGAG AAGAGTCTGA

1551	CAGCAAATTT GTCAGAATTG ATGGTCTGAA CGCTACTACC GAAAAATTGG

1601	ACACACGCTT GGCTTCTGCT GAAAAATCCA TTGCCGATCA CGATACTCGC

1651	CTGAACGGTT TGGATAAAAC AGTGTCAGAC CTGCGCAAAG AAACCCGCCA

1701	AGGCCTTGCA GAACAAGCCG CGCTCTCCGG TCTGTTCCAA CCTTACAACG

1751	TOGGTCGOTT CAATGTAACG GCTGCAGTCG GCGGCTACAA ATCCGAATCG

1801	GCAGTCGCCA TCGGTACCGG CTTCCGCTTT ACCGAAAACT TTGCCGCCAA

1851	AGCAGGCGTG GCAGTCGGCA CTTCGTCCGG TTCTTCCGCA GCCTACCATG

1901	TCGGCGTCAA TTACGAGTGG CTCGAGCACC ACCACCACCA CCACTGA

1	MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT

51	YGNGDSLNTG KGENDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL

101	TAFQTEQIQD SEHSGGHVAK RQPRIGDIAG EHTSFDKLPE GGEATYRGTA

151	FGSDDAGGEL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA

201	VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAARQL

251	EGGGGTGSAT NDDDVKKAAT VAIAAAYANG QEINGFKAGE TIYDIDEDGT

301	ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AEVKAAESEI

351	EKLTTKLADT DAALADTDAA LDATTNALNK AGZNITTFAM ETKTNIVKID

401	EKLEAVADTV DKEAEAFNDI ADSLDETNTK ADEAVKTANE AEQTARETKQ

451	NVDAKVKAAE TAAGKARAAA GTANTAADKA EAVAAKVTDI KADTATNKDN

501	ZAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADEDTE

551	LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYKSES

601	AVAIGTGFRF TENFAAKAGV AVGTSSGSSA AYEVGVNYEW LHHHHHH*

ΔG741-961c

1	ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC

51	GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG

101	TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT

151	TATGGAAACG GTGACAGCCT CAATACGGGC.AAATTGAAGA ACGACAAGGT

201	CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGSG CAGCTCATTA

251	CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA

301	ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT

351	GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT

401	CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG

451	TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC

501	CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA

551	ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC

601	GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC

651	CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG

701	TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC

751	GAGGGTGGCG GAGGCACTGG ATCCGCCACA AACGACGACG ATGTTAAAAA

801	AGCTGCCACT GTGGCCATTG CTGCTGCCTA CAACAATGGC CAAGAAATCA

851	ACGGTTTCAA AGCTGGAGAG ACCATCTACG ACATTGATGA AGACGGCACA

901	ATTACCAAAA AAGACGCAAC TGCAGCCGAT GTTGAAGCCG ACGACTTTAA

951	AGGTCTOGGT CTGAAAAAAG TCGTGACTAA CCTGACCAAA ACCGTCAATG

1001	AAAACAAACA AAACGTCGAT GCCAAAGTAA AAGCTGCAGA ATCTGAAATA

1051	GAAAAGTTAA CAACCAAGTT AGCAGACACT GATGCCGCTT TAGCAGATAC

1101	TGATGCCGCT CTGGATGCAA CCACCAACGC CTTGAATAAA TTGGGAGAAA

1151	ATATAACGAC ATTTGCTGAA GAGACTAAGA CAAATATCGT AAAAATTGAT

1201	GAAAAATTAG AAGCCGTGGC TGATACCGTC GACAAGCATG CCGAAGCATT

1251	CAACGATATC GCCGATTCAT TGGATGAAAC CAACACTAAG GCAGACGARG

1301	CCGICAAAAC CGCCAATGAA GCCAAACAGA CGGCCGAAGA AACCAAACAA

1351	AACGTCGATG CCAAAGTAAA AGCTGCAGAA ACTGCAGCAG GCAAAGCCGA

1401	AGCTGCCGCT GGCACAGCTA ATACTGCAGC CGACAAGGCC GAAGCTGTCG

1451	CTGCAAAAGT TACCGACATC AAAGCTGATA TCGCTACGAA CAAAGATAAT

1501	ATTGCTAAAA AAGCAAACAG TGCCGACGTG TACACCAGAG AAGAGTCTGA

1551	CAGCAAATTT GTCAGAATTG ATGGTCTGAA CGCTACTACC GAAAAATTGG

1601	ACACACGCTT GGCTTCTGCT GA.AAAATCCA TTGCCGATCA CGATACTCGC

1651	CTGAACGGTT TGGATAAAAC AGTGTCAGAC CTGCGCAAAG AAACCCGCCA

1701	AGGCCTTGCA GAACAAGCCG CGCTCTCCGG TCTGTTCCAA CCTTACAACG

1751	TGGGTCTCGA GCACCACCAC CACCACCACT GA

1	MVAADIGAGL ADALTAPLDH KDKGLQSLTL DOSVEKNEKL KLAAQGAEKT

51	YGNGDSLNTG KLENDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL

101	TAFQTEQKID SEHGGKMVAK RQFRIGDIAG EHTSFDELPE GGRATYRGTA

151	FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA

201	VTSGSVLYNQ AEKGSYSLGX FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL

251	EGGGGTGSAT NDDDVKKAAT VAGAAAYNNG QEINGFKAGE TIYDIDEDGT

301	ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNMNYQNVD AKVKAAESEI

351	EKLTTKLADT DAALADTDAA LDATTNALNX LGENITTFAE ETKTNIVKID

401	EKLENVADTV DKHAEAFNDI ADSLDETNTX ADEANTTANE AIMTAEETIM

451	NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN

501	IAKKANSADV YTRFZSDSKF VRIDGLNATT EXADTRLASA EKSIADHDTR

551	LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGLEHHH HHH*

ΔG741-983

1	ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC

51	GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG

101	TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT

151	TATGGAAACG GTGACABCCT CAATACGGGC AAATTGAAGA ACGACAAGGT

201	CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA

251	CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA

301	ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT

351	GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT

401	CTTTIGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG

451	TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC

501	CGCCAAGCAG GGAAACGGCA AAATCGAACA TTIGAAATCG CCAGAACTCA

551	ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC

601	GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC

651	CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG

701	TGAAKACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC

751	GAGGGATCCG GCGGAGGCGG CACTTCTGCG CCCGACTTCA ATGCAGGCGG

801	TACCGGTATC GGCAGCAACA GCAGAGCAAC AACAGCGAAA TCAGCAGCAG

851	TATCTTACGC CGGTATCAAG AACGAAATGT GCAAAGACAG AAGCATGCTC

901	TGTGCCGGTC GGGATGACGT TGCGGTTACA GACAGGGATG CCAAAATCAA

951	TGCCCCCCCC CCGAATCTGC ATACCGGAGA CTTTCCAAAC CCAAATGACG

1001	CATACAAGAA TTTGATCAAC CTCAAACCTG CAATTGAAGC AGGCTATACA

1051	GGACGCGGGG TAGAGGTAGG TATCGTCGAC ACAGGCGAAT CCGTCGGCAG

1101	CATATCCTTT CCCGAACTGT ATGGCAGAAA AGAACACGGC TATAACGAAA

1151	ATTACAAAAA CTATACGGCG TATATGCGGA AGGAAGCGCC TGAAGACGGA

1201	GGCGGTAAAG ACATTGAAGC TTCTTTCGAC GATGAGGCCG TTATAGAGAC

1251	TGAAGCAAAG CCGACGGATA TCCGCCACGT AAAAGAAATC GGACACATCG

1301	ATTTGGTCPC CCATATTATT GGCGGGCGTT CCGTGGACGG CAGACCTGCA

1351	GGCGGTATTG CGCCCGATGC GACGCTACAC ATAATGAATA CGAATGATGA

1401	AACCAAGAAC GAAATGATGG TTGCAGCCAT CCGCAATGCA TGGGTCAAGC

1451	TGGGCGAACG TGGCGTGCGC ATCGTCAATA ACAGTTTTGG AACAACATCG

1501	AGGGCAGGCA CTGCCGACCT TTTCCAAATA GCCAATTCGG AGGAGCAGTA

1551	CCGCCAAGCG TTGCTCGACT ATTCCGGCGG TGATAAAACA GACGAGGGTA

1601	TCCGCCTGAT GCAACAGAGC GATTACGGCA ACCTGTCCTA CCACATCCGT

1651	AATAAAAACA TGCTTTTCAT CTTTTCGACA GGCAATGACG CACAAGCTCA

1701	GCCCAACACA TATGCCCTAT TGCCATTTTA TGAAAAAGAC GCTCAAAAAG

1751	GCATTATCAC AGTCGCAGGC GTAGACCGCA GTGGAGAAAA GTTCAAACGG

1801	GAAATGTATG GAGAACCGGG TACAGAACCG CTTGAGTATG GCTCCAACCA

1851	TTGCGGAATT ACTGCCATGT GGTGCCTGTC GGCACCCTAT GAAGCAAGCG

1901	TCCGTTTCAC CCGTACAAAC CCGATTCAAA TTGCCGGAAC ATCCTTTTCC

1951	GCAcCCATcG TAACCGGCAC GGeGGcTCTG CTGCTGCAGA AATACCCGTG

2001	GATGAGCAAC GACAACCTGC GTACCACGTT GCTGACGACG GCTCAGGACA

2051	TCGGTGCAGT CGGCGTGGAC AGCAAGTTCG GCTGGGGACT GCTGGATGCG

2101	GGTAAGGCCA TGAACGGACC CGCGTCCTTT CCGTTCGGCG ACTTTACCGC

2151	CGATACGAAA GGTACATCCG ATATTGCCTA CTCCTTCCGT AACGACATTT

2201	CAGGCACGGG CGGCCTGATC AAAAAAGGCG GCAGCCAACT GCAAcTGCAC

2251	GGCAACAACA CCTATACGGG CAAAACCATT ATCGAAGGCG GTTCGCTGGT

2301	GTTGTACGGC AACAACAAAT CGGATATGCG CGTCGAAACC AAAGGTGCGC

2351	TGATTTATAA CGGGGCGGCA TCCGGCGGCA GCCTGAACAG CGACGGCATT

2401	GTcTATCTGG cAGATACCGA CCAATCCGGC GCAAACGAAA CCGTACACAT

2451	cAAAGGCAGT cTGCAGcTGG ACGGCAAAGG TACGCTGTAC ACACGTTTGG

2501	GCAAACTGCT GAAAGTGGAC GGTACGGCGA TTATCGGCGG CAAGCTGTAC

2551	ATGTCGGCAC GCGGCAAGGG GGCAGGCTAT CTCAACAGTA CCGGACGACG

2601	TGIICCCTTC CTGAGTGCCG CCAAAATCGG GCAGGATTAT TCTTTCTTCA

2651	CAAACATCGA AACCGACGGC GGCCTGCTGG CTTCCCTCGA CAGCGTCGAA

2701	AAAACAGCGG GCAGTGAAGG cGACACGCTG TCCTATTATG TCCGTCGCGG

2751	CAATGCGGCA CGGACTGCTT CGGCAGCGGC ACATTCCGCG CCCGCCGGTC

2801	TGAAACACGC CGTAGAACAG GGCGGCAGCA ATCTGGAAAA CCTGATGGTC

2851	GAACTGGATG CCTCCGAATC ATCCGCAACA CCCGAGACGG TTGAAACTGC

2901	GGCAGCCGAC CGCACAGATA TGCCGGGCAT CCGCCCCTAC GGCGCAACTT

2951	TCCGCGCAGC GGCAGCCGTA CAGCATGCGA ATGCCGCCGA CGGTGTACGC

3001	ATCTTCAACA GTCTCGCCGC TACCGTCTAT GCCGACAGTA CCGCCGCCCA

3051	TGCCGATATG CAGGGACGCC GCCTGAAAGC CGTATCGGAC GGGTTGGACC

3101	ACAACGGCAC GGGTCTGCGC GTCATCGCGC AAACCCAACA GGACGGTGGA

3151	ACGTGGGAAC AGGGCGGTGT TGAAGGCAAA ATGCGCGGCA GTACCCAAAC

3201	CGTCGGCATT GCCGCGAAAA CCGGCGAAAA TACGACAGCA GCCGCCACAC

3251	TGGGCATGGG AcGCAGCACA TGGAGCGAAA ACAGTGCAAA TGCAAAAACC

3301	GACAGCATTA GTCTGTTTGC AGGCATACGG CACGATGCGG GCGATATCGG

3351	CTATCTCAAA GGCCTGTTCT CCTACGGACG CTACAAAAAC AGCATCAGCC

3401	GcAGCAcCGG TGCGGACGAA CATGCGGAAG GCAGCGTCAA cGocAGGCTG

3451	ATGCAGCTGG GCGCACTGGG CGGTGTCAAC GTTCCGTTTG CCGCAACGGG

3501	AGATTTGACG GTCGAAGGCG GTCTGCGCTA CGACCTGCTC AAACAGGATG

3551	CATTCGCCGA AAAAGGCAGT GCTTTGGGCT GGAGOGGCAA CAGCCTCACT

3601	GAAGGCACGC TGGTCGGACT CGCGGGTCPG AAGCTGTCGC AACCCTTGAG

3651	CGATAAAGCC GTCCTGTTTG CAACGGGGGG CGTGGAACGC CACCTGAACG

3701	GACGCGACTA CACGGTAACG GGCGGCTTTA CCGGCGCGAC TGCAGCAACC

3751	GGCAAGACGG GGGCACGCAA TATGCCGCAC ACCCGTCTGG TTGCCGGCCT

3801	GGGCGCGGAT GTCGAATTCG GCAACGGCTG GAACGGCTTG GCACGTTACA

3851	GCTACGCCGG TTCCAAACAG TACGGCAACC ACAGCGGACG AGTCGGCGTA

3901	GGCTACCGGT TCCTCGAGCA CCACCACCAC CACCACTGA

1	MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT

51	YGNGDSLNTG KTANDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL

101	TAFQTEQIQD SEHSGKEVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA

151	FGSDDAGGKL TTTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRRA

201	VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL

251	EGSGGGGTsA pDFNAGGTGI GSNSRATTAX SAAVSYAGIK NEMCKDRSML

301	CAGRDDVAVT DRDAKINAPP PNGHTGDFPN PNDAYKNLIN LKPAIRAGYT

351	GRGVEVGIVD TGESVGSISF PRLYGRKEHG YNENYKNyTA YMRREAPEDG

401	GGKDIEASPD DEAVIETEAK PTDIREVREI GHIDLVSHII GGRSVDGRPA

451	GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSPGTTS

501	RAGTADLFQI ANSEEQYRQA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR

551	NKNMLFIPST GNDAQAQPNT YALLPFYEKD AQKGIITVAG VDRSGEKFKR

601	EMyGEPGTER LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIQIAGTSPS

651	APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGGLDA

701	GRAMNGPASF PFGDPTADTK GTSDIAYSFR NDISGTGGLI KRQGSQLQGH

751	GNNTYTGRTI IEGGSLVLYG NNKSDQRVET KGALIYNGAA SGGSLNSDGI

801	VYLADTDQSG ANETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY

851	MSARGKGAGY GNSTGRRVPP LSAAKIGQDY SFFTNIETDG GGLASLDSVE

901	KTAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLEHAVEQ GGSNLENLKV

951	ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV QHANAADGVR

1001	IFNSLAATVY ADSTAAHADM QGRRLRAVSD GLDHNGTGLR VIAQTQQDGG

1051	TWEQGGVEGK MRGSTQTVGI AAKTGENTTA AATLGMGRST WSENSANAKT

1101	DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL

1151	MQLGALGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEKGS ALGWSGNSLT

1201	EGTLVGLAGL RLSQPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT

1251	GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSRQ YGNHSGRVGV

1301	GYRFLEHHHH HH*

ΔG741-ORF46.1

1	ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC

51	GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG

101	TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT

151	TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT

201	CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA

251	CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA

301	ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT

351	GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT

401	CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG

451	TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC

501	CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA

551	ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC

601	GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC

651	CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG

701	TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC

751	GACGGTGGCG GAGGCACTGG ATCCTCAGAT TTGGCAAACG ATTCTTTTAT

901	CCGGCAGGTT CTCGACCGTC AGCATTTCGA ACCCGACGGG AAATACCACC

851	TATTCGGCAG CAGGGGGGAA CTTGCCGAGC GCAGCGGCCA TATCGGATTG

901	GGAAAAATAC AAAGCCATCA GTTGGGCAAC CTGATGATTC AACAGGCGGC

951	CATTAAAGGA AATATCGGCT ACATTGTCCG CTTTTCCGAT CACGGGCACG

1001	AAGTCCATTC CCCCTTCGAC AACCATGCCT CACATTCCGA TTCTGATGAA

1051	GCCGGTAGTC CCGTTGACGG ATTTAGCCTT TACCGCATCC ATTGGGACGG

1101	ATACGAACAC CATCCCGCCG ACGGCTATGA CGGGCCACAG GGCGGCGGCT

1151	ATCCCGCTCC CAAAGGCGCG AGGGATATAT ACAGCTACGA CATAAAAGGC

1201	GTTGCCCAAA ATATCCGCCT CAACCTGACC GACAACCGCA GCACCGGACA

1251	ACGGCTTGCC GACCGTTTCC ACAATGCCGG TAGTATGCTG ACGCAAGGAG

1302	TAGGCGACGG ATTCAAACGC GCCACCCGAT ACAGCCCCGA GCTGGACAGA

1351	TCGGGCAATG CCGCCGAAGC CTTCAACGGC ACTGCAGATA TCGTTAAAAA

1401	CATCATCGGC GCGGCAGGAG AAATTGTCGG CGCAGGCGAT GCCGTGCAGG

1451	GCATAAGGGA AGGCTCAAAC ATTGCTGTCA TGCACGGCTT GGGTCTGCTT

1501	TCCACCGAAA ACAAGATGGC GCGCATCAAC GATTTGGCAG ATATGGCGCA

1551	ACTCAAAGAC TATGCCGCAG CAGCCATCCG CGATTGGGCA GTCCAAAACC

1601	CCAATGCCGC ACAAGGCATA GAAGCCGTCA GCAATATCTT TATGGCAGCC

1651	ATCCCCATCA AAGGGATTGG AGCTGTTCGG GGAAAATACG GCPTGGGCG

1701	CATCACGGCA CATCCTATCA AGCGGTCGCA GATGGGCGCG ATCGCATTGC

1751	CGAAAGGGAA ATCCGCCGTC AGCGACAATT TTGCCGATGC GGCATACGCC

1801	AAATACCCGT CCCCTTACCA TTCCCGAAAT ATCCGTTCAA ACTTGGAGCA

1851	GCGTTACGGC AAAGAAAACA TCACCTCCTC AACCGTGCCG CCGTCAAACG

1901	GCAAAAATGT CAAACTGGCA GACCAACGCC ACCCGAAGAC AGGCGTACCG

1951	TTTGACGGTA AAGGGTTTCC GAATTTTGAG AAGCACGTGA AATATGATAC

2001	GCTCGAGCAC CACCACCACC ACCACTGA

1	MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT

51	YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL

101	TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA

151	FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA

201	VISGSVLYNQ ABEGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL

251	DGGGGTGSSD LANDSFIRQV LDRQHFEPDG EYELFGSRGE LAERSGHIGL

301	GKIQSHQLGN LMIQQAAIKG NIGYIVRFSD HGHEVHSPFD NHASHSDSDE

351	AGSPVDGFSL YRIHWDGYER HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG

401	VAQNIKANLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR

451	SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN IAVMHQLGLL

501	STENKMARIN DIADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA

551	IPIKGIGAVR GKYGLGGITA HPIKRSQMGA IALPKGKSAV SDNFADAAYA

601	KYPSPYHSRN IRSNLEQRYG KENITSSTVP PSNGKNVKLA DQRHPKTGVP

651	FDGKGFPNFE KHVKYDTLEH HHHHH*

Example 16—C-Terminal Fusions (‘Hybrids’) with 287/ΔG287

According to the invention, hybrids of two proteins A & B may be either NH₂-A-B-COOH or NH₂-B-A-COOH. The effect of this difference was investigated using protein 287 either C-terminal (in ‘287-His’ form) or N-terminal (in ΔG287 form sequences shown above) to 919, 953 and ORF46.1. A panel of strains was used, including homologous strain 2996. FCA was used as adjuvant:


	287 & 919	287 & 953	287 & ORF46.1

Strain	ΔG287-919	919-287	ΔG287-953	953-287	ΔG287-46.1	46.1-287

2996	128000	16000	65536	8192	16384	8192
BZ232	256	128	128	<4	<4	<4
1000	2048	<4	<4	<4	<4	<4
MC58	8192	1024	16384	1024	512	128
NGH38	32000	2048	>2048	4096	16384	4096
394/98	4096	32	256	128	128	16
MenA (F6124)	32000	2048	>2048	32	8192	1024
MenC (BZ133)	64000	>8192	>8192	<16	8192	2048

Better bactericidal titres are generally seen with 287 at the N-terminus m the ΔG form)
When fused to protein 961 [NH₂-ΔG287-961-COOH sequence shown above], the resulting protein is insoluble and must be denatured and renatured for purification. Following renaturation, around 50% of the protein was found to remain insoluble. The soluble and insoluble proteins were compared, and much better bactericidal titres were obtained with the soluble protein (FCA as adjuvant):


2996	BZ232	MC58	NGH38	F6124	BZ133

Soluble	65536	128	4096	>2048	>2048	4096
Insoluble	8192	<4	<4	16	n.d.	n.d.

