WO2008153429A2 - A protease from staphylococcus aureus, particularly spia or spib, peptides it recognises and their use - Google Patents
A protease from staphylococcus aureus, particularly spia or spib, peptides it recognises and their use Download PDFInfo
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- WO2008153429A2 WO2008153429A2 PCT/PL2008/000042 PL2008000042W WO2008153429A2 WO 2008153429 A2 WO2008153429 A2 WO 2008153429A2 PL 2008000042 W PL2008000042 W PL 2008000042W WO 2008153429 A2 WO2008153429 A2 WO 2008153429A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
Definitions
- a protease from Staphylococcus aureus particularly SpIA or SpIB, peptides it recognises and their use
- the present invention relates to a method of obtaining proteases from Staphylococcus aureus, particularly the proteases SpIA or SpIB, their use in the specific hydrolysis of a polypeptide chain, amino-acid sequence recognized by them and their use.
- Proteases proteinases, peptidases, proteolytic enzymes
- substrate specificity recognizing and hydrolysing only particular peptide bonds
- specific polypeptide hydrolysis primarily the following: enterokinase, factor X, thrombin, TEV protease, PreScissionTM protease, as well as V8 and trypsin, with lesser specificity but also widely used.
- the enzymes described are used primarily, but not solely, for removing the so-called marker polypeptides (also known as tags, fusion tags, polypeptides containing marker polypeptides, affinity tags, polypeptides containing affinity tags; all descriptions are used interchangibly in the following text), recombinant polypeptide fragments useful at various stages of analysis or production (such as ones for detection or purification) but undesirable in the finished product.
- marker polypeptides also known as tags, fusion tags, polypeptides containing marker polypeptides, affinity tags, polypeptides containing affinity tags; all descriptions are used interchangibly in the following text
- recombinant polypeptide fragments useful at various stages of analysis or production (such as ones for detection or purification) but undesirable in the finished product.
- the high specificity of the enzyme used in conjunction with an efficiently recognized site introduced in between the tag and a portion of the polypeptide being the desired final product facilitates the precise removal of the tag, without risking the degradation of the desired product.
- proteases with a narrow substrate specificity, which may find use as precise biotechnological tools (example patent descriptions: US 4 543 329, US 5 013 653, US 6 906 176, US 7 189 540 ).
- proteases of Staphylococcus aureus including proteases SpIB and SpIA
- proteases SpIB and SpIA are known and have been published in J. MoI. Biol. (2006) 358, 270-279.
- This report also revealed a an inefficient, laboratory method of producing recombinant SpIB protease in E. coli and examining the casein lytic activity of the above preparation using zymography.
- substrate specificity of SpIB nor its potential uses nor an efficient method of producing it were described.
- no method has been revealed of producing recombinant SpIA protein, nor of purifying native or recombinant SpIA.
- the proteolytic activity of this enzyme has not been demonstrated directly, and neither has its substrate specificity.
- the state of the art concerning the SpIA protein consists its amino-acid sequence and makes it possible to confirm its homology to the amino-acid sequences of chymotrypsin-like serine proteases.
- no method is known of producing the protein, whether this protein really is a protease, its substrate specificity remains unknown and by the same token its potential uses.
- chymotrypsin-like serine proteases are known (in the case of structural similarity the term "chymotrypsin-like” relates to the same group of proteases as the term “trypsin-like” and herein they are used interchangeably; only for descriptions of activity type are they differentiated, but as such they are not used in the description unless indicated otherwise), to which SpIA and SpIB also belong (SpIB -M ⁇ ROPS: SO 1.282).
- Amino-acid sequence similarity to other proteases of this group made it possible to include both proteases, SpIB and SpIA in the Sl family according to the generally accepted classification using the M ⁇ ROPS database (http://merops.sanger.ac.uk/; Rawlings, N.D., Morton, F.R. & Barrett, AJ. (2006) MEROPS: the peptidase database. Nucleic Acids Res 34, D270-D272).
- M ⁇ ROPS database states that: "All characterised peptidases belonging to the chymotrypsin-like family (family Sl) are endopeptidases.
- the goal of the present invention is to yield a highly specific protease and the method of its manufacture, and the characteristics of its activity facilitating its industrial use.
- SpIA peptidases possess a much higher than expected substrate specificity. Basing on this discovery, the authors propose new, specific substrates for the SpIB or SpIA proteases and methods of hydrolysis and/or producing proteins making use of such peptides (sequence fragments) and new uses for the SpIB or SpIA proteases. The generation of these results was made possible by a novel efficient method of producing the SpIB and SpIA proteases, which constitutes the next aspect of the present invention.
- the subject of the present invention is a polypeptide exhibiting affinity for the active centre of a protease of Staphylococcus aureus, e.g. recognized or recognized and hydrolized, particularly protease SpIA or SpIB, containing the amino-acid sequence Xaal-
- Xaal is an amino-acid selected from among: Trp, Tyr, Phe, VaI, He, Leu,
- Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser,
- Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He,
- Xaa4 is omitted or is any given amino-acid, preferentially selected from among: Ser, Thr,
- Xaa5 is omitted or is any given an amino-acid, whereas in the case of protease SpIB:
- Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser,
- Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg,
- Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy,
- Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn,
- Xaa5 is omitted or is any given an amino-acid, preferentially selected form among: Thr,
- a polypeptide according to the present invention is characterised in that in the case of protease SpIA it contains a sequence selected from among:
- T ⁇ -T ⁇ -Tyr-Thr T ⁇ -T ⁇ -Tyr
- Tyr-T ⁇ -T ⁇ -Tyr Tyr-T ⁇ -T ⁇
- Tyr-T ⁇ -Met-Asn
- Tyr-T ⁇ -Met Tyr-T ⁇ -Leu-Ser, Tyr-T ⁇ -Leu, Tyr-Leu-Phe, Phe-Leu-Phe, T ⁇ -Leu-Phe,
- T ⁇ -Glu-Leu-Gln-Gly T ⁇ -Glu-Leu-Gln-Ser, T ⁇ -Glu-Leu-Gln-Val, T ⁇ -Glu-Leu-Gln-
- AIa T ⁇ -Glu-Leu-Gln-Glu, T ⁇ -Glu-Leu-Gln-Met, T ⁇ -Glu-Leu-Gln-Gln, T ⁇ -Glu-Leu-
- Gln-Asn T ⁇ -Glu-Leu-Gln-Asp, T ⁇ -Glu-Leu-Gln, T ⁇ -Glu-Leu-Thr, T ⁇ -Glu-Val-Gln,
- Val-Glu-Leu-Gln T ⁇ -Gln-Leu-Asp, T ⁇ -Val-Leu-Gln, Phe-Glu-Val-Glu, Gly-Arg-Gly-
- Val-Gly Gly-Arg-Gly-Val
- Val-Glu-Ile-Asp Val-Val-Leu-Gln
- Val-Val-Leu-Gln-Ser He-
- the next subject of the present invention is a protein or peptide recognized by protease
- SpIA or SpIB containing an amino-acid sequence containing a polypeptide according to the present invention defined above.
- nucleotide sequences encoding a polypeptide according to the present invention defined above are also subjects of the present invention.
- the next subject of the present invention is a use of a polypeptide sequence according to the present invention defined above or its derivative in the production of a protein recognized by protease SpIA or SpIB or their derivatives.
- the next subject of the present invention is a use of a nucleotide sequences according to the present invention defined above or its derivatives in the production of a protein recognized by protease SpIA or SpIB or their derivatives.
- Xaal is an amino-acid selected from among: T ⁇ , Tyr, Phe, VaI, He, Leu
- Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser, Tyr, Phe, Asp and Pro,
- Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He, VaI, Thr,
- Xaa4 is omitted or is any given an amino-acid, preferentially selected from among: Ser, Thr, GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr, Xaa5 is omitted or is any given an amino-acid whereas in the case of protease SpIB:
- Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser, Thr or GIy,
- Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg, Lys, Ser or Thr,
- Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy
- Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn.