Titres with the insoluble form were, however, improved by using alum adjuvant instead:


Insoluble	32768	128	4096	>2048	>2048	2048

Example 17—N-Terminal Fusions ‘Hybrids’) to 287

Expression of protein 287 as full-length with a C-terminal His-tag, or without its leader peptide but with a C-terminal His-tag, gives fairly low expression levels. Better expression is achieved using a N-terminal GST-fusion.
As an alternative to using GST as an N-terminal fusion partner, 287 was placed at the C-terminus of protein 919 (‘919-287’), of protein 953 (‘953-287’), and of proteins ORF46.1 (‘ORF46.1-287’). In both cases, the leader peptides were deleted, and the hybrids were direct in-frame fusions.
To generate the 953-287 hybrid, the leader peptides of the two proteins were omitted by designing the forward primer downstream from the leader of each sequence; the stop codon sequence was omitted in the 953 reverse primer but included in the 287 reverse primer. For the 953 gene, the 5′ and the 3′ primers used for amplification included a NdeI and a BamHI restriction sites respectively, whereas for the amplification of the 287 gene the 5′ and the 3′ primers included a BamHI and a XhoI restriction sites respectively. In this way a sequential directional cloning of the two genes in pET21b+, using NdeI-BamHI (to clone the first gene) and subsequently BamHI-XhoI (to clone the second gene) could be achieved.
The 919-287 hybrid was obtained by cloning the sequence coding for the mature portion of 287 into the XhoI site at the 3′-end of the 919-His clone in pET21b+. The primers used for amplification of the 287 gene were designed for introducing a SalI restriction site at the 5′- and a XhoI site at the 3′- of the PCR fragment. Since the cohesive ends produced by the SalI and XhoI restriction enzymes are compatible, the 287 PCR product digested with SalI-XhoI could be inserted in the pET21b-919 clone cleaved with XhoI.
The ORF46.1-287 hybrid was obtained similarly.
The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens:


		Mixture with 287	Hybrid with 287

919	32000	16000
953	8192	8192
ORF461	128	8192

Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287:


	919	953	ORF46.1

Strain	Mixture	Hybrid	Mixture	Hybrid	Mixture	Hybrid

MC58	512	1024	512	1024	—	1024
NGH38	1024	2048	2048	4096	—	4096
BZ232	512	128	1024	16	—	—
MenA (F6124)	512	2048	2048	32	—	1024
MenC (C11)	>2048	n.d.	>2048	n.d.	—	n.d.
MenC (BZ133)	>4096	>8192	>4096	<16	—	2048

Hybrids of ORF46.1 and 919 were also constructed. Best results (four-fold higher titre) were achieved with 919 at the N-terminus.
Hybrids 919-519His, ORF97-225His and 225-ORF97His were also tested. These gave moderate ELISA fitres and bactericidal antibody responses.

Example 18—the Leader Peptide from ORF4

As shown above, the leader peptide of ORF4 can be fused to the mature sequence of other proteins (e.g. proteins 287 and 919). It is able to direct lipidation in E. coli.

Example 19—Domains in 564

The protein ‘564’ is very large (2073aa), and it is difficult to clone and express it in complete form. To facilitate expression, the protein has been divided into four domains, as shown in FIG. 8 (according to the MC58 sequence):


		Domain

	A	B	C	D

Amino Acids	79-360	361-731	732-2044	2045-2073

These domains show the following homologies:

- Domain A shows homology to other bacterial toxins:


gb\|AAG03431.1\|AE004443_9	probable hemagglutinin
	[Pseudomonas aeruginosa] (38%)
gb\|AAC31981.1\|(139897)	HecA
	[Pectobacterium chrysanthemi] (45%)
emb\|CAA36409.1\|(X52156)	filamentous hemagglutinin
	[Bordetella pertussis] (31%)
gb\|AAC79757.1\|(AF057695)	large supernatant protein1
	[Haemophilus ducreyi] (26%)
gb\|AAA25657.1\|(M30186)	HpmA precursor
	[Proteus mirabilis] (29%)

- Domain B shows no homology, and is specific to 564.
- Domain C shows homology to:


gb\|AAF84995.1\|AE004032	HA-like secreted protein
	[Xylella fastidiosa] (33%)
gb\|AAG05850.1\|AE004673	hypothetical protein
	[Pseudomonas aeruginosa] (27%)
gb\|AAF68414.1AF237928	putative FHA
	[Pasteurella multocisida] (23%)
gb\|AAC79757.1\|(AF057695)	large supernatant protein1
	[Haemophilus ducreyi] (23%)
pir\|\|S21010	FHA B precursor
	[Bordetella pertussis] (20%)

- Domain D shows homology to other bacterial toxins:
  - gb|AAF84995.1|AE004032_14 HA-like secreted protein [Xylella fastidiosa] (29%)

Using the MC58 strain sequence, good intracellular expression of 564ab was obtained in the form of GST-fusions (no purification) and his-tagged protein; this domain-pair was also expressed as a lipoprotein, which showed moderate expression in the outer membrane/supernatant fraction.
The b domain showed, moderate intracellular expression when expressed as a his-tagged product (no purification), and good expression as a GST-fusion.
The c domain showed good intracellular expression as a GST-fusion, but was insoluble. The d domain showed moderate intracellular expression as a his-tagged product (no purification). The cd protein domain-pair showed moderate intracellular expression (no purification) as a GST-fusion,
Good bactericidal assay titres were observed using the c domain and the be pair.

Example 20—the 919 Leader Peptide

The 20mer leader peptide from 919 is discussed in example 1 above:

- MKKYLFRAAL YGIAAAYLAA

As shown in example 1, deletion of this leader improves heterologous expression, as does substitution with the ORF4 leader peptide. The influence of the 919 leader on expression was investigated by fusing the coding sequence to the PhoC reporter gene from Morganella morganii [Thaller et al. (1994) Microbiology 140:1341-1350]. The construct was cloned in the pET21-b plasmid between the NdeI and XhoI sites (FIG. 9):

1	MKKYLFRAAL YGIAAAILAA AIPAGNDATT KPDLYYLKNE QAIDSLKLLP

51	PPPEVGSIQF LNDQAMYEKG RMLRNTERGK QAQADADLAA GGVATAFSGA

101	FGYPITEKDS PELYELLTNM IEDAGDLATR SAKEHYMRIR PFAFYGTETC

151	NTKDQKKLST NGSYPSGHTS IGWATALVLA EVNPANQDAI LERGYQLGQS

201	RVICGYHWQS DVDAARIVGS AAVATLHSDP AFQAQLAKAK QEFAQKSQK*

The level of expression of PhoC from this plasmid is >200-fold lower than that found for the same construct but containing the native PhoC signal peptide. The same result was obtained even after substitution of the T7 promoter with the E. coli Plac promoter. This means that the influence of the 919 leader sequence on expression does not depend on the promoter used.
In order to investigate if the results observed were due to some peculiarity of the 919 signal peptide nucleotide sequence (secondary structure formation, sensitivity to RNAases, etc.) or to protein instability induced by the presence of this signal peptide, a number of mutants were generated. The approach used was a substitution of nucleotides of the 919 signal peptide sequence by cloning synthetic linkers containing degenerate codons. In this way, mutants were obtained with nucleotide and/or amino acid substitutions.
Two different linkers were used, designed to produce mutations in two different regions of the 919 signal peptide sequence, in the first 19 base pairs (L1) and between bases 20-36 (S1).

L1:

5′ T ATG AAa/g TAc/t c/tTN TTt/c a/cGC GCC GCC CTG

TAC GGC ATC GCC GCC GCC ATC CTC GCC GCC GCG ATC CC

3′

S1:

5′ T ATG AAA AAA TAC CTA TTC CGa/g GCN GCN c/tTa/g

TAc/t GGc/g ATC GCC GCC GCC ATC CTC GCC GCC CCC ATC

CC

3′

The alignment of some of the mutants obtained is given below.

L1 mutants:

9L1-a	ATGAAGAAGTACCTTTTCAGCGCCGCC---------------------------------

9L1-e	ATGAAATAATACTTTTTCCGCGCCGCC---------------------------------

9L1-d	ATGAAAAAATACTTTTTCCGCGCCGCC---------------------------------

9L1-f	ATGAAAAAATATCTCTTTAGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC

919sp	ATGAAAAAATACCTATTCCGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC

9L1a	MKKYLFSAA--------

9L1e	MKKYFFRAA--------

9L1d	MKKYFFRAA--------

9L1f	MREYLFSAALYGIAAAILAA

919sp	MKKYLFRAALYGIAAAILAA (i.e. native signal peptide)

S1 mutants:

9S1-e	ATGAAAAAATACCTATTC..................ATCGCCGCCGCCATCCTCGCCGCC

9S1-c	ATGAAAAAATACCTATTCCGAGCTGCCCAATACGGCATCGCCGCCGCCATCCTCGCCGCC

9S1-b	ATGAAAAAATACCTATTCCGGGCCGCCCAATACGGCATCGCCGCCGCCATCCTCGCCGCC

9S1-i	ATGAAAAAATACCTATTCCGGGCGGCTTTGTACGGGATCGCCGCCGCCATCCTCGCCGCC

919sp	ATGAAAAAATACCTATTCCGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC

9S1e	MKKYLF......IAAAILAA

9S1c	MKKYLFRAAQYGIAAAILAA

9S1b	MKKYLFRAAQYGIAAAILAA

9S1i	MKKYLFRAALYGIAAAIIAA

919sp	MKKYLFRAALYGIAAAILAA

As shown in the sequences alignments, most of the mutants analysed contain in-frame deletions which were unexpectedly produced by the host cells.
Selection of the mutants was performed by transforming E. coli BL21(DE3) cells with DNA prepared from a mixture of L1 and S1 mutated clones. Single transformants were screened for high PhoC activity by streaking them onto LB plates containing 100 μg/ml ampicillin, 50 μg/ml methyl green, 1 mg/ml PDP (phenolphthaleindiphosphate). On this medium PhoC-producing cells become green (FIG. 10).
A quantitative analysis of PhoC produced by these mutants was carried out in liquid medium using pNPP as a substrate for PhoC activity. The specific activities measured in cell extracts and supernatants of mutants grown in liquid medium for 0, 30, 90, 180 min. were:

Cell Extracts


0	30	90	180

control	0.00	0.00	0.00	0.00
9phoC	1.11	1.11	3.33	4.44
9S1e	102.12	111.00	149.85	172.05
9L1a	206.46	111.00	94.35	83.25
9L1d	5.11	4.77	4.00	3.11
9L1f	27.75	94.35	82.14	36.63
9S1b	156.51	111.00	72.15	28.86
9S1c	72.15	33.30	21.09	14.43
9S1i	156.51	83.25	55.50	26.64
phoCwt	194.25	180.93	149.85	142.08

Supernatants


0	30	90	180

control	0.00	0.00	0.00	0.00
9phoC	0.33	0.00	0.00	0.00
9S1e	0.11	0.22	0.44	0.89
9L1a	4.88	5.99	5.99	7.22
9L1d	0.11	0.11	0.11	0.11
9L1f	0.11	0.22	0.11	0.11
9S1b	1.44	1.44	1.44	1.67
9S1c	0.44	0.78	0.56	0.67
9S1i	0.22	0.44	0.22	0.78
phoCwt	34.41	43.29	87.69	177.60

Some of the mutants produce high amounts of PhoC and in particular, mutant 9L1a can secrete PhoC in the culture medium. This is noteworthy since the signal peptide sequence of this mutant is only 9 amino acids long. This is the shortest signal peptide described to date.

Example 21—C-Terminal Deletions of Maf-Related Proteins

MafB-related proteins include 730, ORF46 and ORF29.
The 730 protein from MC58 has the following sequence:

1	VKPLRRLTNL LAACAVAAAA LIQPALAADL AQDPFITDNA QRQHYEPGGK

51	YHLFGDPRGS VSDRTGKINV IQDYTHQMGN LLIQQANING TIGYHTRFSG

101	HGHEEHAPFD NHAADSASEE KGNVDEGFTV YRLNWEGHEH HPADAYDGPK

151	GGNYPKPTGA RDEYTYHVNG TARSIKLNPT DTRSIRQRIS DNYSNLGSNF

201	SDRADEANRK MFEHNAKLDR WGNSMEFING VAAGALNPFI SAGEALGIGD

251	ILYGTRYAID KAAMRNIAPL PAEGKFAVIG GLGSVAGFEK NTREAVDRWI

301	QENPNAAETV EAVFNVAAAA KVAKLAKAAK PGKAAVSGDF ADSYKKKLAL

351	SDSARQLYQN AKYREALDIH YEDLIRRKTD GSSKFINGRE IDAVTNDALI

401	QAKRTISAID KPKNFLNQKN RKQIKATIEA ANQQGKRAEF WFKYGVHSQV

451	KSYIESKGGI VXTGLGD*

The leader peptide is underlined.
730 shows similar features to ORF46 (see example 8 above):

- as for Orf46, the conservation of the 730 sequence among MenB, MenA and gonococcus is high (>80%) only for the N-terminal portion. The C-terminus, from ˜340, is highly divergent.
- its predicted secondary structure contains a hydrophobic segment spanning the central region of the molecule (aa. 227-247).
- expression of the full-length gene in E. coli gives very low yields of protein. Expression from tagged or untagged constructs where the signal peptide sequence has been omitted has a toxic effect on the host cells. In other words, the presence of the full-length mature protein in the cytoplasm is highly toxic for the host cell while its translocation to the periplasm (mediated by the signal peptide) has no detectable effect on cell viability. This “intracellular toxicity” of 730 is particularly high since clones for expression of the leaderless 730 can only be obtained at very low frequency using a recA genetic background (E. coli strains: HB101 for cloning; HMS174(DE3) for expression).

To overcome this toxicity, a similar approach was used for 730 as described in example 8 for ORF46. Four C-terminal truncated forms were obtained, each of which is well expressed. All were obtained from intracellular expression of His-tagged leaderless 730.
Form A consists of the N-terminal hydrophilic region of the mature protein (aa. 28-226). This was purified as a soluble His-tagged product, having a higher-than-expected MW.
Form B extends to the end of the region conserved between serogroups (aa. 28-340). This was purified as an insoluble His-tagged product.
The C-terminal truncated forms named C1 and C2 were obtained after screening for clones expressing high levels of 730-His clones in strain HMS174(DE3). Briefly, the pET21b plasmid containing the His-tagged sequence coding for the full-length mature 730 protein was used to transform the recA strain HMS174(DE3). Transformants were obtained at low frequency which showed two phenotypes: large colonies and very small colonies. Several large and small colonies were analysed for expression of the 730-His clone. Only cells from large colonies over-expressed a protein recognised by anti-730A antibodies. However the protein over-expressed in different clones showed differences in molecular mass. Sequencing of two of the clones revealed that in both cases integration of an E. coli IS sequence had occurred within the sequence coding for the C terminal region of 730. The two integration events have produced in-frame fusion with 1 additional codon in the case of C1, and 12 additional codons in the case of C2 (FIG. 11). The resulting “mutant” forms of 730 have the following sequences:

730-C1 (due to an IS1 insertion-fig. 11A)

1	MADLAQDPFI TDMAQRQHYE PGGKYHLFGD PRGSVSDRTG KINVIQDYTH

51	QMGNLLIQQA NINGTIGYET RFSGEGHEEM APFDNHAADS ASEEKGNVDE

101	GFTVYRLNWE GHEHMPADAY DGPKGGNYPK PTGARDEYTY HVNGTARSIK

151	LNPTDTRSIR QRISDNYSNL GSNFSDRADE ANRKMFEENA KLDRWGNSME

201	FINGVAAGAL NPFISAGEAL GIGDEDYGTR YAIDKAAMRN IAPLPAEGKF

251	AVIGGLGSVA GFEKNTREAV DRWIQENPNA AETVEAVFNV AAAAKVAKLA

301	KAAKPGKAAV SGDFADSYKK KLALSDSARQ LYQNAKYREA DDIEYEDLIK

351	RKTDGSSKFI NGREIDAVTN DALIQAR*

The additional amino acid produced by the insertion is underlined.

730-C2 (due to an IS5 insertion-Fig. 11B)

1	MADLAQDPFI TDNAQRQHYE PGGKYHLFGD PRGSVSDRTG KINVIQDYTH

51	QMGNLLIQQA NINGTIGYHT RFSGHGHEEH APFDNEAADS ASEEKGNVDE

101	GFTVYRLNWE GBEHHPADAY DGPKGGNYPK PTGARDEYTY HVNGTARSIK

151	LNPTDTRSIR QRISDNYSNL GSNFSDRADE ANRKMFEHNA KLDRWGNSME

201	FINGVAAGAL NPFISAGEAL GIGDILYGTR YAIDKAAMEN IAPLPAEGKE

251	AVIGGLGSVA GFEKNTREAV DRWIQENPNA AETVEAVFNV AAAAKVAKLA

301	KAAKPGKAAV SGDFADSYKK KLALSDSARQ LYQNAKYREA LGKVRISGEI

351	LLG*

The additional amino acids produced by the insertion are underlined.
In conclusion, intracellular expression of the 730-C1 form gives very high level of protein and has no toxic effect on the host cells, whereas the presence of the native C-terminus is toxic. These data suggest that the “intracellular toxicity” of 730 is associated with the C-terminal 65 amino acids of the protein.
Equivalent truncation of ORF29 to the first 231 or 368 amino acids has been performed, using expression with or without the leader peptide (amino acids 1-26; deletion gives cytoplasmic expression) and with or without a His-tag.

Example 22—Domains in 961

As described in example 9 above, the OST-fusion of 961 was the best-expressed in Exoli. To improve expression, the protein was divided into domains (FIG. 12).
The domains of 961 were designed on the basis of YadA (an adhesin produced by Yersinia which has been demonstrated to be an adhesin localized on the bacterial surface that forms oligomers that generate surface projection [Hoiczyk et al. (2000) EMBO J 19:5989-99]) and are: leader peptide, head domain, coiled-coil region (stalk), and membrane anchor domain.
These domains were expressed with or without the leader peptide, and optionally fused either to C-terminal His-tag or to N-terminal GST. Exalt: clones expressing different domains of 961 were analyzed by SDS-PAGE and western blot for the production and localization of the expressed protein, from over-night (o/n) culture or after 3 hours induction with IPTG. The results were:


	Total lysate	Periplasm	Supernatant	OMV
	(Western	(Western	(Western	SDS-
	Blot)	Blot)	Blot)	PAGE

961 (o/n)	−	−	−
961 (IPTG)	+/−	−	−
961-L (o/n)	+	−	−	+
961-L (IPTG)	+	−	−	+
961c-L (o/n)	−	−	−
961c-L (IPTG)	+	+	+
961Δ₁-L (o/n)	−	−	−
961Δ₁-L (IPTG)	+	−	−	+

The results show that in E. coli:

- 961-L is highly expressed and localized on the outer membrane. By western blot analysis two specific bands have been detected: one at ˜45 kDa (the predicted molecular weight) and one at ˜180 kDa, indicating that 961-L can form oligomers. Additionally, these aggregates are more expressed in the over-night culture (without IPTG induction). OMV preparations of this clone were used to immunize mice and serum was obtained. Using overnight culture (predominantly by oligomeric form) the serum was bactericidal; the IPTG-induced culture (predominantly monomeric) was not bactericidal.
- 961Δ₁-L (with a partial deletion in the anchor region) is highly expressed and localized on the outer membrane, but does not form oligomers;
- the 961c-L (without the anchor region) is produced in soluble form and exported in the supernatant.

Titres in ELISA and in the serum bactericidal assay using His-fusions were as follows:


		ELISA	Bactericidal

961a (aa 24-268)	24397	4096
961b (aa 269-405)	7763	64
961c-L	29770	8192
961c (2996)	30774	>65536
961c (MC58)	33437	16384
961d	26069	>65536

E. coli clones expressing different forms of 961 (961, 961-L, 961Δ₁-L and 961c-L) were used to investigate if the 961 is an adhesin (c.f. YadA). An adhesion assay was performed using (a) the human epithelial cells and (b) E. coli clones after either over-night culture or three hours IPTG induction. 961-L grown over-night (961Δ₁-L) and IPTG-induced 961c-L (the clones expressing protein on surface) adhere to human epithelial cells.
961c was also used in hybrid proteins (see above). As 961 and its domain variants direct efficient expression, they are ideally suited as the N-terminal portion of a hybrid protein.

Example 23—Further Hybrids

Further hybrid proteins of the invention are shown below (see also FIG. 14). These are advantageous when compared to the individual proteins:

ORF46.1-741

1	ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA

51	GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC

101	TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG

151	TTGGGCAACC TGATGANTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA

201	CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA

251	ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA

301	TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA

351	CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA

401	GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC

451	AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA

501	CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG

551	CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC

601	TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA

651	AATTGTCGGC GCAGGCGATG CCGTGCAGOG CATAAGCGAA GGCTCAAACA

701	TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG

751	CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC

801	AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG

851	AAGCCGTCAG CAATATCTTT ATGGCASCCA TCCCCATCAA AGGGATTGGA

901	GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA

951	GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA

2001	GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT

1051	TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT

1101	CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG

1151	ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG

1201	AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GGGGTGGTGT

1251	CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA TGCACTAACC GCACCGCTCG

1301	ACCATAAAGA CAAAGGTTTG CAGTCTTTGA CGCTGGATCA GTCCGTCAGG

1351	AAAAACGAGA AACTGAAGCT GGCGGCACAA GGTGCGGAAA AAACTTATGG

1401	AAACGGTGAC AGCCTCAATA CGGGCAAATT GAAGAACGAC AAGGTCAGCC

1451	GTTTCGACTT TATCCGCCAA ATCGAAGTGG ACGGGCAGCT CATTACCTTG

1501	GAGAGTGGAG AGTTCCAAGT ATACAAACAA AGCCATTCCG CCTTAACCGC

1551	CTTTCAGACC GAGCAAATAC AAGATTCGGA GCATTCCGGG AAGATGGTTG

1601	CGAAACGCCA GTTCAGAATC GGCGACATAG CGGGCGAACA TACATCTTTT

1651	GACAAGCTTC CCGAAGGCGG CAGGGCGACA TATCGCGGGA CGGCGTTCGG

1701	TTCAGACGAT GCCGGCGGAA AACTGACCTA CACCATAGAT TTCGCCGCCA

1751	AGCAGGGAAA CGGCAAAATC GAACATTTGA AATCGCCAGA ACTCAANGTC

1801	GACCTGGCCG CCGCCGATAT CAAGCCGGAT GGAAAACGCC ATGCCGTCAT

1851	CAGCGGTTCC GTCCTTTACA ACCAAGCCGA GAAAGGCAGT TACTCCCTCG

1901	GTATCTTTGG CGGAAAAGCC CAGGAAGTTG CCGGCAGCGC GGAAGTGAAA

1951	ACCGTAAACG GCATACGCCA TATCGGCCTT GCCGCCAAGC AACTCGAGCA

2001	CCACCACCAC CACCACTGA

1	MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ

51	AGNIMIQQAA IKGNIGY1VR FSDHGERVHS PFDNHASHSD SDEAGSPVDG

101	FSLYRIHWDG YEHHPAEGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL

151	NATDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA

201	FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTFMKKA

251	RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPEKGIG

301	AVRGKYGIGG ITAEPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH

351	SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP

401	NFEKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR

451	KNERLKLAAQ GAEKTYGNGD SLNTGKLKND KVSREDFIRQ IEVEGQLITL

501	ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF

551	DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGEI EHLKSPELNV

601	DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK

651	TVNGIRHIGL AAKQLEHHHH HH*

ORF46.1-961

1	ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA

51	GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC

101	TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG

151	TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA

201	CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA

251	ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA

301	TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA

351	CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA

401	GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC

451	AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA

501	CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG

551	CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC

601	TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAaA

651	AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA

701	TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG

751	CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC

801	AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCTTAG

851	AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA

901	GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA

951	GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA

1001	GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT

1051	TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT

1101	CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG

1151	ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG

1201	AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GAGGAGGAGC

1251	CACAAACGAC GACGATGTTA AAAAAGCTGC CACTGTGGCC ATTGCTGCTG

1301	CCTACAACAA IGGCCAAGAA ATCAACGGTT TCAAAGCTGG AGAGACCATC

1351	TACGACATTG ATGAAGACGG CACAATTACC AAAAAAGACG CAACTGCAGC

1401	CGATGTTGAA GCCGACGACT TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA

1451	CTAACCTGAC CAAAACCGTC AATGAAAACA AACAAAACGT CGATGCCAAA

1501	GTAAAAGCTG CAGAATCTGA AATAGAAAAG TTAACAACCA AGTTAGCAGA

1551	CACTGATGCC GCTTTAGCAG ATACTGATGC CGCTCTGGAT GCAACCACCA

1601	ACGCCTTGAA TAAATTGGGA GAAAATATAA CGACATTTGC TGAAGAGACT

1651	AAGACAAATA TCGTAAAAAT TGATGAAAAA TTAGAAGCCG TGGCTGATAC

1701	CGTCGACAAG CATGCCGAAG CATTCAACGA TATCGCCGAT TCATTGGATG

1751	AAACCAACAC TAAGGCAGAC GAAGCCGTCA AAACCGCCAA TGAAGCCAAA

1801	CAGACGGCCG AAGAAACCAA ACAAAACGTC GATGCCAAAG TAAAAGCTGC

1851	AGAAACTGCA GCAGGCAAAG CCGAAGCTGC CGCTGGCACA GCTAATACTG

1901	CAGCCGACAA GGCCGAAGCT GTCGCTGCAA AAGTTACCGA CATCAAAGCT

1951	GATANCGCTA CGAACAANGA TAATATTGCT AAAAAAGCAA ACAGTGCCGA

2001	CGTGTACACC AGAGAAGAGT CTGACAGCAA ATTTGTCAGA ATTGATGGTC

2051	TGAACGCTAC TACCGAAAAA TTGGACACAC GCTTGGCTTC TGCTGAAAAA

2101	TCCATTGCCG ATCACGATAC TCGCCTGAAC GGTTTGGATA AAACAGTGTC

2151	AGACCTGCGC AAAGAAACCC GCCAAGGCCT TGCAGAACAA GCCGCGCTCT

2201	CCGGTCTGTT CCAACCTTAC AACGTGGGTC GGTTCAATGT AACGGCTGCA

2251	GTCGGCGGCT ACAAATCCGA ATCGGCAGTC GCCATCGGTA CCGGCTTCCG

2301	CTTTACCGAA AACTTTGCCG CCAAAGCAGG CGTGGCAGTC GGCACTTCGT

2351	CCGGTTCTTC CGCAGCCTAC CATGTCGGCG TCAATTACGA GTGGCTCGAG

2401	CACCACCACC ACCACCACTG A

1	MSDLANDSFI RQVLDRQHFE PDGKYHAFGS RGELAERSGH IGLGK1QSHQ

51	LGNLMIQQAA IKGNIGYIVR FSDHGREVHS PFDNEASRSD SDEAGSPVDG

101	FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQMIRL

151	NLTDNRSTGQ RLADRFRNAG SMLTQGVGDG PKRATRYSPE LDRSGMAAFA

201	FNGTADIVKM IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA

251	EINDLADMAQ LKDYAAAA1R DWAVQNPNAA QGIFAVSNIF MAAIPIKGIG

301	AVRGKYGLGG ITAHPIKRSQ NGAIALPKGK SAVSDNFADA AYAKYPSPYR

351	SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP

401	NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI

451	YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK

501	VKAAESEIEK LTTKLADTDA ALADTDAALD ATTMALNKLG ENITTFARET

551	KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK

601	QTARKTKQNV DAKVKAASTA AGKAEAAAGT ANTAADKAEA VAAKVTDTKA

651	DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK

701	SIADHDTRLN GLDKTVHDLR KETRQGLAEQ AALSGLFQVY NVGRFNVTAA

751	VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY

801	HEHHHH*

ORF46.1-961c

1	ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA

51	GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC

101	TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG

151	TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAPAGGAA ATATCGGCTA

201	CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA

251	ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGAOGGA

301	TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA

351	CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA

401	GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC

451	AACCTGACCG ACAACCGCAG CACCGGACAA CGGCPTGCCG ACCGTTTCCA

501	CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG

551	CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC

601	TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA

651	AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA

701	TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG

751	CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC

801	AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG

851	AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATAGGA

901	GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA

951	GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA

1001	GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT

1051	TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT

1101	CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG

1151	ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG

1201	AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GAGGAGGAGC

1251	CACAAACGAC GACGATGTTA AAAAAGCTGC CACTGTGGCC ATTGCTGCTG

1301	CCTACAACAA TGGCCAAGAA ATCAACGGTT TCAAAGCTGG AGAGACCATC

1351	TACGACATTG ATGAAGACGG CACAATTACC AAAAAAGACG CAACTGCAGC

1401	CGATGTTGAA GCCGACGACT TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA

1451	CTAACCTGAC CAAAACCGTC AATGAAAACA AACAAAACGT CGATGCCAAA

1501	GTAAAAGCTG CAGAATCTGA AATAGAAAAG TTAACAACCA AGTTAGCAGA

1551	CACTGATGCC GCTTTAGCAG ATACTGATGC CGCTCTGGAT GCAACCACCA

1601	ACGCCTTGAA TAAATTGGGA GAAAATATAA CGACATTTGC TGAAGAGACT

1651	AAGACAAATA TCGTAAAAAT TGATGAAAAA TTAGAAGCCG TGGCTGATAC

1701	CGTCGACAAG CATGCCGAAG CATTCAACGA TATCGCCGAT TCATTGGATG

1751	AAACCAACAC TAAGGCAGAC GAAGCCGTCA AAACCGCCAA TGAAGCCAAA

1801	CAGACGGCCG AAGAAACCAA ACAAAACGTC GATGCCAAAG TAAAAGCTGC

1851	AGAAACTGCA GCAGGCAAAG CCGAAGCTGC CGCTGGCACA GCTAATACTG

1901	CAGCCGACAA GGCCGAAGCT GTCGCTGCAA AAGTTACCGA CATCAAAGCT

1951	GATATCGCTA CGAACAAAGA TAATATTGCT AAAAAAGCAA ACAGTGCCGA

2001	CGTGTACACC AGAGAAGAGT CTGACAGCAA ATTTGTCAGA ATTGATGGTC

2051	TGAACGCTAC TACCGAAAAA TTGGACACAC GCTTGGCTTC TGCTGAAAAA

2101	TCCATTGCCG ATCACGATAC TCGCCTGAAC GGTTTGGATA AAACAGTGTC

2151	AGACCTGCGC AAAGAAACCC GCCAAGGCCT TGCAGAACAA GCCGCGCTCT

2201	CCGGTCTGTT CCAACCTTAC AACGTGGGTC TCGAGCACCA CCACCACCAC

2251	CACTGA

1	MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSEQ

51	LGNLMIQQAA IKGNIGYIVR FSDHGREVHS PFDNHASHSD SDEAGSPVDG

101	FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL

151	NLTDNRSTGQ RLADRFHNAG SNLTQGVGDG FKRATRYSPE LDRSGMAAEA

201	FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVNEGL GLLSTENKMA

251	RINDLADNAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG

301	AVRGKYGAGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH

351	SRNIRSNLBQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP

401	NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI

451	YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK

501	VKAAESEIMK LTTKLADTDA ALADTDAALD ATTNALNKLG HNITTFAEET

551	KTNrVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD HAVKTANEAK

601	QTAFETKQNV DAKVKAAETA AGKAFAAAGT ANTAADKAHA VAAKVTDIKA

651	DIATNEDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK

701	SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGLEHHHHH

751	H*

961-ORF46.1

1	ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC

51	TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA

101	CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT

151	GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT

201	CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG

251	CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA

301	GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC

351	CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG

401	AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT

451	GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT

501	GGATCAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG

551	CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA

601	GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA

651	TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA

701	AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT

751	GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA

801	TCGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG

851	AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA

901	GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC

951	GCTCTCCGGT CTGITCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG

1001	CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCCGC

1051	TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC

1101	TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG

1151	GATCCGGAGG AGGAGGATCA GATTTGGCAA ACGATTCTTT TATCCGGCAG

1201	GTTCTCGACC GTCACCATTT CGAACCCGAC GGGAAATACC ACCTATTCGG

1251	CAGCAGGGGG GAACTTGCCG AGCGCAGCGG CCATATCGGA TTGGGAAAAA

1301	TACAAAGCCA TCAGTTGGGC AACCTGATGA TTCAACAGGC GGCCATTAAA

1351	GGAAATATCG GCTACATTGT CCGCTTTTCC GATCACGGGC ACGAAGTCCA

1401	TTCCCCCTTC GACAACCATG CCTCACATTC CGATTCTGAT GAAGCCGGTA

1451	GTCCCGTTGA CGGATTTAGC CTTTACCGCA TCCATTGGGA CGGATACGAA

1501	CACCATCCCG CCGACGGCTA TGACGGGCCA CAGGGCGGCG GCTATCCCGC

1551	TCCCAAAGGC GCGAGGGATA TATACAGCTA CGACATAAAA GGCGTTGCCC

1601	AAAATATCCG CCTCAACCTG ACCGACAACC GCAGCACCGG ACAACGGCTT

1651	GCCGACCGTT TCCACAATGC CGGTAGTATG CTGACGCAAG GAGTAGGCGA

1701	CGGATTCAAA CGCGCCACCC GATACAGCCC CGAGCTGGAC AGATCGGGCA

1751	ATGCCGCCGA AGCCTTCAAC GGCACTGCAG ATATCGTTAA AAACATCATC

1801	GGCGCGOCAG GAGAAATTGT CGGCGCAGGC GATGCCGTGC AGGGCATAAG

1851	CGAAGGCTCA AACATTGCTG TCATGCACGG CTTGGGTCTG CTTTCCACCG

1901	AAAACAAGAT GGCGCGCATC AACGATTTGG CAGATATGGC GCAACTCAAA

1951	GACTATGCCG CAGCAGCCAT CCGCGATIGG GCAGTCCAAA ACCCCAATGC

2001	CGCACAAGGC ATAGAAGCCG TCAGCAATAT CTTTATGGCA GCCATCCCCA

2051	TCAAAGGGAT TGGAGCTGTT CGGGGAAAAT ACGGCTTGGG CGGCATCACG

2101	GCACATCCTA TCAAGCGGTC GCAGATGGGC GCGATCGCAT TGCCGAAAGG

2151	GAAATCCGCC GTCAGCGACA ATTTTGCCGA TGCGGCATAC GCCAAATACC

2201	CGTCCCCTTA CCATTCCCGA AATATCCGTT CAAACTTGGA GCAGCGTTAC

2251	GGCAAAGAAA ACATCACCTC CTCAACCGTG CCGCCGTCAA ACGGCAAAAA

2301	TGTCAAACTG GCAGACCAAC GCCACCCGAA GACAGGCGTA CCGTTTGACG

2351	GTAAAGGGTT TCCGAATTTT GAGAAGCACG TGAAATATGA TACGCTCGAG

2401	CACCACCACC ACCACCACTG A

1	MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT

51	AADVEADDEK GLGLKKVVTN LTKTVNEMKQ NVDAKVKAAE SEIEKLTTEL

101	ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA

151	DTVDEHAEAF NDIADSLDET DTKADEAVKT ANMAKQTAEE TRODVDAKVE

201	AAETAAGEAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIARKANS

251	ADVYTREESD SKPVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT

301	VMDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG

351	FRFTENFAAK AGVAVGTSSG SSANYHVGVN YEWGSGGGGS DLANDSFIRQ

401	VADRQWEEPD GKYELFGSRG ELAERSGHIG LGKIQSHQLG NLMIQQAAIK

451	GNIGYIVRFS DHGEEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE

501	EMPADGYDGP QGGGYPAPKG ARDIYSYDIK GVAQNIRLNL TDNRSTGQRL

551	ADRFMNAGSM LTQGVGDGEK RATRYSPELD RSGNAAEAFN GTADIVENII

601	GAAGEIVGAG DAVQGISEGS NIAVMHGLGL LSTENKMARI NDLADMAQLK

651	DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT

701	AHPIKRSQMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEQRY

751	GRMNITSSTV PPSNGKNVKL ADQRHPKTGV PEDGKGFPNE EKHVKYDTLE

801	HHHHHH*

961-741

1	ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC

51	TGCTGCCTAC AACAATGGCC AAGAAATCAA CGCTPTCAAA GCTGGAGAGA

101	CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT

151	GCAGCCGATG TTGAAGCCGA CGACTITAAA GGTCTGGGTC TGAAAAAAGT

201	CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG

251	CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA

301	GCAGACACTG ATGCCGCITT AGCAGATACT GATGCCGCTC TGGATGCAAC

351	CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG

401	AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT

451	GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT

501	GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG

551	CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA

601	GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA

651	TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA

701	AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT

751	GCCGACGTGT ACACCAGAGA AGASTCTGAC AGCAAATTTG TCAGAATTGA

801	TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG

851	AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA

901	GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC

951	GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG

1001	CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC

1051	TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC

1101	TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG

1151	GATCCGGAGG GGGTGGTGTC GCCGCCGACA TCGGTGCGGG GCTTGCCGAT

1201	GCACTAACCG CACCGCTCGA CCATAAAGAC AAAGGTTTGC AGTCTTTGAC

1251	GCTGGATCAG TCCGTCAGGA AAAACGAGAA ACTGAAGCTG GCGGCACAAG

1301	GTGCGOAAAA AACTTATGGA AACGGTGACA GCCTCAATAC GGGCAAATTG

1351	AAGAACGACA AGGTCAGCCG TTTCGACTTT ATCCGCCAAA TCGAAGTGGA

1401	CGGGCAGCTC ATTACCTTGG AGAGTGGAGA GTTCCAAGTA TACAAACAAA

1451	GCCATTCCGC CTTAACCGCC TTTCAGACCG AGCAAATACA AGATTCGGAG

1501	CATTCCGGGA AGATGGTTGC GAAACGCCAG TTCAGAATCG GCGACATAGC

1551	GGGCGAACAT ACATCTTTTG ACAAGCTTCC CGAADGCGGC AGGGCGACAT

1601	ATCGCGGGAC GGCGTTCGGT TCAGACGATG CCGGCGGAAA ACTGACCTAC

1651	ACCATAGATT TCGCCGCCAA GCAGGGAAAC GGCAAAATCG AACATTTGAA

1701	ATCGCCAGAA CTCAATGTCG ACCTGGCCGC CGCCGATATC AAGCCGGATG

1751	GAAAACGCCA TGCCGTCATC AGCGGTTCCG TCCTTTACAA CCAAGCCGAG

1801	AAAGGCAGTT ACTCCCTCGG TATCTTTGGC GGAAAAGCCC AGGAAGTTGC

1851	CGGCAGCGCG GAAGTGAAAA CCGTAAACGG CATACGCCAT ATCGGCCTTG

1901	CCGCCAAGCA ACTCGAGCAC CACCACCACC ACCACTGA

1	MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT

51	AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL

101	ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA

151	DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK

201	AAETAAGKAE AAAGTANTAA DRAEAVAAKV TDIKADIATN KDNIAKKANS

251	ADVYTREESD SKETRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT

301	VSDLEXETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG

351	FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD

401	ALTAPLDHKD KGLQSINLOQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGKL

451	KNDKVSRFDF IRQIEVDGQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE

501	HSGKMVAKRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY

551	TIDFAAKQGN GKIEHLKSPE LNVDLAAADI KPDGKEHAVI SGSVINNQAE

601	KGSYSLGIFG GRAZEVAGSA EVKTVNGIRH IGLAAKQLEH HHHHH*

961-983

1	ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC

51	TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA

101	CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT

151	GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT

201	CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG

251	CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA

301	GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC

351	CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG

401	AGACTAAGAC AAATATCGTA AAAANTGATG AAAAATTAGA AGCCGTGGCT

451	GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT

501	GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG

551	CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA

601	GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA

651	TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA

701	AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT

751	GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA

801	TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG

851	AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGANAAAACA

901	GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC

951	GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG

1001	CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC

1051	TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC

1101	TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG

1151	GATCCGGCGG AGGCGGCACT TCTGCGCCCG ACTTCAATGC AGGCGGTACC

1201	GGTATCGGCA GCAACAGCAG AGCAACAACA GCGAAATCAG CAGCAGTATC

1251	TTACGCCGGT ATCAAGAACG AAATGTGCAA AGACAGAAGC ATGCTCTGTG

1301	CCGGTCGGGA TGACGTTGCG GTTACAGACA GGGATGCCAA AATCAATGCC

1351	CCCCCCCCGA ATCTGCATAC CGGAGACTTT CCAAACCCAA ATGACGCATA

1401	CAAGAATTTG ATCAACCTCA AACCTGCAAT TGAAGCAGGC TATACAGGAC

1451	GCGGGGTAGA GGTAGGTATC GTCGACACAG GCGAATCCGT CGGCAGCATA

1501	TCCTTTCCCG AACTGTATGG CAGAAAAGAA CACGGCTATA ACGAAAATTA

1551	CAAAAACTAT ACGGCGTATA TGCGGAAGGA AGCGCCTGAA GACGGAGGCG

1601	GTAAAGACAT TGAAGCTTCT TTCGACGATG AGGCCGTTAT AGAGACTGAA

1651	GCAAAGCCGA CGGATATCCG CCACGTAAAA GAAATCGGAC ACATCGATTT

1701	GGTCTCCCAT ATTATTGGCG GGCGTTCCGT GGACGGCAGA CCTGCAGGCG

1751	GTATTGCGCC CGATGCGACG CTACACATAA TGAATACGAA TGATGAAACC

1801	AAGAACGAAA TGATGGTTGC AGCCANCCGC AATGCATGGG TCAAGCTGGG

1851	CGAACGTGGC GTGCGCATCG TCAATAACAG TTTTGGAACA AGATCGAGGG

1901	CAGGCACTGC CGACCTTTTC CAAATAGCCA ATTCGGAGGA GCAGTACCGC

1951	CAAGCGTTGC TCGACTATTC CGGCGGTGAT AAAACAGACG AGGGTATCCG

2001	CCTGATGCAA CAGAGCGATT ACGGCAACCT GTCCTACCAC ATCCGTAATA

2051	AAAACATGCT TTTCATCTTT TCGACAGGCA ATGACGCACA AGCTCAGCCC

2101	AACACATATG CCCTATTGCC ATTTTATGAA AAAGACGCTC AAAAAGGCAT

2151	TATCACAGTC GCAGGCGTAG ACCGCAGTGG AGAAAAGTTC AAACGGGAAA

2201	TOTATGGAGA ACCGGGTACA GAACCGCTTG AGTATGGCTC CAACCATTGC

2251	GGAATTACTG CCATGTGGTG CCTGTCGGCA CCCTATGAAG CAAGCGTCCG

2301	TTTCACCCGT ACAAACCCGA TTCAAATTGC CGGAACATCC TTTTCCGCAC

2351	CCATCGTAAC CGGCACGGCG GCTCTGCTGC TGCAGAAATA CCCGTGGATG

2401	AGCAACGACA ACCTGCGTAC CACGTTGCTG ACGACGGCTC AGGACATCGG

2451	TGCAGTCGGC GTGGACAGCA AGTTCGGCTG GGGACTGCTG GATGCGGGTA

2501	AGGCCATGAA CGGACCCGCG TCCTTTCCGT TCGGCGACTT TACCGCCGAT

2551	ACGAAAGGTA CATCCGATAT TGCCTACTCC TTCCGTAACG ACATTTCAGG

2601	CACGGGCGGC CTGATCAAAA AAGGCGGCAG CCAACTGCAA CTGCACGGCA

2651	ACAACACCTA TACGGGCAAA ACCATTATCG AAGGCGGTTC GCTGGTGTTG

2701	TACGGCAACA ACAAATCGGA TATGCGCGTC GAAACCAAAG GTGCGCTGAT

2751	TTATAACGGG GCGGCATCCG GCGGCAGCCT GAACAGCGAC GGCATTGTCT

2801	ATCTGGCAGA TACCGACCAA TCCGGCGCAA ACGAAACCGT ACACATCAAA

2851	GGCAGTCTGC AGCTGGACGG CAAAGGTACG CTGTACACAC GTTTGGGCAA

2901	ACTGCTGAAA GTGGACGGTA CGGCGATTAT CGGCGGCAAG CTGTACATGT

2951	CGGCACGCGG CAAGGGGGCA GGCTATCTCA ACAGTACCGG ACGACGTGTT

3001	CCCTTCCTGA GTGCCGCCAA AATCGGGCAG GATTATTCTT TCTTCACAAA

3051	CATCGAAACC GACGGCGGCC TGCTGGCTTC CCTCGACAGC GTCGAAAAAA

3101	CAGCGGGCAG TGAAGGCGAC ACGCTGTCCT ATTATGTCCG TCGCGGCAAT

3151	GCGGCACGGA CTGCTTCGGC AGCGGCACAT TCCGCGCCCG CCGGTCTGAA

3201	ACACGCCGTA GAACAGGGCG GCAGCAATCT GGAAAACCTG ATGGTCGAAC

3251	TGGATGCCTC CGAATCATCC GCAACACCCG AGACGGTTGA AACTGCGGCA

3301	GCCGACCGCA CAGATATGCC GGGCATCCGC CCCTACGGCG CAACTTTCCG

3351	CGCAGCGGCA GCCGTACAGC ATGCGAATGC CGCCGACGGT GTACGCATCT

3401	TCAACAGTCT CGCCGCTACC GTCTATGCCG ACAGTACCGC CGCCCATGCC

3451	GATATGCAGG GACGCCGCCT GAAAGCCGTA TCGGACGGGT TGGACCACAA

3501	CGGCACGGGT CTGCGCGTCA TCGCGCAAAC CCAACAGGAC GGTGGAACGT

3551	GGGAACAGGG CGGTGTTGAA GGCAAAATGC GCGGCAGTAC CCAAACCGTC

3601	GGCATTGCCG CGAAAACCGG CGAAAATACG ACAGCAGCCG CCACACTGGG

3651	CATGGGACGC AGCACATGGA GCGAAAACAG TGCAAATGCA AAAACCGACA

3701	GCATTAGTCT GTTTGCAGGC ATACGGCACG ATGCGGGCGA TATCGGCTAT

3751	CTCAAAGGCC TGTTCTCCTA CGGACGCTAC AAAAACAGCA TCAGCCGCAG

3801	CACCGGTGCG GACGAACATG CGGAAGGCAG CGTCAACGGC ACGCTGATGC

3851	AGCTGGGCGC ACTGGGCGGT GTCAACGTTC CGTTTGCCGC AACGGGAGAT

3901	TTGACGGTCG AAGGCGGTCT GCGCTACGAC CTGCTCAAAC AGGATGCATT

3951	CGCCGAAAAA GGCAGTGCTT TGGGCTGGAG CGGCAACAGC CTCACTGAAG

4001	GCACGCTGGT CGGACTCGCG GGTCTGAAGC TGTCGCAACC CTTGAGCGAT

4051	AAAGCCGTCC TGTTTGCAAC GGCGGGCGTG GAACGCGACC TGAACGGACG

4101	CGACTACACG GTAACGGGCG GCTTTACCGG CGCGACTGCA GCAACCGGCA

4151	AGACGGGGGC ACGCAATATG CCGCACACCC GTCTGGTTGC CGGCCTGGGC

4201	GCGGATGTCG AATTCGGCAA CGGCTGGAAC GGCTTGGCAC GTTACAGCTA

4251	CGCCGOTTCC AAACAGTACG GCAACCACAG CGGACGAGTC GGCGTAGGCT

4301	ACCGGTTCCT CGAGCACCAC CACCACCACC ACTGA

1	MATNDDDVKK AAIVATAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT

51	AADVEADDFK GLGLKKVVTN IRKTVNENKQ NVDAKVKAAB SEEEKIRTKL

101	ADTDAALADT DAALDATTNA INKLGENITT FASETETNIV KIDEKLEAVA

151	DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTASE TXQNVDAKVK

201	AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS

251	ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT

301	VSDLREETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG

351	FRPTENFAAK AGVAVGTSSG SSAAYINGVN YEWGSGGGGT SAPDFNAGGT

401	GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MDCAGRDDVA VTDRDAKINA

451	PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI

501	SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS FDDEAVIETE

551	AKPTDIRSVK EIGRIDLVSH IIGGRSVDGR PAGGIAPDAT LHIENTNDET

601	KNEMMVAAIR NAWVKLGERG VRIVANSFGT TSRAGTADLF QIANSEEQYR

651	QALLDYSGGD KTDEGIRLMQ QSDYGELSYM IRNKNELFIF STGEDAQAQF

701	NTYALLPFYE KDAQKGIITV AGVDRSGEKF KREMYGEPGT EPLEYGSNMC

751	GITAMWCLSA PYEASVRFTR TNPIQIAGTS FSAPIVTGTA ALLLQKYPWM

801	SEDNLRTTLL TTAQDIGAVG VDSKFGWGLL DAGKAYXGPA SFPFGDFTAD

851	TKGTSDLAYS FRNDISGTGG LIKKGGSQLQ LHGNNTITGR TIIEGGSLVL

901	YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVTLADTDQ SGANETVHIK

951	GSWILDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGEGA GYLNSTGRKV

1001	PFLSAAKIGQ DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSTYVRRGN

1051	AARTASAAAM SAPAGLKHAV EQGGSNLENL NVELDASESS ATPETVETAA

1101	ADRTDMPGIR PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAHA

1151	DMQGRRLKAV SDGLDHNGTG LRVIAQTQQD GGTWEQGGVE GKERGSTQTV

1201	GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY

1251	LKGLFSYGRY KNSISRSTGA DKHAEGSVNG TLMQLGALGG VIMPFAATGD

1301	LTVEGGLRYD LLKQDAFAEK GSALGWSGNS LTEGTLVGLA GLKLSULSD

1351	KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG

1401	ADVEFGNGWN GLARYSYAGS KQYGNMSGRV GVGYRFLEEH HHHH*

961c-ORF46.1

1	ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC

51	TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA

101	CCATCTACGA CATTGATGAA GACGGCACAA TTAcCAAAAA AGACGCAACT

151	GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT

201	CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG

251	CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA

301	GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC

351	CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG

401	AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT

451	GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT

501	GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG

551	CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA

601	GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA

651	TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACcGACATCA

701	AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT

751	GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA

801	TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG

851	AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA

901	GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC

951	GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGAGGAGGAG

1001	GATCAGATTT GGCAAACGAT TCTTTTATCC GGCAGGTTCT CGACCGTCAG

1051	CATTTCGAAC CCGACGGGAA ATACCACCTA TTCGGCAGCA GGGGGGAACT

1101	TGCCGAGCGC AGCGGCCATA TCGGATTGGG AAAAATACAA AGCCATCAGT

1151	TGGGCAACCT GATGATTCAA CAGGCGGCCA TTAAAGGAAA TATCGGCTAC

1201	ATTGTCCGCT TTTCCGATCA CGGGCACGAA GTCCATTCCC CCTTCGACAA

1251	CCATGCCTCA CATTCCGATT CTGATGAAGC CGGTAGTCCC GTTGACGGAT

1301	TTAGCCTTTA CCGCATCCAT TGGGACGGAT ACGAACACCA TCCCGCCGAC

1351	GGCTATGACG GGCCACAGGG CGGCGGCTAT CCCGCTCCCA AAGGCGCGAG

1401	GGATATATAC AGCTACGACA TAAAAGGCGT TGCCCAAAAT ATCCGCCTCA

1451	ACCTGACCGA CAACCGCAGc ACCGGACAAC GGCTTGCCGA CCGTTTCCAC

1501	AATGCCGGTA GTATGCTGAC GCAAGGAGTA GGCGACGGAT TCAAACGCGC

1551	CACCCGATAC AGCCCCGAGC TGGACAGATC GGGCAATGCC GCCGAAGCCT

1601	TCAACGGCAC TGCAGATATC GTTAAAAACA TCATCGGCGC GGCAGGAGAA

1651	ATTGTCGGCG CAGGCGATGC CGTGCAGGGC ATAAGCGAAG GCTCAAACAT

1701	TGCTGTCATG CACGGCTTGG GTCTGCTTTC CACCGAAAAC AAGATGGCGC

1751	GCATAAACGA TTTGGCAGAT ATGGCGCAAC TCAAAGACTA TGcCGCAGCA

1801	GCCATCCGCG ATTGGGCAGT CCAAAACCCC AATGCCGCAC AAGGCATAGA

1851	AGCCGTCAGC AATATCTTTA TGGCAGCCAT CCCCATCAAA GGGATTGGAG

1901	CTGTTCGGGG AAAATACGGC TTGGGCGGCA TCACGGCACA TCCTATCAAG

1951	CGGTCGCAGA TGGGCGCGAT CGCATTGCCG AAAGGGAAAT CCGCCGTCAG

2001	CGACAATTTT GCCGATGCGG CATACGCCAA ATACCCGTCC CCTTACCATT

2051	CCOGAAATAT CCGTTCAAAC TTGGAGCAGC GTTACGGCAA AGAAAACATC

2101	ACCTCCTCAA CCGTGCCGCC GTCAAACGGC AAAAATGTCA AACTGGCAGA

2151	CCAACGCCAC CCGAAGACAG GCGTACCGTT TGACGGTAAA GGGTTTCCGA

2201	ATTTTGAGAA GCACGTGAAA TATGATACGC TCGAGCACCA CCACCACCAC

2251	CACTGA

1	MATNDDEVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT

51	AADVEADDFK GIGLENVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL

101	ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA

151	NDIADSLDET NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK

201	AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS

251	ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT

301	VSDAREETRQ GLAEQAALSG LFQPYNVGGS GGGGSDLAND SFIRQVLDRQ

351	HFEPDGKYDL FGSRGELAER SGHIGLGKIQ SHQLGNLNIQ QAAIKGNIGY

401	IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGESLYRIE WEGYESHPAD

451	GYDGPQGGGY PAPKGARDZY SYDIKGVAQN IRLNLTDNRS TGORLADRFH

501	NAGEKLTQGV GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE

551	IVGAGDAVQG ISEGSNIAVM HGLGLASTEN KMARINDLAD MAQLKDYAAA

601	AIRDWAVQNP NAAQGIEAVS NIEMAAIPIK GIGAVRGEYG LGGITAHPIK

651	RSQMGAIALP KGKSAVSDNF ADANYAKYPS PYRSRNIRSN LEQRYGKENI

701	TSSTVPPSNG KNVKLADQRH PKTGVPFDGK GFPNFEEHVK YDTLEREEHM

751	H*

961c-741

1	ATGGCCACAA ACGACGACGA TGTTAAAAAR GCTGCCACTG TGGCCATTGC

51	TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA

101	CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT

151	GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT

201	CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG

251	CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA

301	GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC

351	CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG

401	AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT

451	GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT

501	GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG

551	CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA

601	GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA

651	TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA

701	AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT

751	GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA

901	TGGTCTGAAC GCTACTACCG APAAATTGGA CACACGCTTG GCTTCTGCTG

851	AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA

901	GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC

951	GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGAGGGGGTG

1001	GTGTCGCCGC CGACATCGGT GCGGGGCTTG CCGATGCACT AACCGCACCG

1051	CTCGACCATA AAGACAAAGG TTTGCAGTCT TTGACGCTGG ATCAGTCCGT

1101	CAGGAAAAAC GAGAAACTGA AGCTGGCGGC ACAAGGTGCG GAAAAAACTT

1151	ATGGAAACGG TGACAGCCTC AATACGGGCA AATTGAAGAA CGACAAGGTC

1201	AGCCGTTTCG ACTTTATCCG CCAAATCGAA GTGGAGGGGC AGCTCATTAC

1251	CTTGGAGAGT GGAGAGTTCC AAGTATACAA ACAAAGCCAT TCCGCCTTAA

1301	CCGCCTTTCA GACCGAGCAA ATACAAGATT CGGAGCATTC CGGGAAGATG

1351	GTTGCGAAAC GCCAGTTCAG AATCGGCGAC ATADCGGGCG AACATACATC

1401	TTTTGACAAG CTTCCCGAAG GCGGCAGGGC GACATATCGC GGGACGGCGT

1451	TCGGTTCAGA CGATGCCGGC GGAAAACTGA CCTACACCAT AGATTTCGCC

1501	GCCAAGCAGG GAAACGGCAA AATCGAACAT TTGAAATCGC CAGAACTCAA

1551	TGTCGACCTG GCCGCCGCCG ATATCAAGCC GGATGGAAAA CGCCATGCCG

1601	TCATCAGCGG TTCCGTCCTT TACAACCAAG CCGAGAAAGG CAGTTACTCC

1651	CTCGGTATCT TTGGCGGAAA AGCCCAGGAA GTTGCCGGCA GCGCGGAAGT

1701	GAAAACCGTA AACGGCATAC GCCATATCGG CCTTGCCGCC AAGCAACTCG

1751	AGCACCACCA CCACCACCAC TGA

1	MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT

51	AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKATTKL

101	ADTDAALADT DAALDATTNA LNXLGENITT FASETKTNIV KIDEKLEAVA

151	DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAKE TKQNVQAKVX

201	AAHTAAGXAE AAAGTANTAA DKAENVAAKV TDIKADIATN KDNIAKKMRS

251	ADVYTREESD SXFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT

301	VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP

351	LDERDXGLQS LTLDQSVRXN FKLKLAAQGA EKTYGNGDSL NTGKLKNDKV

401	SRFDFIRQIE VDGQLITLES GEFQVYXQSE SALTAFQTEQ TQGSERSGEM

451	VAKROFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTIDFA

501	AKQGNGKIEH LKSPELNVDL AAADIXPDGK RHAVISGSVL YNQAEKGSYS

551	LGIEGGKAQE VAGSAHVXTV NGIRHIGLAA KQLEIMEEHH *

961c-983

1	ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC

51	TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA

101	CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT

151	GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT

201	CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG

251	CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA

301	GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC

351	CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG

401	AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT

451	GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT

501	GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG

551	CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA

601	GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA

651	TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA

701	AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT

751	GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA

801	TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG

851	AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA

901	GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC

951	GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGCGGAGGCG

1001	GCACTTCTGC GCCCGACTTC AATGCAGGCG GTACCGGTAT CGGCAGCAAC

1051	AGCAGAGCAA CAACAGCGAA ATCAGCAGCA GTATCTTACG CCGGTATCAA

1101	GAACGAAATG TGCAAAGACA GAAGCATGCT CTGTGCCGGT CGGGATGACG

1151	TTGCGGTTAC AGACAGGGAT GCCAAAATCA ATGCCCCCCC CCCGAATCTG

1201	CATACCGGAG ACTTTCCAAA CCCAAATGAC GCATACAAGA ATTTGATCAA

1251	CCTCAAACCT GCAATTGAAG CAGGCTATAC AGGACGCGGG GTAGAGGTAG

1301	GTATCGTCGA CACAGGCGAA TCCGTCGGCA GCATATCCTT TCCCGAACTG

1351	TATGGCAGAA AAGAACACGG CTATAACGAA AATTACAAAA ACTATACGGC

1401	GTATATGCGG AAGGAAGCGC CTGAAGACGG AGGCGGTAAA GACATTGAAG

1451	CTTCTTTCGA CGATGAGGCC GTTATAGAGA CTGAAGCAAA GCCGACGGAT

1501	ATCCGCCACG TAAAAGAAAT CGGACACATC GATTTGGTCT CCCATATTAT

1551	TGGCGGGCGT TCCGTGGACG GCAGACCTGC AGGCGGTATT GCGCCCGATG

1601	CGACGCTACA CATAATGAAT ACGAATGATG AAACCAAGAA CGAAATGATG

1651	GTTGCAGCCA TCCGCAATGC ATGGGTCAAG CTGGGCGAAC GTGGCGTGCG

1701	CATCGTCAAT AACAGTTTTG GAACAACATC GAGGGCAGGC ACTGCCGACC

1751	TTTTCCAAAT AGCCAATTCG GAGGAGCAGT ACCGCCAAGC GTTGCTCGAC

1801	TATTCCGGCG GTGATAAAAC AGACGAGGGT ATCCGCCTGA TGCAACAGAG

1851	CGATTACGGC AACCTGTCCT ACCACATCCG TAATAAAAAC ATGCTTTTCA

1901	TCTTTTCGAC AGGCAATGAC GCACAAGCTC AGCCCAACAC ATATGCCCTA

1951	TTGCCATTTT ATGAAAAAGA CGCTCAAAAA GGCATTATCA CAGTCGCAGG

2001	CGTAGACCGC AGTGGAGAAA AGTTCAAACG GGAAATGTAT GGAGAACCGG

2051	GTACAGAACC GCTTGAGTAT GGCTCCAACC ATTGCGGAAT TACTGCCATG

2101	TGGTGCCTGT CGGCACCCTA TGAAGCAAGC GTCCGTTTCA CCCGTACAAA

2151	CCCGATTCAA ATTGCCGGAA CATCCTTTTC CGCACCCATC GTAACCGGCA

2201	CGGCGGCTCT GCTGCTGCAG AAATACCCGT GGATGAGCAA CGACAACCTG

2251	CGTACCACGT TGCTGACGAC GGCTCAGGAC ATCGGTGCAG TCGGCGTGGA

2301	CAGCAAGTTC GGCTGGGGAC TGCTGGATGC GGGTAAGGCC ATGAACGGAC

2351	CCGCGTCCTT TCCGTTCGGC GACTTTACCG CCGATACGAA AGGTACATCC

2401	GATATTGCCT ACTCCTTCCG TAACGACATT TCAGGCACGG GCGGCCTGAT

2451	CAAAAAAGGC GGCAGCCAAC TGCAACTGCA CGGCAACAAC ACCTATACGG

2501	GCAAAACCAT TATCGAAGGC GGTTCGCTGG TGTTGTACGG CAACAACAAA

2551	TCGGATATGC GCGTCGAAAC CAAAGGTGCG CTGATTTATA ACGGGGCGGC

2601	ATCCGGCGGC AGCCTGAACA GCGACGGCAT TGTCTATCTG GCAGATACCG

2651	ACCAATCCGG CGCAAACGAA ACCGTACACA TCAAAGGCAG TCTGCAGCTG

2701	GACGGCAAAG GTACGCTGTA CACACGTTTG GGCAAACTGC TGAAAGTGGA

2751	CGGTACGGCG ATTATCGGCG GCAAGCTGTA CATGTCGGCA CGCGGCAAGG

2801	G3GCAGGCPA TCTCAACAGT ACCGGACGAC GTGTTCCCTT CCTGAGTGCC

2851	GCCAAAATCG GGCAGGATTA TTCTTTCTTC ACAAACATCG AAACCGACGG

2901	CGGCCTGCTG GCTTCCCTCG ACAGCGTCGA AAAAACAGCG GGCAGTGAAG

2951	GCGACACGCT GTCCTATTAT GTCCGTCGCG GCAATGCGGC ACGGACTGCT

3001	TCGGCAGCGG CACATTCCGC GCCCGCCGGT CTGAAACACG CCGTAGAACA

3051	GGGCGGCAGC AATCTGGAAA ACCTGATGGT CGAACTGGAT GCCTCCGAAT

3101	CATCCGCAAC ACCCGAGACG GTTGAAACTG CGGCAGCCGA CCGCACAGAT

3151	ATGCCGGGCA TCCGCCCCTA CGGCGCAACT TTCCGCGCAG CGGCAGCCGT

3201	ACAGCATGCG AATGCCGCCG ACGGTGTACG CATCTTCAAC AGTCTCGCCG

3251	CTACCGTCTA TGCCGACAGT ACCGCCGCCC ATGCCGATAT GCAGGGACGC

3301	CGCCTGAAAG CCGTATCGGA CGGGTTGGAC CACAACGGCA CGGGTCTGCG

3351	CGTCATCGCG CAAACCCAAC AGGACGGTGG AACGTGGGAA CAGGGCGGTG

3401	TTGAAGOCAA AATGCGCCGC AGTACCCAAA CCGTCGGCAT TGCCGCGAAA

3451	ACCGGCGAAA ATACGACAGC AGCCGCCACA CTGGGCATGG GACGCAGCAC

3501	ATGGAGCGAA AACAGTGCAA ATGCAAAAAC CGACAGCATT AGTCTGTTTG

3551	CAGGCATACG GCACGATGCG GGCGATATCG GCTATCTCAA AGGCCTGTTC

3601	TCCTACGGAC GCTACAAAAA CAGCATCAGC CGCAGCACCG GTGCGGACGA

3651	ACATGCGGAA GGCAGCGTCA ACGGCACGCT GATGCAGCTG GGCGCACTGG

3701	GCGGTGTCAA CGTTCCGTTT GCCGCAACGG GAGATTTGAC GGTCGAAGGC

3751	GGTCTGCGCT ACGACCTGCT CAAACAGGAT GCATTCGCCG AAAAAGGCAG

3801	TGCTTTGGGC TGGAGCGGCA ACAGCCTCAC TGAAGGCACG CTGGTCGGAC

3851	TCGCGGGTCT GAAGCTGTCG CAACCCTTGA GCGATAAAGC CGTCCTGTTT

3901	GCAACGGCGG GCGPGOAACG CGACCTGAAC GGACGCGACT ACACGGTAAC

3951	GGGCGGCTTT ACCGGCGCGA CTGCAGCAAC CGGCAAGACG GGGGCACGCA

4001	ATATGCCGCA CACCCGTCTG GTTGCCGGCC TGGGCGCGGA TGTCGAATTC

4051	GGCAACGGCT GGAACGGCTT GGCACGTTAC AGCTACGCCG GTTCCAAACA

4101	GTACGGCAAC CACAGCGGAC GAGTCGGCGT AGGCTACCGG TTCCTCGAGC

4151	ACCACCACCA CCACCACTGA

1	MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITEKDAT

51	AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKI

101	ADTDAALADT DAALDATTNA LNKLGENITT PAEETKTNIV KIDEKLEAVA

151	DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAKE TKQNVDAKVK

201	AARTAAGNAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS

251	ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSLADH DTRLNGLDKT

301	VSDLRKETRQ GLAEQAALSG LFQPYVVGGS GGGGTSAPDF NAGGTGIGSN

351	SRATTAKSAA VSYAGIKNEM CEDRSELCAG RDDVAVTDRD AKINAPPPNL

401	HIGDFPNFND AYKNLINLKP AIKAGYTGEG VEVGIVDTGE SVGSISFPEL

451	YGRKEHGYNE NYKNYTAYER KEAPEDGGGK DIEASFDDEA VIETEAKPTD

501	IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETKNEMM

551	VAAIRNAAWK LGERGVRIVN NSFGTTSRAG TADLFQIANS EEQYRQALLD

601	YSGGDKTDEG IRLMQQSDYG NLSYMIRNKN MLFIFSTGAM AQAQPNTYAL

651	LPFYEKDAQK GIITVAGVDR SGEKFKREMY GEPGTEPLEY GSNECGITAK

701	WCLSAFYEAS VRFTWFATIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNDNL

751	RTTLLTTAQD IGAVGVDSKF GWGLIZAGKA MNGPASFPFG DFTADTKGTS

901	DIAYSFRNDI SGTGGLIKKG GSQLQAHGNN TYTGKTIIEG GSLVLYGNNK

951	SDERVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSGANE TVHIKGSLQL

901	DGKGTLYTRL GRIZKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA

951	AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA

1001	SAAAHSAPAG LKHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD

1051	MPGIRPYGAT FRAAAAVQHA NAADGVRIFN SLAATVYADS TAARADMQGR

1101	RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGEHRG STQTVGIAAK

1151	TGENTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIREDA GDIGYLKGLF

1201	SYGRYKNSIS RSTGADEHAE GSVNGTLMQL GALGGVNVPF AATGDLTVEG

1251	GLRYDLLKQD ARARKGSALG WSGNSLTEGT LVGLAGLKLS QPDSDKAVLF

1301	ATAGVERDLN GRDYTVTGGF TGATAATGKT GARNMPHTRL VAGLGADVEF

1351	GNGWNGLARY SYAGSKQYGN RSGRVGVGYR FLEHHHHHH*

961cL-ORF46.1

1	ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT

51	CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG

101	CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT

151	TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC

201	CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC

251	TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC

301	AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA

351	GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG

401	CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA

451	ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA

501	ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG

551	ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC

601	AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT

651	CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG

701	CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA

751	AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC

801	TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA

851	AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA

901	CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA

951	CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC

1001	TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT

1051	GGATCCGGAG GAGGAGGATC AGATTTGGCA AACGATTCTT TTATCCGGCA

1101	GGTTCTCGAC CGTCAGCATT TCGAACCCGA CGGGAAATAC CACCTATTCG

1151	GCAGCAGGGG GGAACTTGCC GAGCGCAGCG GCCATATCGG ATTGGGAAAA

1201	ATACAAAGCC ATCAGTTGGG CAACCTGATG ATTCAACAGG CGGCCATTAA

1251	AGGAAATATC GGCTACATTG TCCGCTTTTC CGATCACGGG CACGAAGTCC

1301	ATTCCCCCTT CGACAACCAT GCCTCACATT CCGATTCTGA TGAAGCCGGT

1351	AGTCCCGTTG ACGGATTTAG CCTTTACCGC ATCCATTGGG ACGGATACGA

1401	ACACCATCCC GCCGACGGCT ATGACGGGCC ACAGGGCGGC GGCTATCCCG

1451	CTCCCAAAGG CGCGACGGAT ATATACACCT ACGACATAAA AGGCGTTGCC

1501	CAAAATATCC GCCTCAACCT GACCGACAAC CGCAGCACCG GACAACGGCT

1551	TGCCGACCGT TTCCACAATG CCGGTAGTAT GCTGACGCAA GGAGTAGGCG

1601	ACGGATTCAA ACGCGCCACC CGATACAGCC CCGAGCTGGA CAGATCGGGC

1651	AATGCCGCCG AACCCTTCAA CGGCACTGCA GATATCGTTA AAAACATCAT

1701	CGGCGCGGCA GGAGAAATTG TCGGCGCAGG CGATGCCGTG CAGGGCATAA

1751	GCGAAGGCTC AAACATTGCT GTCATGCACG GCTTGGGTCT GCATTCCACC

1801	GAAAACAAGA TGGCGCGCAT CAACGATTTG GCAGATATGG CGCAACTCAA

1851	AGACTATGCC GCAGCAGCCA TCCGCGATTG GGCAGTCCAA AACCCCAATG

1901	CCGCACAAGG CATAGAAGCC GTCAGCAATA TCTTTATGGC AGCCATCCCC

1951	ATCAAAGGGA TTGGAGCTGT TCGGGGAAAA TACGGCTTGG GCGGCATCAC

2001	GGCACATCCT ATCAAGCGGT CGCACATGGG CGCGATCGCA TTGCCGAAAG

2051	GGAAATCCGC CGTCAGCGAC AATTTTGCCG ATGCGGCATA CGCCAAATAC

2101	CCGTCCCCTT ACCATTCCCG AAATATCCGT TCAAACTTGG AGCAGCGTTA

2151	CGGCAAAGAA AACATCACCT CCTCAACCGT GCCGCCGTCA AACGGCAAAA

2201	ATGTCAAACT GGCAGACCAA CGCCACCCGA AGACAGGCGT ACCGTTTGAC

2251	GGTAAAGGGT TTCCGAATTT TGAGAAGCAC GTGAAATATG ATACGTAACT

2301	CGAG

1	MKHEPSKVAT TAILATFCSG ALAATNDDDV KKAATVAIAK AYNNGQEING

51	FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNATKTVNEN

101	KQNVDAKVKA AESEIEMATT KLADTDAALA DTDAALDATT NALNKLGENI

151	TTFAEETKTN IVEIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV

201	KTANEAKQTA EETYQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA

251	KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKEVRIDG ANATTEKLDT

301	RLASAEKSIA DRDTRANGLD KTVGDARKET RQGLAEQAAL SGLFQPYNVG

351	GSGGGGSDLA NDSFIRQVAD RQHFEPDGKY ELFGSRGELA ERSGHIGLGK

401	IQSHQAGNLM IQQAAIKGNI GYIVRESDHG REVHSPFDNH ASHSDSDEAG

451	SPVDGFSLYR IHWDGYEHHP ADGYDGFQGG GYPAPKGARD IYSYDIKGVA

501	QNIRDNLTDN RSTGQRLADR FENAGSKLTQ GVGDGFKRAT RYSPELDRSG

551	NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VHHGLGLAST

601	ENKNARINDA ADHAQLYDYA AAAIRDWAVQ NPNAAQGIEA VSNIFMAAIP

651	IKGIGAVRGK YGLGGITAHP IKRSQMGAIA APKGKEAVSD NFADAAYAKY

701	PSPYHSRNIR SNLEQRYGKE NITSSTVPPS NGENVKLADQ RHPKTGVPFD

751	GEGFPNFEKR VKYDT*

961cL-741

1	ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT

51	CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG

101	CCACTGTGGC CATTGCTGCT GCCTACAACK ATGGCCAAGA AATCAACGGT

151	TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC

201	CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC

251	TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC

301	AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA

351	GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG

401	CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA

451	ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA

501	ATTAGAAGCC GTCGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG

551	ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC

601	AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT

651	CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG

701	CCGCTGCCAC ACCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA

751	AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC

801	TAAAAATGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA

851	AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA

901	CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA

951	CCGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC

1001	TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGCGT

1051	GGATCCGGTG GGGGTGGTGT CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA

1101	TGCACTAACC GCACCGCTCG ACCATAAAGA CAAAGGTTTG CAGTCTTTGA

1151	CGCTGGATCA GTCCGTCAGG AAAAACGAGA AACTGAAGCT GGCGGCACAA

1201	GGTGCGGAAA AAACTTATGG AAACGGTGAC AGCCTCAATA CGGGCAAATT

1251	GAAGAACGAC AAGGTCAGCC GTTTCGACTT TATCCGCCAA ATCGAAGTGG

1301	ACGGGCAGCT CATTACCTTG GAGAGTGGAG AGTTCCAAGT ATACAAACAA

1351	AGCCATTCCG CCTTAACCGC CTTTCAGACC GAGCAAATAC AAGATTCGGA

1401	GCATTCCGGG AAGATGGTTG CGAAACGCCA GTTCAGAATC GGCGACATAG

1451	CGGGCGAACA TACATCTTTT GACAAGCTTC CCGAAGGCGG CAGGGCGACA

1501	TATCGCGGGA CGGCGTTCGG TTCAGACGAT GCCGGCGGAA AACTGACCTA

1551	CACCATAGAT TTCGCCGCCA AGCAGGGAAA CGGCAAAATC GAACATTTGA

1601	AATCGCCAGA ACTCAATGTC GACCTGGCCG CCGCCGATAT CAAGCCGGAT

1651	GGAAAACGCC ATGCCGTCAT CAGCGGTTCC GTCCTTTACA ACCAAGCCGA

1701	GAAAGGCAGT TACTCCCTCG GTATCTTTGG CGGAAAAGCC CACGAAGTTG

1751	CCGGCAGCGC GGAAGTGAAA ACCGTAAACG GCATACGCCA TATCGGCCTT

1801	GCCGCCAAGC AACTCGAGCA CCACCACCAC CACCACTGA

1	MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA ANNNGQEING

51	FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN

101	KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NAINKLGENI

151	TTFAEETKTN IVKIDEKLEA VADTVDKRAE AFNDIADSLD ETNTKADEAV

201	KTANEKKQTA EETKQNVDAK VFAAETAAGY AFAAAGTANT AADKAEAVAA

251	KVTDIKADIA TNYDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT

301	RLASAEKSIA DEDTRINGLD KTVSDLRKET RQGLAEQAAL SGLFQPYVVG

351	QSGCCGVAAD IGAGLADALT APLDEKDKGL QSLTLDQSVR KNEKLKLAAQ

401	GAEKTYGNGD SLNTGKLKND KVSREDFIRQ IEVDGQLITL ESGMFQVYKQ

451	SMSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT

501	YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD

551	GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL

601	AAKQLEHHHH HH*

961cL-883

1	ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT

51	CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG

101	CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT

151	TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC

201	CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGOTC

251	TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC

301	AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA

351	GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG

401	CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA

451	ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA

501	ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG

551	ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC

601	AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT

651	CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG

701	CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA

751	AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC

801	TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA

851	AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA

901	CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA

951	CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC

1001	TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT

1051	GGATCCGGCG GAGGCGGCAC TTCTGCGCCC GACTTCAATG CAGGCGGTAC

1101	CGGTATCGGC AGCAACAGCA GAGCAACAAC AGCGAAATCA GCAGCAGTAT

1151	CTTACGCCGG TATCAAGAAC GAAATGTGCA AAGACAGAAG CATGCTCTGT

1201	GCCGGTCGGG ATGACGTTGC GGTTACAGAC AGGGATGCCA AAATCAATGC

1251	CCCCCCCCCG AATCTGCATA CCGGAGACTT TCCAAACCCA AATGACGCAT

1301	ACAAGAATTT GATCAACCTC AAACCTGCAA TTGAAGCAGG CTATACAGGA

1351	CGCGGGGTAG AGGTAGGTAT CGTCGACACA GGCGAATCCG TCGGCAGCAT

1401	ATCCTTTCCC GAACTGTATG GCAGAAAAGA ACACGGCTAT AACGAAAATT

1451	ACAAAAACTA TACGGCGTAT ATGCGGAAGG AAGCGCCTGA AGACGGAGGC

1501	GGTAAAGACA TTGAAGCTTC TTTCGACGAT GAGGCCGTTA TAGAGACTGA

1551	AGCAAAGCCG ACGGATATCC GCCACGTAAA AGAAATCGGA CACATCGATT

1601	TGGTCTCCCA TATTATTGGC GGGCGTTCCG TGGACGGCAG ACCTGCAGGC

1651	GaTATTGCGC CCGATGCGAC GCTACACATA ATGAATACGA ATGATGAAAC

1701	CAAGAACGAA ATGATGGTTG CAGCCATCCG CAATGCATGG GTCAAGCTGG

1751	GCGAACGTGG CGTGCGCATC GTCAATAACA GTTTTGGAAC AACATCGAGG

1801	GCAGGCACTG CCGACCTTTT CCAAATAGCC AATTCGGAGG AGCAGTACCG

1851	CCAAGCGTTG CTCGACTATT CCGGCGGTGA TAAAACAGAC GAGGGTATCC

1901	GCCTGATGCA ACAGAGCGAT TACGGCAACC TGTCCTACCA CATCCGTAAT

1951	AAAAACATGC TTTTCATCTT TTCGACAGGC AATGACGCAC AAGCTCAGCC

2001	CAACACATAT GCCCTATTGC CATTTTATGA AAAAGACGCT CAAAAAGGCA

2051	TTATCACAGT CGCAGGCGTA GACCGCAGTG GAGAAAAGTT CAAACGGGAA

2101	ATGTATGGAG AACCGGGTAC AGAACCGCTT GAGTATGGCT CCAACCATTG

2151	CGGAATTACT GCCATGTGGT GCCTGTCGGC ACCCTATGAA GCAAGCGTCC

2201	GTTTCACCCG TACAAACCCG ATTCAAATTG CCGGAACATC CTTTTCCGCA

2251	CCCATCGTAA CCGGCACGGC GGCTCTGCTG CTGCAGAAAT ACCCGTGGAT

2301	GAGCAACGAC AACCTGCGTA CCACGTTGCT GACGACGGCT CAGGACATCG

2351	GTGCAGTCGG CGTGGACAGC AAGTTCGGCT GGGGACTGCT GGATGCGGGT

2401	AAGGCCATGA ACGGACCCGC GTCCTTTCCG TTCGGCGACT TTACCGCCGA

2451	TACGAAAGGT ACATCCGATA TTGCCTACTC CTTCCGTAAC GACATTTCAG

2501	GCACGGGCGG CCTGATCAAA AAAGGCGGCA GCCAACTGCA ACTGCACGGC

2551	AACAACACCT ATACGGGCAA AACCATTATC GAAGGCGGTT CGCTGGTGTT

2601	GTACGGCAAC AACAAATCGG ATATGCGCGT CGAAACCAAA GGTGCGCTGA

2651	TTTATAACGG GGCGGCATCC GGCGGCAGCC TGAACAGCGA CGGCATTGTC

2701	TATCTGGCAG ATACCGACCA ATCCGGCGCA AACGAAACCG TACACATCAA

2751	AGGCAGTCTG CAGCTGGACG GCAAAGGTAC GCTGTACACA CGTTTGGGCA

2801	AACTGCTGAA AGTGGACGGT ACGGCGATTA TCGGCGGCAT GCTGTACATG

2851	TCGGCACGCG GCAAGGGGGC AGGCTATCTC AACAGTACCG GACGACGTGT

2901	TCCCTTCCTG AGTGCCGCCA AAATCGGGCA GGATTATTCT TTCTTCACAA

2951	ACATCGAAAC CGACGGCGGC CTGCTGGCTT CCCTCGACAG CGTCGAAAAA

3001	ACAGCGGGCA GTGAAGGCGA CACGCTGTCC TATTATGTCC GTCGCGGCAA

3051	TGCGGCACGG ACTGCTTCGG CAGCGGCACA TTCCGCGCCC GCCGGTCTGA

3101	AACACGCCGT AGAACAGGGC GGCAGCAATC TGGAAAACCT GATGGTCGAA

3151	CrGGATGCCT CCGAATCATC CGCAACACCC GAGACGGTTG AAACTGCGGC

3201	AGCCGACCGC ACAGATATGC CGGGCATCCG CCCCTACGGC GCAACTTTCC

3251	GCGCAGCOGC AGCCGTACAG CATGCGAATG CCGCCGACGG TGTACGCATC

3301	TTCAACAGTC TCGCCGCTAC CGTCTATGCC GACAGTACCG CCGCCCATGC

3351	CGATATGCAG GGACGCCGCC TGAAAGCCGT ATCGGACGGG TTGGACCACA

3401	ACGGCACGGG TCTGCGCGTC ATCGCGCAAA CCCAACAGGA CGGTGGAACG

3451	TGGGAACAGG GCGGTGTTGA AGGCAAAATG CGCGGCAGTA CCCAAACCGT

3501	CGGCATTGCC GCGAAAACCG GCGAAAATAC GACAGCAGCC GCCACACTGG

3551	GCATGGGACG CAGCACATGG AGCGAAAACA GTQCAAATGC AAAAACCGAC

3601	AGCATTAGTC TGTTTGCAGG CATACGGCAC GATGCGGGCG ATATCGGCTA

3651	TCTCAAAGGC CTGTTCTCCT ACGGACGCTA CAAAAACAGC ATCAGCCGCA

3701	GCACCGGTGC GGACGAACAT GCGGAAGGCA GCGTCAACGG CACGCTGATG

3751	CAGCTGGGCG CACTGGGCGG TGTCAACGTT CCGTTTGCCG CAACGGGAGA

3801	TTTGACGGTC GAAGGCGGTC TGCGCTACGA CCTGCTCAAA CAGGATGCAT

3851	TCGCCGAAAA AGGCAGTGCT TTGGGCTGGA GCGGCAACAG CCTCACTGAA

3901	GGCACGCTGG TCGGACTCGC GGGTCTGAAG CTGTCGCAAC CCTTGAGCGA

3951	TAAAGCCGTC CTGTTTGCAA CGGCGGGCGT GGAACGCGAC CTGAACGGAC

4001	GCGACTACAC GGTAACGGGC GGCTTTACCG GCGCGACTGC AGCAACCGGC

4051	AAGACGGGGG CACGCAATAT GCCGCACACC CGTCTGGTTG CCGGCCTGGG

4101	CGCGGATGTC GAATTCGGCA ACGGCTGGAA CGGCTTGGCA CGTTACAGCT

4151	ACGCCGGTTC CAAACAGTAC GGCAACCACA GCGGACGAGT CGGCGTAGGC

4201	TACCGGTTCT GACTCGAG

1	MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING

51	FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN

101	KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI

151	TTFABETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADRAV

201	KTANEAKQTA EETKQNVDAK VKAABTAAGK AFAAAGTANT AADKARAVAA

251	KVTDIKADIA TNKDNIAKKA NSADVYTRBE SDSKFVRIEG LNATTEKLDT

301	RDASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG

351	GSGGGGTSAP DFNAGGTGIG SNSRATTAKS ANVSYAGIKN EFKKDRSELC

401	AGRDDVAVTD RDAKTNAPPP NLHTGDFPNP NDAYENDINL KPAIRAGYTG

451	RGVEVGIVDT GESVGSISFP ELYGRKBHGY NENYKNYTAY MRKEAPEDGG

501	GKDIEASFDD EAVIETRAKP TDIRHVKEIG RIDLVSHIIG GRSVDGRPAG

551	GIAPDATLHI MNTNDRTXNE EEVAAIRNAW VKLGERGVRI VNNSFGTTSR

601	AGTADLFQIA NSEEQYRQAL LDYSGGDKTD EGIRLMQQSD YGNL5YH1RN

651	KNMDFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIITVAGV DRSGEKFKRB

701	MYGSPGTRPL RYGSNHCGIT AMWCLSAPYE ASVRPTRTNP IQIAGTSFSA

751	PIVTGTAALL LQXYPWRSND NLRTTLLTTA QDIGAVGVDS KPGWGLLDAG

801	KAMNGPASFP FGDFTADTKG TSDIAYSFRN DISGTGGLIK KGGSQLQLHQ

851	NNTYTGKTII EGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV

901	YLADTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDG TAIIGGKLMY

951	SARGKGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDOG LLASLDSVEK

1001	TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEQG GSNLENLMVE

1051	LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAADGVRI

1101	FNSLAATVYA DSTAAHADMQ GRRLKAVSDG LDHNGTGLRV IAQTQQDGGT

1151	WEQGGVEGKM RGSTQTVGIA AKTGERPTAA ATLGMGRSTW SENSANAKTD

1201	SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM

1251	QLGALGGVNV PFAATGDLTV EGGLRYDLLK QDAFAEKGSA LGWSGNSLTE

1301	GTLVGLAGLR LSQPLSDKAV LFATAGVERD LNGRDYTVTG GRTGATAATG

1351	KTGARNMPHT RLVAGLGADV EFGNGWNGLA RYSYAGSKQY GNHSGRVGVG

1401	YRF*

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. For instance, the use of proteins from other strains is envisaged [e.g. see WO00/66741 for polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953].

EXPERIMENTAL DETAILS

FPLC Protein Purification

The following table summarises the FPLC protein purification that was used:


Protein	PI	Column	Buffer	pH	Protocol

121.1^untagged	6.23	Mono Q	Tris	8.0	A
128. 1^untagged	5.04	Mono Q	Bis-Tris propane	6.5	A
406.1L	7.75	Mono Q	Diethanolamine	9.0	B
576.1L	5.63	Mono Q	Tris	7.5	B
593^untagged	8.79	Mono S	Hepes	7.4	A
726^untagged	4.95	Hi-trap S	Bis-Tris	6.0	A
919^untagged	10.5(-leader)	Mono S	Bicine	8.5	C
919Lorf4	10.4(-leader)	Mono S	Tris	8.0	B
920L	6.92(-leader)	Mono Q	Diethanolamine	8.5	A
953L	7.56(-leader)	Mono S	MES	6.6	D
982^untagged	4.73	Mono Q	Bis-Tris propane	6.5	A
919-287	6.58	Hi-trap Q	Tris	8.0	A
953-287	4.92	Mono Q	Bis-Tris propane	6.2	A

Buffer solutions included 20-120 mM NaCl, 5.0 mg/ml CHAPS and 10% v/v glycerol. The dialysate was centrifuged at 13000 g for 20 min and applied to either a mono Q or mono S FPLC ion-exchange resin. Buffer and ion exchange resins were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual [Pharmacia: FPLC Ion Exchange and Chromatofocussing; Principles and Methods. Pharmacia Publication]. Proteins were eluted using a step-wise NaCl gradient. Purification was analysed by SDS-PAGE and protein concentration determined by the Bradford method.
The letter in the ‘protocol’ column refers to the following:
FPLC-A:
Clones 121.1, 128.1, 593, 726, 982, periplasmic protein 920L and hybrid proteins 919-287, 953-287 were purified from the soluble fraction of E. coli obtained after disruption of the cells. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD₅₅₀reached 0.6-08. Expression of recombinant protein was induced with/PTO at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed on ice or at 4° C. For cytosolic proteins (121.1, 128.1, 593, 726 and 982) and periplasmic protein 920L, bacteria were resuspended in 25 ml of PBS containing complete protease inhibitor (Boehringer-Mannheim). Cells were lysed by by sonication using a Branson Sonifier 450. Disrupted cells were centrifuged at 8000 g for 30 min to sediment unbroken cells and inclusion bodies and the supernatant taken to 35% v/v saturation by the addition of 3.9 M (NH₄)₂SO₄. The precipitate was sedimented at 8000 g for 30 minutes. The supernatant was taken to 70% v/v saturation by the addition of 3.9 M (NH₄)₂SO₄and the precipitate collected as above. Pellets containing the protein of interest were identified by SDS-PAGE and dialysed against the appropriate ion-exchange buffer (see below) for 6 hours or overnight. The periplasmic fraction from E. coli expressing 953L was prepared according to the protocol of Evans et. al. [Infect. Immun. (1974) 10:1010-1017] and dialysed against the appropriate ion-exchange buffer. Buffer and ion exchange resin were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual (Pharmacia). Buffer solutions included 20 mM NaCl, and 10% (v/v) glycerol. The dialysate was centrifuged at 13000 g for 20 min and applied to either a mono Q or mono S FPLC ion-exchange resin. Buffer and ion exchange resin were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual (Pharmacia). Proteins were eluted from the ion-exchange resin using either step-wise or continuous NaCl gradients. Purification was analysed by SDS-PAGE and protein concentration determined by Bradford method. Cleavage of the leader peptide of periplasmic proteins was demonstrated by sequencing the NH₂-terminus (see below).
FPLC-B:
These proteins were purified from the membrane fraction of E. coli, Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium. Clones 406.1L and 919LOrf4 were grown at 30° C. and Orf25L and 576.1L at 37° C. until the OD₅₅₀reached 0.6-0.8. In the case of 919LOrf4, growth at 30° C. was essential since expression of recombinant protein at 37° C. resulted in lysis of the cells. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. AU subsequent procedures were performed at 4° C. Bacteria were resuspended in 25 ml of PBS containing complete protease inhibitor (Boehringer-Mannheim) and lysed by osmotic shock with 2-3 passages through a French Press. Unbroken cells were removed by centrifugation at 5000 g for 15 min and membranes precipitated by centrifugation at 100000 g (Beckman Ti50, 38000 rpm) for 45 minutes. A Dounce homogenizer was used to re-suspend the membrane pellet in 7.5 ml of 20 mM Tris-HCl (pH 8.0), 1.0 M NaCl and complete protease inhibitor. The suspension was mixed for 2-4 hours, centrifuged at 100000 g for 45 min and the pellet resuspended in 7.5 ml of 20 mM Tris-HCl (pH 8.0), 1.0M NaCl, 5.0 mg/ml CHAPS, 10% (v/v) glycerol and complete protease inhibitor. The solution was mixed overnight, centrifuged at 100000 g for 45 minutes and the supernatant dialysed for 6 hours against an appropriately selected buffer. In the case of Orf25L, the pellet obtained after CHAPS extraction was found to contain the recombinant protein. This fraction, without further purification, was used to immunise mice.
FPLC-C:
Identical to FPLC-A, but purification was from the soluble fraction obtained after permeabilising E. coli with polymyxin B, rather than after cell disruption.
FPLC-D:
A single colony harbouring the plasmid of interest was grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at 30° C. until the OD₅₅₀reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed on ice or at 4° C. Cells were resuspended in 20 mM Bicine (pH 8.5), 20 mM NaCl, 10% (v/v) glycerol, complete protease inhibitor (Boehringer-Mannheim) and disrupted using a Branson Sonifier 450. The sonicate was centrifuged at 8000 g for 30 rain to sediment unbroken cells and inclusion bodies. The recombinant protein was precipitated from solution between 35% v/v and 70% v/v saturation by the addition of 3.9M (NH₄)₂SO₄. The precipitate was sedimented at 8000 g for 30 minutes, resuspended in 20 mM Bicine (pH 8.5), 20 mM NaCl, 10% (v/v) glycerol and dialysed against this buffer for 6 hours or overnight. The dialysate was centrifuged at 13000 g for 20 min and applied to the FPLC resin. The protein was eluted from the column using a step-wise NaCl gradients. Purification was analysed by SDS-PAGE and protein concentration determined by Bradford method.