- Xaa5 is omitted or is any given an amino-acid.
- Both in the case of SpIB protease and SpIA protease Zl and Z2 are polypeptide(s) containing one or more amino-acids, where one of them denotes a polypeptide containing a desirable protein or peptide and the other a polypeptide containing a marker polypeptide, b) the fusion protein is isolated, preferentially using a chromatography technique using a medium exhibiting an affinity for the marker polypeptide, c) a hydrolysis reaction of the fusion protein is performed using a protease exhibiting the enzymatic activity of protease SpIA or protease SpIB and preferentially the desired protein is purified from the reaction mixture.
- a polypeptide containing a marker polypeptide also known as a tag, an affinity tag or marker polypeptide
- affinity chromatography describes a separation (fractionation) method of proteins or peptides containing marker polypeptide based on its affinity for any particular suitable media and therefore is used in a broader meening than the general use of the term.
- a specialist will be able to propose a series of sequences of this type based on common knowledge, which may be used to design an isolation system for the protein produced, particularly through affinity chromatography.
- the introduction of a sequence recognized by an antibody facilitates the isolation of a protein containing it using said antibody.
- Another example are amino-acid sequences containing an affinity for glutathione.
- the next example are techniques based on the well known formation of complexes of certain metal ions and some amino-acid residues. The best known example of this system is complex formation between nickel ions and the imidazole rings of histidines introduced into the polypeptide chain to be isolated.
- the next example are amino-acid sequences containing affinity for streptavidin or its derivatives.
- the next example are amino-acid sequences containing affinity for particular selected sugars.
- the next example are amino-acid sequences containing charged amino-acids having affinity for charged media.
- amino-acid sequences containing hydrophobic amino-acids having affinity for hydrophobic media consist of a marker amino-acid sequence and a substance for which such a sequence has a strong enough affinity make it possible to design a purification system for proteins containing the marker sequence. Usually this will be an affinity chromatography technique on a medium containing said substance.
- a desired protein being a part of a fusion protein according to the present invention described above may be, though without limitation, any given known protein, whose amino-acid sequence or coding sequence is known. For example, this may be a therapeutic protein, whose production is desirable due to its therapeutic properties.
- amino-acid sequences or sequences encoding known proteins may, for example but without limitation, be obtained from the GenBank database accessible at the URL http://www.ncbi.nlm.nih.gov/Genbank/index.html, which contains the sequences of known genes and amino-acid sequences of known proteins.
- a bacterial system or other system of choice To increase the level of expression of the fusion protein in a bacterial system or other system of choice, one may use known methods of increasing expression levels in bacterial cells or other cells of choice, which encompass by the way of example but without limitation the use of strong promoters, the use of transcription enhancing sequences or the use of preferentially used codons by a given bacterial cell or other cells of choice.
- a method according to the present invention is characterised in that in the case of protease SpIA a fusion protein contains a sequence selected from among: Zl -Trp- Leu-Tyr-Z2, Z 1 -Trp-Leu-Tyr-Ser-Z2, Z 1 -Tyr-Glu-Tyr-Ala-Z2, Z 1 -Tyr-Glu-Tyr-Ser-Z2, Zl-Tyr-Glu-Tyr-Z2, Zl-Tyr-Met-Tyr-Z2, Zl-Tyr-Met-Tyr-Ser-Z2, Zl-Tyr-Ala-Tyr-Ser- Z2, Zl-Tyr-Ala-Tyr-Z2, Zl-Tyr-Thr-Tyr-Ser-Z2, Zl-Tyr-Thr-Tyr-Z2, Zl-Tyr-Leu-Tyr- Gly-Z2, Zl-
- the hydrolysis is performed at 0°C to 45°C, and at a pH from 5.0 to 8.0 in the case of protease SpIA or at a pH from 5.0 to 9.0 in the case of protease SpIB.
- the hydrolysis is performed in a buffer with a concentration of 1 to 50OmM, wherein in the case of protease SpIA the buffer is an N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, TAPS, CHES, MOPS, Bis-Tris, phosphate, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine or Tris buffer, whereas in the case of protease SpIB the buffer is a N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, MOPS, Bis-
- the next the subject of the present invention is a variant of protease SpIA or SpIB, characterised in that it contains an amino-acid sequence containing at least one of the following modifications:
- a known secretory sequence a known bacterial secretory sequence, a known fungal secretory sequence, a sequence containing a methionine residue, a sequence of a polypeptide exhibiting affinity for the active centre of protease SpIA or SpIB according to the present invention as defined above, a sequence recognized by a proteolytic enzyme, a known marker polypeptide sequence, or a sequence of a polypeptide exhibiting the properties of a marker polypeptide.
- a protease variant according to the present invention is characterised in that the secretory sequence is a bacterial secretory sequence recognized by Bacillus subtilis or other gram positive bacterium or other gram negative bacterium or a strain of fungi.
- a protease variant according to the present invention is characterised in that it contains a sequence selected from among: SEQ ID No.: 4, SEQ ID NO: 6, SEQ
- the next the subject of the present invention is a nucleotide sequence encoding a protease variant according to the present invention as defined above.
- this nucleotide sequence contains a nucleotide sequence selected from among: SEQ ID No.: 3, SEQ ID NO: 5, SEQ ID No.: 9, SEQ ID NO: 11, SEQ ID NO: 13,
- the next the subject of the present invention is a method of producing protease SpIA or
- SpIB or their variants characterised in that: a) the expression of a protein according to the present invention, as defined above, is carried out in the cells of a bacteria or other host, e.g. fungal host cells, preferentially the protein is encoded by a nucleotide sequence according to the present invention defined above, and subsequently; b) the desired enzyme or a fraction containing it is isolated.
- the bacterial host is a strain of Bacillus subtilis or other gram positive bacterium or other gram negative bacterium or a strain of a fungi expressing a protein encoded by a nucleotide sequence designated SEQ ID No.: 3 or SEQ ID No.: 9.
- stage b) the fermentation broth is separated from the bacterial mass through centrifugation, and the secretory proteins in the medium separated from the bacteria are precipitated out with ammonium sulphate or other suitable agent, the precipitated proteins are separated and dissolved in a small volume of buffer and dialysed against a buffer with pH 5.5 ( ⁇ 1.5).
- the isolated protein is additionally purified using affinity chromatography, ion exchange chromatography, hydrophobic chromatography, reversed phase chormatography, and/or molecular sieving, and finally, the purified preparation is possibly concentrated and crystallised.
- the catalytic ability of the same enzyme or of another enzyme may be used to precisely hydrolyse the polypeptide chain.
- an enzyme is produced with an N-terminal or C-terminal fusion tag selected preferentially from the large pool of tags described or a new peptide with desirable tag qualities.
- a tag can be constituted by a histidine tag (His-tag), but is not limited to it.
- the fusion tag and the sequence of the protease according to the present invention are separated by an inserted sequence recognized and cleaved by a protease according to the present invention or a sequence recognized and cleaved by another enzyme capable of precisely hydrolysing the polypeptide chain. Following the production of said fusion protein, it is isolated using the properties of the tag and then the tag is cleaved off using catalytic properties of the protease according to the present invention or another enzyme capable of precisely hydrolysing the polypeptide chain.
- the protease of the present invention liberated from the tag increases the pool of active enzyme increasing the rate of cleavage.
- Cleavage of the tag can be performed directly in or on the media (column) used to isolate the fusion protein or following elution, where the former method facilitates the simultaneous purification of the protease from the fusion tag, whereas in the latter case, it is desirable to perform another purification step.
- the additional purification step is also desirable during the use of an enzyme capable of precisely hydrolysing the polypeptide chain other than the protease according to the present invention or when SpIB is prepared using SpIA or SpIA is prepared using SpIB to separate said enzyme from the protease according to the present invention.