Cloning Strategy and Oligonucleotide Design

Genes coding for antigens of interest were amplified by PCR, using oligonucleotides designed on the basis of the genomic sequence of N. meningitidis B MC58. Genomic DNA from strain 2996 was always used as a template in PCR reactions, unless otherwise specified, and the amplified fragments were cloned in the expression vector pET21b+(Novagen) to express the protein as C-terminal His-tagged product, or in pET-24b+(Novagen) to express the protein in ‘untagged’ form (e.g. AG 287K).
Where a protein was expressed without a fusion partner and with its own leader peptide (if present), amplification of the open reading frame (ATG to STOP codons) was performed.
Where a protein was expressed in ‘untagged’ form, the leader peptide was omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.
The melting temperature of the primers used in PCR depended on the number and type of hybridising nucleotides in the whole primer, and was determined using the formulae:
T _m1=4(G+C)+2(A+T) (tail excluded)
T _m2=64.9+0.41(% GC)−600/N (whole primer)
The melting temperatures of the selected oligonucleotides were usually 65-70° C. for the whole oligo and 50-60° C. for the hybridising region alone.
Oligonucleotides were synthesised using a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2.0 ml NH₄OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were centrifuged and the pellets resuspended in water.


			Restriction
		Sequences	site

Orf1L	Fwd	CGCGATCCGCTAGC-AAAACAACCGACAAACGG	NheI
	Rev	CCCGCTCGAG-TTACCAGCGGTAGCCTA	XhoI

Orf1	Fwd	CTAGCTAGC-GGACACACTTATTTCGGCATC	NheI
	Rev	CCCGCTCGAG-TTACCAGCGGTAGCCTAATTTG	XhoI

Orf1LOmpA	Fwd		NdeI-(NheI)
	Rev	CCCGCTCGAG-	XhoI

Orf4L	Fwd	CGCGGATCCCATATG-AAAACCTTCTTCAAAACC	NdeI
	Rev	CCCGCTCGAG-TTATTTGGCTGCGCCTTC	XhoI

Orf7-1L	Fwd	GCGGCATTAAT-ATGTTGAGAAAATTGTTGAAATGG	AseI
	Rev	GCGGCCTCGAG-TTATTTTTTCAAAATATATTTGC	XhoI

Orf9-L	Fwd	GCGGCCATATG-TTACCTAACCGTTTCAAAATGT	Ndel
	Rev	GCGGCCTCGAG-TTATTTCCGAGGTTTTCGGG	XhoI

Orf23L	Fwd	CGCGGATCCCATATG-ACACGCTTCAAATATTC	NdeI
	Rev	CCCGCTCGAG-TTATTTAAACCGATAGGTAAA	XhoI

Orf25-1 His	Fwd	CGCGGATCCCATATG-GGCAGGGAAGAACCGC	NdeI
	Rev	GCCCAAGCTT-ATCGATGGAATAGCCGCG	HindIII

Orf29-1 b-His-	Fwd	CGCGGATCCGCTAGC-AACGGTTTGGATGCCCG	NheI
(MC58)	Rev	CCCGCTCGAG-TTTGTCTAAGTTCCTGATAT	XhoI
		CCCGCTCGAG-ATTCCCACCTGCCATC

Orf29-1 b-L	Fwd	CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT	NheI
(MC58)	Rev	CCCGCTCGAG-TTAATTCCCACCTGCCATC	XhoI

Orf29-1 c-His	Fwd	CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT	NheI
(MC58)	Rev	CCCGCTCGAG-TTGGACGATGCCCGCGA	XhoI

Orf29-1 c-L	Fwd	CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT	NheI
(MC58)	Rev	CCCGCTCGAG-TTATTGGACGATGCCCGC	XhoI

Orf25L	Fwd	CGCGGATCCCATATG-TATCGCAAACTGATTGC	NdeI
	Rev	CCCGCTCGAG-CTAATCGATGGAATAGCC	XhoI

Orf37L	Fwd	CGCGGATCCCATATG-AAACAGACAGTCAAATG	NdeI
	Rev	CCCGCTCGAG-TCAATAACCCGCCTTCAG	XhoI

Orf38L	Fwd	CGCGGATCCCATATG-	NdeI
		TTACGTTTGACTGCTTTAGCCGTATGCACC
	Rev	CCCGCTCGAG-	XhoI
		TTATTTTGCCGCGTTAAAAGCGTCGGCAAC

Orf40L	Fwd	CGCGGATCCCATATG-AACAAAATATACCGCAT	NdeI
	Rev	CCCGCTCGAG-TTACCACTGATAACCGAC	XhoI

Orf40.2-His	Fwd	CGCGGATCCCATATG-ACCGATGACGACGATTTAT	NdeI
	Rev	GCCCAAGCTT-CCACTGATAACCGACAGA	HindIII

Orf40.2L	Fwd	CGCGGATCCCATATG-AACAAAATATACCGCAT	NdeI
	Rev	GCCCAAGCTT-TTACCACTGATAACCGAC	HindIII

Orf46-2L	Fwd	GGGAATTCCATATG-GGCATTTCCCGCAAAATATC	NdeI
	Rev	CCCGCTCGAG-TTATTTACTCCTATAACGAGGTCTCTTAAC	XhoI

Orf46-2	Fwd	GGGAATTCCATATG-TCAGATTTGGCAAACGATTCTT	NdeI
	Rev	CCCGCTCGAG-TTATTTACTCCTATAACGAGGTCTCTTAAC	XhoI

Orf46.1L	Fwd	GGGAATTCCATATG-GGCATTTCCCGCAAAATATC	NdeI
	Rev	CCCGCTCGAG-TTACGTATCATATTTCACGTGC	XhoI

orf46.	Fwd	GGGAATTCCATATGCACGGAAATATGATACGAAG	BamHI-NdeI
(His-GST)	Rev	CCCGCTCGAGTTTACTCCTATAACGAGGTCTCTTAAC	XhoI

rf46.1-His	Fwd	GGGAATTCCATATGTCAGATTTGGCAAACGATTCTT	NdeI
	Rev	CCCGCTCGAGCGTATCATATTTCACGTGC	XhoI

orf46.2-His	Fwd	GGGAATTCCATATGTCAGATTTGGCAAACGATTCTT	NdeI
	Rev	CCCGCTCGAGTTTACTCCTATAACGAGGTCTCTTAAC	XhoI

Orf65-1-(His/	Fwd	CGCGGATCCCATATG-CAAAATGCGTTCAAAATCCC	BamHI-NdeI
GST) (MC58)	Rev	CGCGGATCCCATATG-AACAAAATATACCGCAT	XhoI
		CCCGCTCGAG-TTTGCTTTCGATAGAACGG

Orf2-1L	Fwd	GCGGCCATATG-GTCATAAAATATACAAATTTGAA	NdeI
	Rev	GCGGCCTCGAG-TTAGCCTGAGACCTTTGCAAATT	XhoI

Orf76-1L	Fwd	GCGGCCATATG-AAACAGAAAAAAACCGCTG	NdeI
	Rev	GCGGCCTCGAG-TTACGGTTTGACACCGTTTTC	XhoI

Orf83.1L	Fwd	CGCGGATCCCATATG-AAAACCCTGCTCCTC	NdeI
	Rev	CCCGCTCGAG-TTATCCTCCTTTGCGGC	XhoI

Orf85-2L	Fwd	GCGGCCATATG-GCAAAAATGATGAAATGGG	NdeI
	Rev	GCGGCCTCGAG-TTATCGGCGCGGCGGGCC	XhoI

Orf91L	Fwd	GCGGCCATATGAAAAAATCCTCCCTCATCA	NdeI
(MC58)	Rev	GCGGCCTCGAGTTATTTGCCGCCGTTTTTGGC	XhoI

Orf91-His	Fwd	GCGGCCATATGGCCCCTGCCGACGCGGTAAG	NdeI
(MC580	Rev	GCGGCCTCGAGTTTGCCGCCGTTTTTGGCTTTC	XhoI

Orf97-1L	Fwd	GCGGCCATATG-AAACACATACTCCCCCTGA	NdeI
	Rev	GCGGCCTCGAG-TTATTCGCCTACGGTTTTTTG	XhoI

Orf119L	Fwd	GCGGCCATATGATTTACATCGTACTGTTTC	NdeI
(MC58)	Rev	GCGGCCTCGAGTTAGGAGAACAGGCGCAATGC	XhoI

Orf119-His	Fwd	GCGGCCATATGTACAACATGTATCAGGAAAAC	NdeI
(MC58)	Rev	GCGGCCTCGAGGGAGAACAGGCGCAATGCGG	XhoI

Orf137.1 (His-	Fwd	CGCGGATCCGCTAGCTGCGGCACGGCGGG	BamHI-NdeI
GST) (MC58)	Rev	CCCGCTCGAGATAACGGTATGCCGCCAG	XhoI

Orf143-1L	Fwd	CGCGGATCCCATATG-GAATCAACACTTTCAC	NdeI
	Rev	CCCGCTCGAG-TTACACGCGGTTGCTGT	XhoI

008	Fwd	CGCGGATCCCATATG-AACAACAGACATTTTG	NdeI
	Rev	CCCGCTCGAG-TTACCTGTCCGGTAAAAG	XhoI

050-1(48)	Fwd	CGCGGATCCGCTAGC-ACCGTCATCAAACAGGAA	NheI
	Rev	CCCGCTCGAG-TCAAGATTCGACGGGGA	XhoI

105	Fwd	CGCGGATCCCATATG-TCCGCAAACGAATACG	NdeI
	Rev	CCCGCTCGAG-TCAGTGTTCTGCCAGTTT	XhoI

111L	Fwd	CGCGGATCCCATATG-CCGTCTGAAACACG	NdeI
	Rev	CCCGCTCGAG-TTAGCGGAGCAGTTTTTC	XhoI

117-1	Fwd	CGCGGATCCCATATG-ACCGCCATCAGCC	NdeI
	Rev	CCCGCTCGAG-TTAAAGCCGGGTAACGC	XhoI

121-1	Fwd	GCGGCCATATG-GAAACACAGCTTTACATCGG	NdeI
	Rev	GCGGCCTCGAG-TCAATAATAATATCCCGCG	XhoI

122-1	Fwd	GCGGCCATATG-ATTAAAATCCGCAATATCC	NdeI
	Rev	GCGGCCTCGAG-TTAAATCTTGGTAGATTGGATTTGG	XhoI

128-1	Fwd	GCGGCCATATG-ACTGACAACGCACTGCTCC	NdeI
	Rev	GCGGCCTCGAG-TCAGACCGCGTTGTCGAAAC	XhoI

148	Fwd	CGCGGATCCCATATG-GCGTTAAAAACATCAAA	NdeI
	Rev	CCCGCTCGAG-TCAGCCCTTCATACAGC	XhoI

149.1L	Fwd	CCCGCTCGAG-TCAGCCCTTCATACAGC	XhoI
(MC58)	Rev	GCGGCATTAATGGCACAAACTACACTCAAACC	AseI

149.1His	Fwd	GCGGCATTAATGCATGAAACTGAGCAATCGGTGG	AseI
(MC58)	Rev	GCGGCCTCGAGAAACTTCACGTTCACGCCGCCGGTAAA	XhoI

205 (His-GST)	Fwd	CGCGGATCCCATATGGGCAAATCCGAAAATACG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGATAATGGCGGCGGCGG	XhoI

206L	Fwd	CGCGGATCCCATATG-TTTCCCCCCGACAA	NdeI
	Rev	CCCGCTCGAG-TCATTCTGTAAAAAAAGTATG	XhoI

214 (His-GST)	Fwd	CGCGGATCCCATATGCTTCAAAGCGACAGCAG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGTTCGGATTTTTGCGTACTC	XhoI

216	Fwd	CGCGGATCCCATATG-GCAATGGCAGAAAACG	NdeI
	Rev	CCCGCTCGAG-CTATACAATCCGTGCCG	XhoI

225-1L	Fwd	CGCGGATCCCATATG-GATTCTTTTTTCAAACC	NdeI
	Rev	CCCGCTCGAG-TCAGTTCAGAAAGCGGG	XhoI

235L	Fwd	CGCGGATCCCATATG-AAACCTTTGATTTTAGG	NdeI
	Rev	CCCGCTCGAG-TTATTTGGGCTGCTCTTC	XhoI

243	Fwd	CGCGGATCCCATATG-GTAATCGTCTGGTTG	NdeI
	Rev	CCCGCTCGAG-CTACGACTTGGTTACCG	XhoI

247-1L	Fwd	GCGGCCATATG-AGACGTAAAATGCTAAAGCTAC	NdeI
	Rev	GCGGCCTCGAG-TCAAAGTGTTCTGTTTGCGC	XhoI

264-His	Fwd	GCCGCCATATG-TTGACTTTAACCCGAAAAA	NdeI
	Rev	GCCGCCTCGAG-GCCGGCGGTCAATACCGCCCGAA	XhoI

270 (His-GST)	Fwd	CGCGGATCCCATATGGCGCAATGCGATTTGAC	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGTTCGGCGGTAAATGCCG	XhoI

274L	Fwd	GCGGCCATATG-GCGGGGCCGATTTTTGT	NdeI
	Rev	GCGGCCTCGAG-TTATTTGCTTTCAGTATTATTG	XhoI

283L	Fwd	GCGGCCATATG-AACTTTGCTTTATCCGTCA	NdeI
	Rev	GCGGCCTCGAG-TTAACGGCAGTATTTGTTTAC	XhoI

285-His	Fwd	CGCGGATCCCATATGGGTTTGCGCTTCGGGC	BamHI
	Rev	GCCCAAGCTTTTTTCCTTTGCCGTTTCCG	HindIII

286-His	Fwd	CGCGGATCCCATATG-GCCGACCTTTCCGAAAA	NdeI
(MC58)	Rev	CCCGCTCGAG-GAAGCGCGTTCCCAAGC	XhoI

286L	Fwd	CGCGGATCCCATATG-CACGACACCCGTAC	NdeI
	Rev	CCCGCTCGAG-TTAGAAGCGCGTTCCCAA	XhoI

287L	Fwd	CTAGCTAGC-TTTAAACGCAGCGTAATCGCAATGG	NheI
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

287	Fwd	CTAGCTAGC-GGGGGCGGCGGTGGCG	NheI
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

287LOrf4	Fwd	CTAGCTAGCGCTCATCCTCGCCGCC-	NheI
		TGCGGGGGCGGCGGT
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

287-fu	Fwd	CGGGGATCC-GGGGGCGGCGGTGGCG	BamHI
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

287-His	Fwd	CTAGCTAGC-GGGGGCGGCGGTGGCG	NheI
	Rev	CCCGCTCGAG-ATCCTGCTCTTTTTTGCC*	XhoI

287-His	Fwd	CTAGCTAGC-TGCGGGGGCGGCGGTGGCG	NheI
(2996)	Rev	CCCGCTCGAG-ATCCTGCTCTTTTTTGCC	XhoI

Δ1 287-His	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC^§	NheI

Δ2 287-His	Fwd	CGCGGATCCGCTAGC-CAAGATATGGCGGCAGT^§	NheI

Δ3 287-His	Fwd	CGCGGATCCGCTAGC-GCCGAATCCGCAAATCA^§	NheI

Δ4 287-His	Fwd	CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG^§	NheI

Δ4 287MC58-His	Fwd	CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG^§	NheI

287a-His	Fwd	CGCCATATG-TTTAAACGCAGCGTAATCGC	NdeI
	Rev	CCCGCTCGAG-AAAATTGCTACCGCCATTCGCAGG	XhoI

287b-His	Fwd	CGCCATATG-GGAAGGGTTGATTTGGCTAATGG	NdeI

287b-2996-His	Rev	CCCGCTCGAG-CTTGTCTTTATAAATGATGACATATTTG	XhoI

287b-MC58-His	Rev	CCCGCTCGAG-TTTATAAAAGATAATATATTGATTGATTCC	XhoI

287c-2996-His	Fwd	CGCGCTAGC-ATGCCGCTGATTCCCGTCAATC^§	NheI

′287^untagged,	Fwd	CTAGCTAGC-GGGGGCGGCGGTGGCG	NheI
(2996)	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

ΔG287-His*	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
	Rev	CCCGCTCGAG-ATCCTGCTCTTTTTTGCC	XhoI

ΔG287K (2996)	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

ΔG 287-L	Fwd	CGCGGATCCGCTAGC-	NheI
		TTTGAACGCAGTGTGATTGCAATGGCTTGTATTTTTGCC
		CTTTCAGCCTGT TCGCCCGATGTTAAATCGGCG
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

ΔG 287-Orf4L	Fwd	CGCGGATCCGCTAGC-	NheI
		AAAACCTTCTTCAAAACCCTTTCCGCCGCCGCACTCGCG
		CTCATCCTCGCCGCCTGC TCGCCCGATGTTAAATCG
	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

292L	Fwd	CGCGGATCCCATATG-AAAACCAAGTTAATCAAA	NdeI
	Rev	CCCGCTCGAG-TTATTGATTTTTGCGGATGA	XhoI

308-1	Fwd	CGCGGATCCCATATG-TTAAATCGGGTATTTTATC	NdeI
	Rev	CCCGCTCGAG-TTAATCCGCCATTCCCTG	XhoI

401L	Fwd	GCGGCCATATG-AAATTACAACAATTGGCTG	NdeI
	Rev	GCGGCCTCGAG-TTACCTTACGTTTTTCAAG	XhoI

406L	Fwd	CGCGGATCCCATATG-CAAGCACGGCTGCT	NdeI
	Rev	CCCGCTCGAG-TCAAGGTTGTCCTTGTCTA	XhoI

502-1L	Fwd	CGCGGATCCCATATG-ATGAAACCGCACAAC	NdeI
	Rev	CCCGCTCGAG-TCAGTTGCTCAACACGTC	XhoI

502-A	Fwd	CGCGGATCCCATATGGTAGACGCGCTTAAGCA	BamHI-NdeI
(His-GST)	Rev	CCCGCTCGAGAGCTGCATGGCGGCG	XhoI

503-1L	Fwd	CGCGGATCCCATATG-GCACGGTCGTTATAC	NdeI
	Rev	CCCGCTCGAG-CTACCGCGCATTCCTG	XhoI

519-1L	Fwd	GCGGCCATATG-GAATTTTTCATTATCTTGTT	NdeI
	Rev	GCGGCCTCGAG-TTATTTGGCGGTTTTGCTGC	XhoI

525-1L	Fwd	GCGGCCATATG-AAGTATGTCCGGTTATTTTTC	NdeI
	Rev	GCGGCCTCGAG-TTATCGGCTTGTGCAACGG	XhoI

529-(His/GST)	Fwd	CGCGGATCCGCTAGC-TCCGGCAGCAAAACCGA	BamHI-NdeI
(MC58)	Rev	GCCCAAGCTT-ACGCAGTTCGGAATGGAG	HindIII

552L	Fwd	GCCGCCATATGTTGAATATTAAACTGAAAACCTTG	NdeI
	Rev	GCCGCCTCGAGTTATTTCTGATGCCTTTTCCC	XhoI

556L	Fwd	GCCGCCATATGGACAATAAGACCAAACTG	NdeI
	Rev	GCCGCCTCGAGTTAACGGTGCGGACGTTC	XhoI

557L	Fwd	CGCGGATCCCATATG-AACAAACTGTTTCTTAC	NdeI
	Rev	CCCGCTCGAG-TCATTCCGCCTTCAGAAA	XhoI

564ab-(His/GST)	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(MC58)		CAAGGTATCGTTGCCGACAAATCCGCACCT
	Rev	CCCGCTCGAG-	XhoI
		AGCTAATTGTGCTTGGTTTGCAGATAGGAGTT

564abL	Fwd	CGCGGATCCCATATG-	NdeI
(MC58)		AACCGCACCCTGTACAAAGTTGTATTTAACAAACATC
	Rev	CCCGCTCGAG-	XhoI
		TTAAGCTAATTGTGCTTGGTTTGCAGATAGGAGTT

564b-(His/GST)	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(MC58)		ACGGGAGAAAATCATGCGGTTTCACTTCATG
	Rev	CCCGCTCGAG-	XhoI
		AGCTAATTGTGCTTGGTTTGCAGATAGGAGTT

564c-(His/GST)	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(MC58)		GTTTCAGACGGCCTATACAACCAACATGGTGAAATT
	Rev	CCCGCTCGAG-	XhoI
		GCGGTAACTGCCGCTTGCACTGAATCCGTAA

564bc-(His/GST)	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(MC58)		ACGGGAGAAAATCATGCGGTTTCACTTCATG
	Rev	CCCGCTCGAG-	XhoI
		GCGGTAACTGCCGCTTGCACTGAATCCGTAA

564d-(His/GST)	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(MC58)		CAAAGCAAAGTCAAAGCAGACCATGCCTCCGTAA
	Rev	CCCGCTCGAG-	XhoI
		TCTTTTCCTTTCAATTATAACTTTAGTAGGTTCAATTTTG
		GTCCCC

564cd-(His/GST)	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(MC58)		GTTTCAGACGGCCTATACAACCAACATGGTGAAATT
	Rev	CCCGCTCGAG-	XhoI
		TCTTTTCCTTTCAATTATAACTTTAGTAGGTTCAATTTTG
		GTCCCC

570L	Fwd	GCGGCCATATG-ACCCGTTTGACCCGCG	NdeI
	Rev	GCGGCCTCGAG-TCAGCGGGCGTTCATTTCTT	XhoI

576-1L	Fwd	CGCGGATCCCATATG-AACACCATTTTCAAAATC	NdeI
	Rev	CCCGCTCGAG-TTAATTTACTTTTTTGATGTCG	XhoI

580L	Fwd	GCGGCCATATG-GATTCGCCCAAGGTCGG	NdeI
	Rev	GCGGCCTCGAG-CTACACTTCCCCCGAAGTGG	XhoI

583L	Fwd	CGCGGATCCCATATG-ATAGTTGACCAAAGCC	NdeI
	Rev	CCCGCTCGAG-TTATTTTTCCGATTTTTCGG	XhoI

593	Fwd	GCGGCCATATG-CTTGAACTGAACGGACT	NdeI
	Rev	GCGGCCTCGAG-TCAGCGGAAGCGGACGATT	XhoI

650 (His-GST)	Fwd	CGCGGATCCCATATGTCCAAACTCAAAACCATCG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGGCTTCCAATCAGTTTGACC	XhoI