- protease according to the present invention to specifically hydrolyse a polypeptide containing the amino-acid sequence Xaal-Xaa2-Xaa3-Xaa4-Xaa5 (ie. Yl-Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Y2, where
- Yl and Y2 denotes a polypeptide containing one or more amino-acids), where: in the case of protease SpIA:
- Xaal is an amino-acid selected from among: Trp, Tyr, Phe, VaI, He, Leu,
- Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser,
- Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He,
- Xaa4 is omitted or is any given an amino-acid, preferentially selected from among: Ser,
- Xaa5 is omitted or is any given amino-acid, whereas in the case of protease SpIB:
- Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser,
- Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg,
- Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy,
- Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn, where, preferentially,
- Xaa5 is omitted or is any given amino-acid preferentially selected form among: Thr, Ser,
- the hydrolysed polypeptide preferentially contains a sequence selected from among: Trp-Leu-Tyr, Trp-Leu-Tyr-Ser, Tyr-Glu-Tyr-Ala, Tyr-Glu-Tyr-Ser,
- Tyr-Glu-Tyr Tyr-Met-Tyr, Tyr-Met-Tyr-Ser, Tyr-Ala-Tyr-Ser, Tyr-Ala-Tyr, Tyr-Thr-Tyr-
- the hydrolysis is performed in a solution containing from 0 to 50OmM NaCl.
- the next the subject of the present invention is a protease exhibiting the activity of protease SpIA characterised in that it possesses an active centre encompassing a catalytic triad containing at least one from among the amino-acids: His, Asp and Ser, wherein the RMSD of Ca carbons of the main chain being a part of the amino-acids forming the catalytic triad is no greater than 2,2A, preferentially no greater than 1,8 A, in comparison with the Ca carbons of the main chain being a part of the amino-acids His39, Asp78 and Ser 154 contained in protease SpIA with a tertiary structure defined in Table 1.
- a protease according to the present invention is characterised in that the RMSD Ca carbons of the main chain within well defined secondary structures of the molecular core (thus not including loops, other mobile elements, fragments exposed to the outside of the molecule and its other features poorly defined in the state of the art) is no greater than 2A, preferentially no greater than 1,5 A, in combination with their corresponding structural Ca carbon atoms of the main chain contained in the protease SpIA with the tertiary structure defined in Table 1.
- a protease according to the present invention is characterised in that the well defined secondary structure of the molecular core contains fragments corresponding to the structure of the protein SpIA selected from among the following sequences: Val4 to Glu6, Asnl ⁇ to Ala20, Gly24 to Val29, Thr33 to Asn37, Val51 to Ala53, Asn64 to Val67, Ile70 to Glu72, Leu79 to His85, Argl l2 to Ilel l ⁇ , Metl28 to Ilel35, Phel42 to Phel45, Serl54 to Leul59, Glyl67 to Alal71, Asnl81 to Tyrl85, Glul92 to Glnl95.
- a protease according to the present invention is characterised in that it contains a fragment forming an ⁇ -helix corresponding to the structure of the fragment of the SpIA protein selected preferentially from among the following sequences: Lys38 to Ala41, Glul92 to Asnl96.
- a protease according to the present invention is characterised in that it contains a fragment forming an ⁇ - strandcorresponding to the structure of the fragment of the SpIA protein selected preferentially from among the following sequences: Val4 to Lys5, VaI 18 to Ala20, Thr25 to Val28, Thr33 to Thr36, Val51 to Ala53, Asn64 to Val67, Asp69 to Glu72, Ala80 to Val84, Argl 12 to Del 16, Phel29 to Glyl33, Phel42 to Phel45, VaI 158 to Leul59, GIy 167 to Alal71, Asnl81 to VaI 184.
- a protease according to the present invention is characterised in that it possesses a tertiary structure for which the RMSD of Ca carbons of the main chain is no larger than 2,5 A, preferentially no larger than 1 ,8 A, in combination with the Ca carbons of the main chain contained in protease SpIA with a tertiary structure defined in Table 1.
- a protease according to the present invention is characterised in that it contains structural elements preferentially selected from among:
- Val28 at the position corresponding to Val28 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- Val29 at the position corresponding to Val29 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- Val35 at the position corresponding to Val35 it contains an amino-acid selected from among: VaI, Leu, He, Ala; - at the position corresponding to Thr36 it contains an amino-acid selected from among: Ser, Thr;
- Val67 at the position corresponding to Val67 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- Val82 at the position corresponding to Val82 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- Val98 contains an amino-acid selected from among: VaI, Leu, He, Ala, Ser, Thr;
- Met 128 at the position corresponding to Met 128 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- Tyrl70 at the position corresponding to Tyrl70 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- GIu 177 at the position corresponding to GIu 177 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- protease exhibiting protease SpIB activity, characterised in that it contains an active centre formed by, among others, the catalytic triad His, Asp and Ser, wherein the RMSD of all atoms forming the amino-acids of the catalytic triad is no greater than 3.0A, preferentially no greater than 2 A, in combination with His 39, Asp 77 and Ser 157 contained in protease SpIB with a tertiary structure defined in Table 2.
- a protease exhibiting protease SpIB activity is characterised in that the RJVlSD of Ca carbons of the main chain within well defined secondary structures of the molecular core (thus not including loops, other mobile elements, fragments exposed to the outside of the molecule and its other features poorly defined in the state of the art) is no greater than 2 A, preferentially no greater than 1.5 A, in combination with their corresponding structural Ca carbon atoms of the main chain contained in the protease SpIB with the tertiary structure defined in Table 2.
- a protease exhibiting protease SpIB activity is characterised in that the well-defined secondary structure of the molecular core contains fragments corresponding to the structure of the protein SpIB selected from among the following sequences: VaW to Lys ⁇ , Thrl ⁇ to Ala20, Ala24 to Val29, Thr33 to Val40, Ile50 to Ala52, Ile63 to Asn71, Val78 to Glu84, Argl l5 to Ilel l9, Leul31 to Vall38, Serl45 to Tyrl48, Thrl52 to Leul62, Glyl70 to Serl75, Alal85 to Tyrl89, Lysl96 to Alal99.
- a protease exhibiting protease SpIB activity is characterised in that it contains a fragment forming an ⁇ -helix corresponding to the structure of the fragment of the SpIB protein selected preferentially from among the following sequences: Lys38 to Ser41, Lysl96 to Glu200.
- a protease exhibiting protease SpIB activity is characterised in that it contains a fragment forming a ⁇ - strand corresponding to the structure of the fragment of the SpIB protein selected preferentially from among the following sequences: Val4 to Thr5, VaI 18 to Ala20, Thr25 to Val28, Thr33 to Thr36, Arg49 to Ala52, Ile63 to Asn71, Ser79 to Val83, Argl l5 to Ilel l9, Tyrl32 to Glyl36, Serl45 to Tyrl48, Vall ⁇ l to Leul62, Glyl70 to Serl75, Alal85 to Vall88.
- a protease exhibiting protease SpIB activity is characterised in that it possesses a tertiary structure for which the RMSD of Ca carbons of the main chain is no larger than 3A, preferentially no larger than 2.5A, in comparison with the Ca carbons of the main chain contained in protease SpIB with a tertiary structure defined in Table 2.