652	Fwd	GCGGCCATATG-AGCGCAATCGTTGATATTTTC	NdeI
	Rev	GCGGCCTCGAG-TTATTTGCCCAGTTGGTAGAATG	XhoI

664L	Fwd	GCGGCCATATG-GTGATACATCCGCACTACTTC	NdeI
	Rev	GCGGCCTCGAG-TCAAAATCGAGTTTTACACCA	XhoI

726	Fwd	GCGGCCATATG-ACCATCTATTTCAAAAACGG	NdeI
	Rev	GCGGCCTCGAG-TCAGCCGATGTTTAGCGTCCATT	XhoI

741-His	Fwd	CGCGGATCCCATATG-AGCAGCGGAGGGGGTG	NdeI
(MC58)	Rev	CCCGCTCGAG-TTGCTTGGCGGCAAGGC	XhoI

ΔG741-His	Fwd	CGCGGATCCCATATG-GTCGCCGCCGACATCG	NdeI
(MC58)	Rev	CCCGCTCGAG-TTGCTTGGCGGCAAGGC	XhoI

686-2-(His/GST)	Fwd	CGCGGATCCCATATG-GGCGGTTCGGAAGGCG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-TTGAACACTGATGTCTTTTCCGA	XhoI

719-(His/GST)	Fwd	CGCGGATCCGCTAGC-AAACTGTCGTTGGTGTTAAC	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-TTGACCCGCTCCACGG	XhoI

730-His	Fwd	GCCGCCATATGGCGGACTTGGCGCAAGACCC	NdeI
(MC58)	Rev	GCCGCCTCGAGATCTCCTAAACCTGTTTTAACAATGCCG	XhoI

730A-His	Fwd	GCCGCCATATGGCGGACTTGGCGCAAGACCC	NdeI
(MC58)	Rev	GCGGCCTCGAGCTCCATGCTGTTGCCCCAGC	XhoI

730B-His	Fwd	GCCGCCATATGGCGGACTTGGCGCAAGACCC	NdeI
(MC58)	Rev	GCGGCCTCGAGAAAATCCCCGCTAACCGCAG	XhoI

741-His	Fwd	CGCGGATCCCATATG-AGCAGCGGAGGGGGTG	NdeI
(MC58)	Rev	CCCGCTCGAG-TTGCTTGGCGGCAAGGC	XhoI

ΔG741-His	Fwd	CGCGGATCCCATATG-GTCGCCGCCGACATCG	NdeI
(MC58)	Rev	CCCGCTCGAG-TTGCTTGGCGGCAAGGC	XhoI

743 (His-GST)	Fwd	CGCGGATCCCATATGGACGGTGTTGTGCCTGTT	BamHI-NdeI
	Rev	CCCGCTCGAGCTTACGGATCAAATTGACG	XhoI

757 (His-GST)	Fwd	CGCGGATCCCATATGGGCAGCCAATCTGAAGAA	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGCTCAGCTTTTGCCGTCAA	XhoI

759-His/GST	Fwd	GGCGGATCCGCTAGC-TACTCATCCATTGTCCGC	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-CCAGTTGTAGCCTATTTG	XhoI

759L	Fwd	CGCGGATCCGCTAGC-ATGCGCTTCACACACAC	NdeI
(MC58)	Rev	CCCGCTCGAG-TTACCAGTTGTAGCCTATTT	XhoI

760-His	Fwd	GCCGCCATATGGCACAAACGGAAGGTTTGGAA	NdeI
	Rev	GCCGCCTCGAGAAAACTGTAACGCAGGTTTGCCGTC	XhoI

769-His	Fwd	GCGGCCATATGGAAGAAACACCGCGCGAACCG	NdeI
(MC58)	Rev	GCGGCCTCGAGAACGTTTTATTAAACTCGAC	XhoI

907L	Fwd	GCGGCCATATG-AGAAAACCGACCGATACCCTA	NdeI
	Rev	GCGGCCTCGAG-TCAACGCCACTGCCAGCGGTTG	XhoI

911L	Fwd	CGCGGATCCCATATG-AAGAAGAACATATTGGAATTTTGGGTCGGACTG	NdeI
	Rev	CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG	XhoI

911LOmpA	Fwd	GGGAATTCCATATGAAAAAGACAGCTATCGCGATTGCA	NdeI-(NheI)
		GTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGC
		TAGC-GCTTTCCGCGTGGCCGGCGGTGC
	Rev	CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG	XhoI

911LPelB	Fwd	CATGCCATGG-CTTTCCGCGTGGCCGGCGGTGC	NcoI
	Rev	CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG	XhoI

913-His/GST	Fwd	CGCGGATCCCATATG-TTTGCCGAAACCCGCC	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-AGGTTGTGTTCCAGGTTG	XhoI

913L	Fwd	CGCGGATCCCATATG-AAAAAAACCGCCTATG	NdeI
(MC58)	Rev	CCCGCTCGAG-TTAAGGTTGTGTTCCAGG	XhoI

916L	Fwd	CGCGGATCCCATATG-AAAAAATACCTATTCCGC	NdeI
	Rev	CCCGCTCGAG-TTACGGGCGGTATTCGG	XhoI

919	Fwd	CGCGGATCCCATATG-CAAAGCAAGAGCATCCAAA	NdeI
	Rev	CCCGCTCGAG-TTACGGGCGGTATTCGG	XhoI

919L Orf4	Fwd	GGGAATTCCATATGAAAACCTTCTTCAAAACCCTTTCCG	NdeI-(NheI)
		CCGCCGCGCTAGCGCTCATCCTCGCCGCC-
		TGCCAAAGCAAGAGCATC
	Rev	CCCGCTCGAG-TTACGGGCGGTATTCGGGCTTCATACCG	XhoI

(919)-287	Fwd	CGCGGATCCGTCGAC-TGTGGGGGCGGCGGTGGC	SalI
fusion	Rev	CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC	XhoI

920-1L	Fwd	GCGGCCATATG-AAGAAAACATTGACACTGC	NdeI
	Rev	GCGGCCTCGAG-TTAATGGTGCGAATGACCGAT	XhoI

925-His/GST	Fwd	ggggacaagtttgtacaaaaaagcaggctTGCGGCAAGGATGCCGG	attB1
(MC58)^GATE	Rev	ggggaccactttgtacaagaaagctgggtCTAAAGCAACAATGCCGG	attB2

926L	Fwd	CGCGGATCCCATATG-AAACACACCGTATCC	NdeI
	Rev	CCCGCTCGAG-TTATCTCGTGCGCGCC	XhoI

927-2-(His/GST)	Fwd	CGCGGATCCCATATG-AGCCCCGCGCCGATT	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-TTTTTGTGCGGTCAGGCG	XhoI

932-His/GST	Fwd	ggggacaagtttgtacaaaaaagcaggctTGTTCGTTTGGGGGATTTAA	attB1
(MC58)^GATE		ACCAAACCAAATC

935 (His-GST)	Fwd	CGCGGATCCCATATGGCGGATGCGCCCGCG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGAAACCGCCAATCCGCC	XhoI

936-1L	Rev	ggggaccactttgtacaagaaagctgggtTCATTTTGTTTTTCCTTCTTCT	attB2
		CGAGGCCATT
	Fwd	CGCGGATCCCATATG-AAACCCAAACCGCAC	NdeI
	Rev	CCCGCTCGAG-TCAGCGTTGGACGTAGT	XhoI

953L	Fwd	GGGAATTCCATATG-AAAAAAATCATCTTCGCCG	NdeI
	Rev	CCCGCTCGAG-TTATTGTTTGGCTGCCTCGAT	XhoI

953-fu	Fwd	GGGAATTCCATATG-GCCACCTACAAAGTGGACG	NdeI
	Rev	CGGGGATCC-TTGTTTGGCTGCCTCGATTTG	BamHI

954 (His-GST)	Fwd	CGCGGATCCCATATGCAAGAACAATCGCAGAAAG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAGTTTTTTCGGCAAATTGGCTT	XhoI

958-His/GST	Fwd	ggggacaagtttgtacaaaaaagcaggctGCCGATGCCGTTGCGG	attB1
(MC58)^GATE	Rev	ggggaccactttgtacaagaaagctgggtTCAGGGTCGTTTGTTGCG	attB2

961L	Fwd	CGCGGATCCCATATG-AAACACTTTCCATCC	NdeI
	Rev	CCCGCTCGAG-TTACCACTCGTAATTGAC	XhoI

961	Fwd	CGCGGATCCCATATG-GCCACAAGCGACGAC	NdeI
	Rev	CCCGCTCGAG-TTACCACTCGTAATTGAC	XhoI

961 c (His/GST)	Fwd	CGCGGATCCCATATG-GCCACAAACGACG	BamHI-NdeI
	Rev	CCCGCTCGAG-ACCCACGTTGTAAGGTTG	XhoI

961 c-(His/GST)	Fwd	CGCGGATCCCATATG-GCCACAAGCGACGACGA	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-ACCCACGTTGTAAGGTTG	XhoI

961 c-L	Fwd	CGCGGATCCCATATG-ATGAAACACTTTCCATCC	NdeI
	Rev	CCCGCTCGAG-TTAACCCACGTTGTAAGGT	XhoI

961 c-L	Fwd	CGCGGATCCCATATG-ATGAAACACTTTCCATCC	NdeI
(MC58)	Rev	CCCGCTCGAG-TTAACCCACGTTGTAAGGT	XhoI

961d (His/GST)	Fwd	CGCGGATCCCATATG-GCCACAAACGACG	BamHI-NdeI
	Rev	CCCGCTCGAG-GTCTGACACTGTTTTATCC	XhoI

961 Δ1-L	Fwd	CGCGGATCCCATATG-ATGAAACACTTTCCATCC	NdeI
	Rev	CCCGCTCGAG-TTATGCTTTGGCGGCAAAG	XhoI

fu 961-...	Fwd	CGCGGATCCCATATG-GCCACAAACGACGAC	NdeI
	Rev	CGCGGATCC-CCACTCGTAATTGACGCC	BamHI

fu 961-...	Fwd	CGCGGATCCCATATG-GCCACAAGCGACGAC	NdeI
(Mc58)	Rev	CGCGGATCC-CCACTCGTAATTGACGCC	BamHI

fu
961 c-...	Fwd	CGCGGATCCCATATG-GCCACAAACGACGAC	NdeI
	Rev	CGCGGATCC-ACCCACGTTGTAAGGTTG	BamHI

fu
961 c-L-...	Fwd	CGCGGATCCCATATG-ATGAAACACTTTCCATCC	NdeI
	Rev	CGCGGATCC-ACCCACGTTGTAAGGTTG	BamHI

fu (961)-	Fwd	CGCGGATCC-GGAGGGGGTGGTGTCG	BamHI
741 (MC58)-His	Rev	CCCGCTCGAG-TTGCTTGGCGGCAAGGC	XhoI

fu (961)-983-	Fwd	CGCGGATCC-GGCGGAGGCGGCACTT	BamHI
His	Rev	CCCGCTCGAG-GAACCGGTAGCCTACG	XhoI

fu (961)-	Fwd	CGCGGATCCGGTGGTGGTGGT-	BamHI
Orf46.1-His		TCAGATTTGGCAAACGATTC
	Rev	CCCGCTCGAG-CGTATCATATTTCACGTGC	XhoI

fu (961 c-L)-	Fwd	CGCGGATCC-GGAGGGGGTGGTGTCG	BamHI
741 (MC58)	Rev	CCCGCTCGAG-TTATTGCTTGGCGGCAAG	XhoI

fu (961 c-L)-	Fwd	CGCGGATCC-GGCGGAGGCGGCACTT	BamHI
983	Rev	CCCGCTCGAG-TCAGAACCGGTAGCCTAC	XhoI

fu (961c-L)-	Fwd	CGCGGATCCGGTGGTGGTGGT-	BamHI
Orf46.1		TCAGATTTGGCAAACGATTC
	Rev	CCCGCTCGAG-TTACGTATCATATTTCACGTGC	XhoI

961-(His/GST)	Fwd	CGCGGATCCCATATG-GCCACAAGCGACGACG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-CCACTCGTAATTGACGCC	XhoI

961 ΔA-His	Fwd	CGCGGATCCCATATG-GCCACAAACGACGAC	NdeI
	Rev	CCCGCTCGAG-TGCTTTGGCGGCAAAGTT	XhoI

961a-(His/GST)	Fwd	CGCGGATCCCATATG-GCCACAAACGACGAC	BamHI-NdeI
	Rev	CCCGCTCGAG-TTTAGCAATATTATCTTTGTTCGTAGC	XhoI

961b-(His/GST)	Fwd	CGCGGATCCCATATG-AAAGCAAACCGTGCCGA	BamHI-NdeI
	Rev	CCCGCTCGAG-CCACTCGTAATTGACGCC	XhoI

961-His/GST^GATE	Fwd	ggggacaagtttgtacaaaaaagcaggctGCAGCCACAAACGACGACG	attB1
		ATGTTAAAAAAGC
	Rev	ggggaccactttgtacaagaaagctggggTTACCACTCGTAATTGACGC	attB2
		CGACATGGTAGG

982	Fwd	GCGGCCATAG-GCAGCAAAAGACGTACAGTT	NdeI
	Rev	GCGGCCTCGAG-TTACATCATGCCGCCCATACCA	XhoI

983-His	Fwd	CGCGGATCCGCTAGC-TTAGGCGGCGGCGGAG	NheI
(2996)	Rev	CCCGCTCGAG-GAACCGGTAGCCTACG	XhoI

ΔG983-His	Fwd	CCCCTAGCTAGC-ACTTCTGCGCCCGACTT	NheI
(2996)	Rev	CCCGCTCGAG-GAACCGGTAGCCTACG	XhoI

983-His	Fwd	CGCGGATCCGCTAGC-TTAGGCGGCGGCGGAG	NheI
	Rev	CCCGCTCGAG-GAACCGGTAGCCTACG	XhoI

ΔG983-His	Fwd	CGCGGATCCGCTAGC-ACTTCTGCGCCCGACTT	NheI
	Rev	CCCGCTCGAG-GAACCGTAGCCTACG	XhoI

983L	Fwd	CGCGGATCCGCTAGC-	NheI
		CGAACGACCCCAACCTTCCCTACAAAAACTTTCAA
	Rev	CCCGCTCGAG-TCAGAACCGACGTGCCAAGCCGTTC	XhoI

987-His	Fwd	GCCGCCATATGCCCCCACTGGAAGAACGGACG	NdeI
(MC48)	Rev	GCCGCCTCGAGTAATAAACCTTCTATGGGCAGCAG	XhoI

989-(His/GST)	Fwd	CGCGGATCCCATATG-TCCGTCCACGCATCCG	BamHI-NdeI
(MC58)	Rev	CCCGCTCGAG-TTTGAATTTGTAGGTGTATTG	XhoI

989L	Fwd	CGCGGATCCCATATG-ACCCCTTCCGCACT	NdeI
(MC58)	Rev	CCCGCTCGAG-TTATTTGAATTTGTAGGTGTAT	XhoI

CrgA-His	Fwd	CGCGGATCCCATATG-AAAACCAATTCAGAAGAA	NdeI
(MC58)	Rev	CCCGCTCGAG-TCCACAGAGATTGTTTCC	XhoI

PilC1-ES	Fwd	GATGCCCGAAGGGCGGG	XhoI
(MC58)	Rev	GCCCAAGCTT-TCAGAAGAAGACTTCACGC

PilC1-His	Fwd	CGCGGATCCCATATG-CAAACCCATAAATACGCTATT	NdeI
(MC58)	Rev	GCCCAAGCTT-GAAGAAGACTTCACGCCAG	HindIII

Δ1PilC1-His	Fwd	CGCGGATCCCATATG-GTCTTTTTCGACAATACCGA	NdeI
(MC58)	Rev	GCCCAAGCTT-	HindIII

PilC1L	Fwd	CGCGGATCCCATATG-AATAAAACTTTAAAAAGGCGG	NdeI
(MC58)	Rev	GCCCAAGCTT-TCAGAAGAAGACTTCACGC	HindIII

ΔGTbp2-His	Fwd	CGCGAATCCCATATG-TTCGATCTTGATTCTGTCGA	NdeI
(MC58)	Rev	CCCGCTCGAG-TCGCACAGGCTGTTGGCG	XhoI

Tbp2-His	Fwd	CGCGAATCCCATATG-TTGGGCGGAGGCGGCAG	NdeI
(MC58)	Rev	CCCGCTCGAG-TCGCACAGGCTGTTGGCG	XhoI

Tbp2-His	Fwd	CGCGAATCCCATATG-TTGGGCGGAGGCGGCAG	NdeI
(MC58)	Rev	CCCGCTCGAG-TCGCACGGCTGTTGGCG	XhoI

NMB0109-	Fwd	CGCGGATCCCATATG-GCAAATTTGGAGGTGCGC	BamHI-NdeI
(His/GST)	Rev	CCCGCTCGAG-TTCGGAGCGGTTGAAGC	XhoI
(MC58)

NMB0109L	Fwd	CGCGGATCCCATATG-CAACGTCGTATTATAACCC	NdeI
(MC58)	Rev	CCCGCTCGAG-TTATTCGGAGCGGTTGAAG	XhoI

NMB0207-	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(His/GST)		GGCATCAAAGTCGCCATCAACGGCTAC
(MC58)	Rev	CCCGCTCGAG-TTTGAGCGGGCGCACTTCAAGTCCG	XhoI

NMB0462-	Fwd	CGCGGATCCCATATG-GGCGGCAGCGAAAAAAAC	BamHI-NdeI
(His/GST)	Rev	CCCGCTCGAG-GTTGGTGCCGACTTTGAT	XhoI
(MC58)

NMB0623-	Fwd	CGCGGATCCCATATG-GGCGGCGGAAGCGATA	BamHI-NdeI
(His/GST)	Rev	CCCGCTCGAG-TTTGCCCGCTTTGAGCC	XhoI
(MC58)

NMB0625 (His-	Fwd	CGCGGATCCCATATGGGCAAATCCGAAAATACG	BamHI-NdeI
GST)(MC58)	Rev	CCCCGCTCGAGCATCCCGTACTGTTTCG	XhoI

NMB0634	Fwd	ggggacaagtttgtacaaaaaagcaggctCCGACATTACCGTGTACAAC	attB1
(His/GST)		GGCCAACAAAGAA
(MC58)	Rev	ggggaccactttgtacaagaaagctgggtCTTATTTCATACCGGCTTGCT	attB2
		CAAGCAGCCGG

NMB0776-	Fwd	ggggacaagtttgtacaaaaaagcaggctGATACGGTGTTTTCCTGTAA	attB1
His/GST		AACGGACAA
(MC58)^GATE	Rev	ggggaccactttgtacaagaaagctgggtCTAGGAAAAATCGTCATCGT	attB2
		TGAAATTCCC

NMB1115-	Fwd	ggggacaagtttgtacaaaaaagcaggctATGCACCCCATCGAAACC	attB1
His/GST	Rev	ggggaccactttgtacaagaaagctggggtCTAGTCTTGCAGTGCCTC	attB2
(MC58)^GATE

NMB1343-	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(His/GST)		GGAAATTTCTTATATAGAGGCATTAG	XhoI
(MC58)	Rev	CCCGCTCGAG-
		GTTAATTTCTATCAACTCTTTAGCAATAAT

NMB1369	Fwd	CGCGGATCCCATATGGCCTGCCAAGACGACA	BamHI-NdeI
(His-GST)	Rev	CCCGCTCGAGCCGCCTCCTGCCGAAA	XhoI
(MC58)

NMB1551	Fwd	CGCGGATCCCATATGGCAGAGATCTGTTTGATAA	BamHI-NdeI
(His-GST)	Rev	CCCGCTCGAGCGGTTTTCCGCCCAATG	XhoI
(MC58)

NMB1899	Fwd	CGCGGATCCCATATGCAGCCGGATACGGTC	BamHI-NdeI
(His-GST)	Rev	CCCGCTCGAGAATCACTTCCAACACAAAAT	XhoI
(MC58)

NMB2050-	Fwd	CGCGGATCCCATATG-TGGTTGCTGATGAAGGGC	BamHI-NdeI
(His/GST)	Rev	CCCGCTCGAG-GACTGCTTCATCTTCTGC	XhoI
(MC58)

NMB2050L	Fwd	CGCGGATCCCATATG-GAACTGATGACTGTTTTGC	NdeI
(MC58)	Rev	CCCGCTCGAG-TCAGACTGCTTCATCTTCT	XhoI

NMB2159-	Fwd	CGCGGATCCCATATG-	BamHI-NdeI
(His/GST)		AGCATTAAAGTAGCGATTAACGGTTTCGGC
(MC58)	Rev	CCCGCTCGAG-	XhoI
		GATTTTGCCTGCGAAGTATTCCAAAGTGCG

fu-ΔG287...-	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
His	Rev	CGGGGATCC-ATCCTGCTCTTTTTTGCCGG	BamHI

fu-(ΔG287)-	Fwd	CGCGGATCCGGTGGTGGTGGT-	NheI
919-His		CAAAGCAAGAGCATCCAAACC	BamHI
	Rev	CCCAAGCTT-TTCGGGCGGTATTCGGGCTTC

fu-(ΔG287)-	Fwd	CGCGGATCCGGTGGTGGTGGT-	BamHI
953-His	Rev	GCCACCTACAAAGTGGAC
		GCCCAAGCTT-TTGTTTGGCTGCCTCGAT	HindIII

fu-(ΔG287)-	Fwd	CGCGGATCCGGTGGTGGTGGT-ACAAGCGACGACG	BamHI
961-His	Rev	GCCCAAGCTT-CCACTCGTAATTGACGCC	HindIII

fu-(ΔG287)-	Fwd	CGCGGATCCGGTGGTGGTGGT-	BamHI
Orf46.1-His		TCAGATTTGGCAAACGATTC
	Rev	CCCAAGCTT-CGTATCATATTTCACGTGC	HindIII

fu-(ΔG287-	Fwd	CCCAAGCTTGGTGGTGGTGGTGGT-	HindIII
919)-Orf46.1-	Rev	TCAGATTTGGCAAACGATTC
His		CCCGCTCGAG-CGTATCATATTTCACGTGC	XhoI

fu-(ΔG287-	Fwd	CCCAAGCTTGGTGGTGGTGGTGGT-	HindIII
Orf46.1)-	Rev	CAAAGCAAGAGCATCCAAACC
His		CCCGCTCGAG-CGGGCGGTATTCGGGCTT	XhoI

fu ΔG287	Fwd	CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC	NheI
(394.98)-...	Rev	CGGGGATCC-ATCCTGCTCTTTTTTGCCGG	BamHI

fu Orf1-	Fwd	CGCGGATCCGCTAGC-GGACACACTTATTTCGGCATC	NheI
(Orf46.1)-	Rev	CGCGGATCC-CCAGCGGTAGCCTAATTTGAT
His

fu (Orf1)-	Fwd	CGCGGATCCGGTGGTGGTGGT-	BamHI
Orf46.1-His	Rev	TCAGATTTGGCAAACGATTC
		CCCAAGCTT-CGTATCATATTTCACGTGC	HindIII

fu (919)-	Fwd1	GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAG	SalI
Orf46.1-His	Fwd2	GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
	Rev	CCCGCTCGAG-CGTATCATATTTCACGTGC	XhoI

fu orf46-...	Fwd	GGAATTCCATATGTCAGATTTGGCAAACGATTC	NdeI
	Rev	CGCGGATCCCGTATCATATTTCACGTGC	BamHI

Fu (orf46)-	Fwd	CGGGGATCCGGGGGCGGCGGTGGCG	BamHI
287-His	Rev	CCCAAGCTTATCCTGCTCTTTTTTGCCGGC	HindIII

Fu (orf46)-	Fwd	CGCGGATCCGGTGGTGGTGGTCAAAGCAAGAGCATCCA	BamHI
919-His		AACC
	Rev	CCCAAGCTTCGGGCGGTATTCGGGCTTC	HindIII

Fu (orf46-919)-	Fwd	CCCCAAGCTTGGGGGCGGCGGTGGCG	HindIII
287-His	Rev	CCCGCTCGAGATCCTGCTCTTTTTTGCCGGC	XhoI

Fu (orf46-	Fwd	CCCAAGCTTGGTGGTGGTGGTGGTCAAAGCAAGAGCAT	HindIII
287)-919-His		CCAAACC
	Rev	CCCGCTCGAGCGGGCGGTATTCGGGCTT	XhoI