- a protease exhibiting protease SpIB activity is characterised in that possesses the following structural elements:
- Val29 at the position corresponding to Val29 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- - at the position corresponding to Leu35 it contains an amino-acid selected from among: VaI, Leu, He, Ala; - at the position corresponding to Thr36 it contains an amino-acid selected from among: Ser, Thr;
- Val78 at the position corresponding to Val78 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- Val80 at the position corresponding to Val80 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- VaIl 18 at the position corresponding to VaIl 18 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Ser, Thr;
- Leul31 at the position corresponding to Leul31 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- the next the subject of the present invention is a method of storage of protease exhibiting protease SpIA activity according to the invention, as defined above, or of protease exhibiting protease SpIB activity according to the invention, as defined above, wherein the said enzymes are preferentially stored for more than 12 hours: a.) in solution preferentially at temperatures from +25 0 C to O 0 C b.) in solution containing antifreezing agent at temperatures from +25°C to -85 0 C c.) frozen at the temperatures form O 0 C to -200 0 C preferentially in a -2O 0 C freezer, -7O 0 C freezer, dry ice or liquid nitrogen d.) liophylized (freeze-dried) preferentially in moisture free containers or moisture free, vacuum sealed containers or moisture free, inert gas filled containers.
- Met methionine
- S Ser, serine
- T Thr
- C Cys, cysteine
- Y
- Tyr tyrosine
- N Asn, asparagine
- Q GIn glutamine
- D Asp, aspartic acid
- E GIu
- K Lys, lysine
- R Arg, arginine
- H His, histidine
- Xaa5 in the case of protease SpIB and Xaa4 and Xaa5 in the case of protease SpIA may be omitted or be any given amino-acid since, unexpectedly, SpIA and SpIB differ from other proteases characterised by high substrate specificity, which usually also exhibit specificity towards the amino-acid immediately following the hydrolysed bond (at the newly formed
- N-terminus arising from the hydrolysis of the peptide bond, meaning the Pl' position according to the nomenclature scheme proposed by: Schechter, L, and Berger, A. (1967)
- Another aspect of the present invention relates to proteins exhibiting a defined protease activity according to the present invention due to the retention by these proteins of the tertiary structure of the protease defined in Table 1 or 2 respectively.
- This parameter is root mean square distance (deviation), RMSD, or simply RMS (the notations should be treated as synonymous and were used as such in the description).
- the value of the RMSD parameter is calculated based on a comparison of the orientation of corresponding atoms following the superimposition of compared structures for best fit. The value of the parameter is expressed in Angstr ⁇ ms (A) and was used as such throughout the text. In general, the lower the value of the parameter, the higher the similarity of the structures.
- the subject of the present invention are proteins exhibiting proteolytic activity according to the present inventionbecause their tertiary structure is sufficiently similar to that of protease SpIA or SpIB. Said similarity is measured using the RMSD parameter for significant structural components of protease SpIA or SpIB in relation to corresponding structural components of the compared protein.
- the subject of the present invention is an enzyme which preferentially fulfills at least one of the structural criteria defined in the Claims.
- the subject of the present invention are thus proteins possessing residues corresponding with the following key amino-acid residues in the SpIA or SpIB sequences: a) residues of the so-called catalytic triad: S 154, H39 and D78 in SpIA, and S 157, H39 and D77 in SpIB. Substitution of these residues results in the complete loss of catalytic ability. For example, for the SpIB protease it has been shown that mutant S 157— >A completely lacks proteolytic activity. b) residues responsible for substrate recognition: for SpIA:
- Pl primarily S175, H172, T152 to N156, A174,
- P2 primarily F173, H39 and D77
- P4 primarily F173 and Y186, c) glutamic acid residue at the N-terminal end of the polypeptide chain, which is responsible for the stabilisation of the N-terminus of the protein through hydrogen bonding and by the same token facilitates the expression of full proteolytic activity.
- This residue may be substituted by an aspartic acid residue with similar physico-chemical properties or by glutamine or by asparagine.
- SpIB proteases makes it possible to compare their sequences via a structural comparison, and thereby to compare sequences of corresponding structural elements.
- the structure is compared, and where they are similar, the sequences are compared, even if they are not homologous in the classic sense.
- Such a solution carries much more information in comparison to an ordinary sequence alignment, because it indicates elements significant in the functioning of a protein.
- Such a comparison has been shown in Fig. 2, where appropriate fragments are grouped on the basis of structural similarity. This approach facilitates the differentiation of evidently conserved regions which are essential for protein functioning.