(ΔG741)-961c-	Fwd1	GGAGGCACTGGATCCGCAGCCACAAACGACGACGA	XhoI
His	Fwd2	GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
	Rev	CCCGCTCGAG-ACCCAGCTTGTAAGGTTG	XhoI

(ΔG741)-961-	Fwd1	GGAGGCACTGGATCCGCAGCCACAAACGACGACGA	XhoI
His	Fwd2	GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
	Rev	CCCGCTCGAG-CCACTCGTAATTGACGCC	XhoI

(ΔG741)-983-	Fwd	GCGGCCTCGAG-	XhoI
His	Rev	GGATCCGGCGGAGGCGGCACTTCTGCG
		CCCGCTCGAG-GAACCGGTAGCCTACG	XhoI

(ΔG741)-	Fwd1	GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC	SalI
orf46.1-His	Fwd2	GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA
	Rev	CCCGCTCGAG-CGTATCATATTTCACGTGC	XhoI

(ΔG983)-	Fwd	GCGGCCTCGAG-GGATCCGGAGGGGGTGGTGTCGCC	XhoI
741(MC58)-	Rev	CCCGCTCGAG-TTGCTTGGCGGCAAG	XhoI
His

(ΔG983)-	Fwd1	GGAGGCACTGGATCCGCAGCCACAAACGACGACGA	XhoI
961c-His	Fwd2	GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG	XhoI
	Rev	CCCGCTCGAG-ACCCAGCTTGTAAGGTTG	XhoI

(ΔG983)-	Fwd1	GGAGGCACTGGATCCGCAGCCACAAACGACGACGA	XhoI
961-His	Fwd2	GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG	XhoI
	Rev	CCCGCTCGAG-CCACTCGTAATTGACGCC	XhoI

(ΔG983)-	Fwd1	GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC	XhoI
Orf46.1-His	Fwd2	GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA	SalI
	Rev	CCCGCTCGAG-CGTATCATATTTCACGTGC	XhoI

*This primer was used as a Reverse primer for all the C terminal fusions of 287 to the His-tag.
^§Forward primers used in combination with the 287-His Reverse primer.
NB-All PCR reactions use strain 2996 unless otherwise specified (e.g. strain MC58)

In all constructs starting with an ATG not followed by a unique NheI site, the ATG codon is part of the NdeI site used for cloning. The constructs made using NheI as a cloning site at the 5° end (e.g. all those containing 287 at the N-terminus) have two additional codons (GCT AGC) fused to the coding sequence of the antigen.
Preparation of Chromosomal DNA Templates N. meningitidis strains 2996, MC58, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% w/v sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8) After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml of lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one CHCl₃/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes of ethanol, and collected by centrifugation. The pellet was washed once with 70% (v/v) ethanol and redissolved in 4.0 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA concentration was measured by reading OD₂₆₀.

PCR Amplification

The standard PCR protocol was as follows: 200 ng of genomic DNA from 2996, MC581000, or BZ232 strains or 10 ng of plasmid DNA preparation of recombinant clones were used as template in the presence of 40 μM of each oligonucletide primer, 400-800 μM dNTPs solution, lx PCR buffer (including 1.5 mM NgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, Boerhingher Mannheim Expand Long Template).
After a preliminary 3 minute incubation of the whole mix at 95° C., each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (T_m1). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (T_m2). Elongation times, performed at 68° C. or 72° C., varied according to the length of the Orf to be amplified. In the case of Orf1 the elongation time, starting from 3 minutes, was increased by 15 seconds each cycle. The cycles were completed with a 10 minute extension step at 72° C.
The amplified DNA was either loaded directly on a 1% agarose gel. The DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen Gel Extraction Kit, following the manufacturer's protocol.

Digestion of PCR Fragments and of the Cloning Vectors

The purified DNA corresponding to the amplified fragment was digested with the appropriate restriction enzymes for cloning into pET-21b+, pET22b+ or pET-24b+. Digested fragments were purified using the QIAquick PCR purification kit (following the manufacturer's instructions) and eluted with either H₂O or 10 mM Tris, pH 8.5. Plasmid vectors were digested with the appropriate restriction enzymes, loaded onto a 1.0% agarose gel and the band corresponding to the digested vector purified using the Qiagen QiAquick Gel Extraction Kit.

Cloning

The fragments corresponding to each gene, previously digested and purified, were ligated into pET21b+, pET22b+ or pET-24b+. A molar ratio of 3:1 fragment/vector was used with T4 DNA ligase in the ligation buffer supplied by the manufacturer.
Recombinant plasmid was transformed into competent E. coli DH5 or HB101 by incubating the ligase reaction solution and bacteria for 40 minutes on ice, then at 37° C. for 3 minutes. This was followed by the addition of 800 μl LB broth and incubation at 37° C. for 20 minutes. The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 2000 of the supernatant and plated onto LB ampicillin (100 mg/ml) agar.
Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37° C. in 4.0 ml of LB broth+100 μg/ml ampicillin. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately 1 μg of each individual miniprep was digested with the appropriate restriction enzymes and the digest loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 kb DNA Ladder, GIBCO). Positive clones were selected on the basis of the size of insert.

Expression

After cloning each gene into the expression vector, recombinant plasmids were transformed into E. coli strains suitable for expression of the recombinant protein. 1 μl of each construct was used to transform E. coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, to give an OD₆₀₀between 0.1 and 0.2. The flasks were incubated at 30° C. or at 37° C. in a gyratory water bath shaker until OD₆₀₀indicated exponential growth suitable for induction of expression (0.4-0.8 OD). Protein expression was induced by addition of 1.0 mM IPTG. After 3 hours incubation at 30° C. or 37° C. the OD₆₀₀was measured and expression examined. 1.0 ml of each sample was centrifuged in a microfuge, the pellet resuspended in PBS and analysed by SDS-PAGE and Coomassie Blue staining.

Gateway Cloning and Expression

Sequences labelled GATE were cloned and expressed using the GATEWAY Cloning Technology (GIBCO-BRL). Recombinational cloning (RC) is based on the recombination reactions that mediate the integration and excision of phage into and from the E. coli genome, respectively. The integration involves recombination of the attP site of the phage DNA within the attB site located in the bacterial genome (BP reaction) and generates an integrated phage genome flanked by attL and attR sites. The excision recombines attL and attR sites back to attP and attB sites (LR reaction). The integration reaction requires two enzymes [the phage protein Integrase (Int) and the bacterial protein integration host factor (IHF)] (BP clonase). The excision reaction requires Int, IHF, and an additional phage enzyme, Excisionase (His) (LR clonase), Artificial derivatives of the 25-bp bacterial attB recombination site, referred to as B1 and B2, were added to the 5 end of the primers used in PCR reactions to amplify Neisserial ORFs. The resulting products were BP cloned into a “Donor vector” containing complementary derivatives of the phage attP recombination site (P1 and P2) using BP clonase. The resulting “Entry clones” contain ORFs flanked by derivatives of the attL site (L1 and L2) and were subcloned into expression “destination vectors” which contain derivatives of the attL-compatible attR sites (R1 and R2) using LR clonase. This resulted in “expression clones” in which ORFs are flanked by B1 and B2 and fused in frame to the GST or His N terminal tags.
The E. coli strain used for GATEWAY expression is BL21-SI. Cells of this strain are induced for expression of the T7 RNA polymerase by growth in medium containing salt (0.3 M NaCl).
Note that this system gives N-terminus His tags.

Preparation of Membrane Proteins.

Fractions composed principally of either inner, outer or total membrane were isolated in order to obtain recombinant proteins expressed with membrane-localisation leader sequences. The method for preparation of membrane fractions, enriched for recombinant proteins, was adapted from Filip et. al. [J. Bact. (1973) 115:717-722] and Davies et. al. [J. Immunol. Meth. (1990) 143:215-225]. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD₅₅₀reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. and resuspended in 20 ml of 20 mM Tris-HCl (pH 7.5) and complete protease inhibitors (Boehringer-Mannheim). All subsequent procedures were performed at 4° C. or on ice.
Cells were disrupted by sonication using a Branson Sonifier 450 and centrifuged at 5000 g for 20 min to sediment unbroken cells and inclusion bodies. The supernatant, containing membranes and cellular debris, was centrifuged at 50000 g (Beckman Ti50, 29000 rpm) for 75 min, washed with 20 mM Bis-tris propane (pH 6.5), 1.0 M NaCl, 10% (v/v) glycerol and sedimented again at 50000 g for 75 minutes. The pellet was resuspended in 20 mM Tris-HCl (pH 7.5), 2.0% (v/v) Sarkosyl, complete protease inhibitor (1.0 mM EDTA, final concentration) and incubated for 20 minutes to dissolve inner membrane. Cellular debris was pelleted by centrifugation at 5000 g for 10 min and the supernatant centrifuged at 75000 g for 75 minutes (Beckman Ti50, 33000 rpm). Proteins 0081, and 519L were found in the supernatant suggesting inner membrane localisation. For these proteins both inner and total membrane fractions (washed with NaCl as above) were used to immunise mice. Outer membrane vesicles obtained from the 75000 g pellet were washed with 20 mM Tris-HCl (pH 7.5) and centrifuged at 75000 g for 75 minutes or overnight. The OMV was finally resuspended in 500 μl of 20 mM Tris-HCl (pH 7.5), 10% v/v glycerol. Orf1L and Orf40L were both localised and enriched in the outer membrane fraction which was used to immunise mice. Protein concentration was estimated by standard Bradford Assay (Bio-Rad), while protein concentration of inner membrane fraction was determined with the DC protein assay (Bio-R ad). Various fractions from the isolation procedure were assayed by SDS-PAGE.
Purification of his-Tagged Proteins
Various forms of 287 were cloned from strains 2996 and MC58. They were constructed with a C-terminus His-tagged fusion and included a mature form (aa 18-427), constructs with deletions (Δ1, Δ2, Δ3 and Δ4) and clones composed of either B or C domains. For each clone purified as a His-fusion, a single colony was streaked and grown overnight at 37° C. on a LB/Amp (100 μg/ml) agar plate. An isolated colony from this plate was inoculated into 20 ml of LB/Amp (100 μg/ml) liquid medium and grown overnight at 37° C. with shaking. The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 μg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37° C.) until the OD₅₅₀reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration 1.0 mM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000 g for 15 min at 4° C. The bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8.0) for soluble proteins or (ii) buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8 and, optionally, 8M urea) for insoluble proteins. Proteins purified in a soluble form included 287-His, Δ1, Δ2, Δ3 and Δ4287-His, Δ4287MC58-His, 287c-His and 287cMC58-His. Protein 287bMC58-His was insoluble and purified accordingly. Cells were disrupted by sonication on ice four times for 30 sec at 40 W using a Branson sonifier 450 and centrifuged at 13000×g for 30 ruin at 4° C. For insoluble proteins, pellets were resuspended in 2.0 ml buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5 and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000 g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 150 μl Ni²⁺-resin (previously equilibrated with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 min. The resin was Chelating Sepharose Fast Flow (Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700 g for 5 min at 4° C. and the supernatant discarded. The resin was washed twice (batch-wise) with 10 ml buffer A or B for 10 min, resuspended in 1.0 nil buffer A or B and loaded onto a disposable column. The resin continued to be washed with either (i) buffer A at 4° C. or (ii) buffer B at room temperature, until the OD₂₈₀of the flow-through reached 0.02-0.01. The resin was further washed with either (i) cold buffer C (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8.0) or (ii) buffer D (10 mM Tris-Ha, 100 mM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD₂₈₀of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8.0) or (ii) elution buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5 and, optionally, 8M urea) and fractions collected until the OD₂₈₀indicated all the recombinant protein was obtained. 20 μl aliquots of each elution fraction were analysed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.
Renaturation of Denatured his-Fusion Proteins.
Denaturation was required to solubilize 287bMC8, so a renaturation step was employed prior to immunisation. Glycerol was added to the denatured fractions obtained above to give a final concentration of 10% v/v. The proteins were diluted to 200 μg/ml using dialysis buffer I (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, 2.0M urea, pH 8.8) and dialysed against the same buffer for 12-14 hours at 4° C. Further dialysis was performed with buffer II (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was estimated using the formula:
Protein (mg/ml)=(1.55×OD₂₈₀)−(0.76×OD₂₆₀)

Amino Acid Sequence Analysis.

Automated sequence analysis of the NH₂-terminus of proteins was performed on a Beckman sequencer (LF 3000) equipped with an on-line phenylthiohydantoin-amino acid analyser (System Gold) according to the manufacturer's recommendations.

Immunization

Balb/C mice were immunized with antigens on days 0, 21 and 35 and sera analyzed at day 49.

Sera Analysis—ELISA

The acapsulated MenB M7 and the capsulated strains were plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO₂. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and bacteria were washed twice with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer; 1% BSA, 0.1% Tween-20, 0.1% NaN₃in PBS) were added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H₂O₂) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl 12.5% H₂SO₄was added to each well and OD₄₉₀was followed. The ELISA titers were calculated abitrarely as the dilution of sera which gave an OD₄₉₀value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD₄₉of 0.4 was higher than 1:400.

Sera Analysis—FACS Scan Bacteria Binding Assay

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO₂. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 025% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA in PBS, 0.4% NaN₃) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD₆₂₀of 0.05. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:100, 1:200, 1:400) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well, 100 μl of R-Phicoerytrin conjugated F(ab)₂goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power 15 mW) setting were: FL2 on; FSC-H threshold: 92; FSC PMT Voltage: E 01; SSC PMT: 474; Amp. Gains 6.1; FL-2 PMT: 586; compensation values: 0.

Sera Analysis—Bactericidal Assay

N. meningitidis strain 2996 was grown overnight at 37° C. on chocolate agar plates (starting from a frozen stock) with 5% CO₂. Colonies were collected and used to inoculate 7 ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD₆₂₀of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD₆₂₀reached the value of 0.23-0.24. Bacteria were diluted in 50 mM Phosphate buffer pH 7.2 containing 10 mM MgCl₂, 10 mM CaCl₂and 0.5% (w/v) BSA (assay buffer) at the working dilution of 10⁵CFU/ml. The total volume of the final reaction mixture was 50 μl with 25 μl of serial two fold dilution of test serum, 12.5 μl of bacteria at the working dilution, 12.5 μl of baby rabbit complement (final concentration 25%).
Controls included bacteria incubated with complement serum, immune sera incubated with bacteria and with complement inactivated by heating at 56° C. for 30′. Immediately after the addition of the baby rabbit complement, 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). The 96-wells plate was incubated for 1 hour at 37° C. with rotation. 7 μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 1). Agar plates were incubated for 18 hours at 37 degrees and the colonies corresponding to time 0 and time 1 were counted.

Sera Analysis—Western Blots

Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded onto a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., using transfer buffer (0.3% Tris base, 1.44% glycine, 20% (v/v) methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
The OMVs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO₂on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20 mM Tris-HCl pH 7.5, 2 mM EDTA. Heat inactivation was performed at 56° C. for 45 minutes and the bacteria disrupted by sonication for 5 minutes on ice (50% duty cycle, 50% output, Branson sonifier 3 mm microtip). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000 g at the temperature of 4° C. The pellet containing the membranes was resuspended in 2% sarkosyl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA and incubated at room temperature for 20 minutes to solubilise the inner membranes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000 g for 3 hours. The pellet, containing the outer membranes was washed in PBS and resuspended in the same buffer. Protein concentration was measured by the D.C. Bio-Rad Protein assay (Modified Lowry method), using BSA as a standard′.
Total cell extracts were prepared as follows: N. meningitidis strain 2996 was grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

961 Domain Studies

Cellular Fractions Preparation
Total lysate, periplasm, supernatant and OMV of E. coli clones expressing different domains of 961 were prepared using bacteria from over-night cultures or after 3 hours induction with IPTG. Briefly, the periplasm were obtained suspending bacteria in saccarose 25% and Tris 50 mM (pH 8) with polimixine 100 μg/ml. After 1 hr at room temperature bacteria were centrifuged at 13000 rpm for 15 min and the supernatant were collected. The culture supernatant were filtered with 0.2 μm and precipitated with TCA 50% in ice for two hours. After centrifugation (30 min at 13000 rp) pellets were rinsed twice with ethanol 70% and suspended in PBS. The OMV preparation was performed as previously described. Each cellular fraction were analyzed in SUS-PAGE or in Western Blot using the polyclonal anti-serum raised against GST-961.
Adhesion Assay
Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva) were maintained, in DMEM (Gibco) supplemented with 10% heat-inactivated FCS, 15 mM L-glutamin′ e and antibiotics.
For the adherence assay, sub-confluent culture of Chang epithelial cells were rinsed with PBS and treated with trypsin-EDTA (Gibco), to release them from the plastic support. The cells were then suspended in PBS, counted and dilute in PBS to 5×10⁵cells/ml.
Bacteria from over-night cultures or after induction with IPTG, were pelleted and washed twice with PBS by centrifuging at 13000 for 5 min. Approximately 2-3×10⁸(cfu) were incubated with 0.5 mg/ml FITC (Sigma) in 1 ml buffer containing 50 mM NaHCO₃and 100 mM NaCl pH 8, for 30 min at room temperature in the dark. FITC-labeled bacteria were wash 2-3 times and suspended in PBS at 1-1.5×10⁹/ml. 200 μl of this suspension (2-3×10⁸) were incubated with 2000 (1×10⁵) epithelial cells for 30 min a 37° C. Cells were than centrifuged at 2000 rpm for 5 min to remove non-adherent bacteria, suspended in 200 μl of PBS, transferred to FACScan tubes and read

Claims

1. A method of inducing a bactericidal immune response in an animal, comprising administering to the animal a composition comprising a non-lipidated polypeptide, which comprises an amino acid sequence having greater than 99% sequence identity to the amino acid sequence of ΔG741 from Neisseria meningitidis strain MC58, wherein the non-lipidated polypeptide is present in an immunologically effective amount that is effective to elicit bactericidal antibodies against a Neisseria meningitidis serogroup B strain in the animal.

2. The method according to claim 1, wherein the immunologically effective amount is at least 20 μg.

3. The method according to claim 2, wherein the immunologically effective amount is 50-200 μg.

4. The method according to claim 2, wherein said composition additionally comprises an effective amount of an adjuvant.

5. The method according to claim 4, wherein the adjuvant is aluminum hydroxide or aluminum phosphate.

6. The method according to claim 2, wherein said non-lipidated polypeptide does not comprise an N-terminal amino acid residue site for lipidation.

7. The method according claim 2, further comprising a pharmaceutically acceptable carrier, adjuvant, diluent or buffer.

8. The method according to claim 2, wherein the non-lipidated polypeptide elicits a bactericidal immune response against a heterologous Neisseria meningitidis strain in the animal.

9. The method according to claim 2, consisting essentially of the non-lipidated polypeptide.

10. The method according to claim 2, wherein the composition does not comprise a protein having an amino acid sequence of greater than 80% sequence identity to SEQ ID NO: 2534 in WO99/57280.

11. The method according to claim 2, wherein the non-lipidated polypeptide is a recombinant polypeptide.

12. The method according to claim 2, wherein the non-lipidated polypeptide is a fusion polypeptide.

13. The method according to claim 2, wherein the non-lipidated polypeptide is a purified polypeptide.

14. The method according to claim 2, wherein the non-lipidated polypeptide is conjugated to a carrier.

15. The method according to claim 2, wherein the non-lipidated polypeptide was expressed in E. coli.

16. The method according to claim 1, wherein the composition comprises

(a) the non-lipidated polypeptide; and

(b) an immunostimulatory effective amount of an aluminum hydroxide adjuvant.

17. The method according to claim 16, wherein the immunologically effective amount of the non-lipidated polypeptide is at least 20 μg.

18. The method according to claim 16, wherein the immunologically effective amount of the non-lipidated polypeptide is 50-200 μg.

19. The method according to claim 16, wherein said non-lipidated polypeptide does not comprise an N-terminal amino acid residue site for lipidation.

20. The method according to claim 16, further comprising a pharmaceutically acceptable carrier, diluent or buffer.

21. The method according to claim 16, wherein the non-lipidated polypeptide elicits a bactericidal immune response against a heterologous Neisseria meningitidis strain in the animal.

22. The method according to claim 16, consisting essentially of (a) and (b).

23. The method according to claim 16, wherein the composition does not comprise a protein having an amino acid sequence of greater than 80% sequence identity to SEQ ID NO: 2534 of WO99/57280.

24. The method according to claim 16, wherein the non-lipidated polypeptide is a recombinant polypeptide.

25. The method according to claim 16, wherein the non-lipidated polypeptide is a fusion polypeptide.

26. The method according to claim 16, wherein the non-lipidated polypeptide is a purified polypeptide.

27. The method according to claim 16, wherein the non-lipidated polypeptide is conjugated to a carrier.

28. The method according to claim 16, wherein the non-lipidated polypeptide comprises the amino acid sequence of ΔG741 from Neisseria meningitidis strain MC58.

29. The method according to claim 16, wherein the non-lipidated polypeptide was expressed in E. coli.