- a protein according to the present invention possessing the functional characteristics of SpIA should possess the following residues in locations corresponding to the following amino-acids in the SpIA sequence:
- Val28 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Val29 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Ile34 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Val35 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Thr36 - this optimally consists of amino-acids selected from among Ser, Thr; His39 - histidine of the catalytic triad; this optimally consists of His;
- Val67 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Ile70 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Asp78 - aspartic acid of the catalytic triad this optimally consists of Asp;
- Ile ⁇ l - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala, Met;
- Glnl50 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
- Asnl53 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
- Serl54 - serine of the catalytic triad described earlier, must be Ser;
- Glyl72 - this optimally consists of GIy
- Glul77 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
- Phel93 - this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
- Ilel94 this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala.
- a protein according to the present invention which possesses the functional characteristics of the SpIB possess the following residues in locations corresponding to the following amino-acids in the SpIB sequence:
- Glul - this optimally consists of amino-acids selected from among: GIu, Asp, GIn, Asn
- Val28 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Val29 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Ile34 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Ile66 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Ile69 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- Val78 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
- VaIl 18 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala,
- Tyrl21- this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
- Serl57 - serine of the catalytic triad described earlier, must be Ser;
- Ilel71 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala;
- Phel97 - this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
- Ilel98 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala.
- a particular embodiment of the present invention though without limitation is a protein possessing the structure of protease SpIA described in Table 1 or a protein possessing the structure of protease SpIB described in Table 2.
- both protease SpIA and protease SpIB recognise specific amino-acid sequences and hydrolyse the polypeptide chain immediately behind or within the recognised sequence. Due to the length of the recognised sequence (three or four sequential amino-acids for SpIA or SpIB respectively) the number of identical sequences in the human proteome and other proteomes is small, thus the enzyme is suitable for removing fusion tags during the production of the remaining majority of human proteins and proteins of other organisms.
- the present invention encompasses the amino-acid sequences of a polypeptide chain specifically recognised and hydrolysed by protease SpIA or SpIB, nucleotide sequences encoding said amino-acid sequences (thus facilitating the production of polypeptides containing them using recombinant protein technology) as well as a method of specifically hydrolysing polypeptides containing said amino-acid sequences using protease SpIA or SpIB.
- the present invention also encompasses proteases SpIA and
- SpIB themselves and enzymes posessing SpIA and SpIB activity due to close structural similarity as defined herein, as enzymes recognizing or recognizing and hydrolysing selected amino-acid sequences as well as methods of producing protease SpIA or SpIB in a recombinant system. Furthermore, the present invention encompasses synthetic substrates based on sequences specifically recognised and hydrolysed by protease SpIA or SpIB.
- the most preferential advantages of the present invention can be of use in the following processes: a) recognition of a specific amino-acid sequence in a polypeptide chain (particularly in the sequence of a recombinant protein) and its specific hydrolysis at a precisely determined site within the recognised sequence or a small distance away from it, b) the highly efficient production of the proteases SpIA and SpIB.
- Sequence No. 1 (SEQ ID NO 1) represents the sequence encoding protease SpIA from
- Staphylococcus aureus along with its native signal peptide.
- Sequence No. 2 (SEQ ID NO 2) represents the amino-acid sequence of protease SpIA from
- Staphylococcus aureus (mature protein: amino-acids from 1 to 200) along with its native signal peptide (amino-acids from -35 to -1).
- Sequence No. 3 represents the sequence encoding variant protease SpIA from Staphylococcus aureus, whose sequence encoding the native signal peptide has been replaced with a sequence encoding a signal peptide recognised by Bacillus subtilis.
- Sequence No. 4 represents the amino-acid sequence of variant of protease
- SpIA from Staphylococcus aureus, in which the native signal peptide sequence has been replaced with signal peptide recognised by Bacillus subtilis (amino-acids from -29 to -1).
- Sequence No. 5 represents the sequence encoding a fusion protein containing the sequence of mature SpIA from S. aureus with a histidine tag and a sequence recognized by SpIA whereas sequence No. 6 (SEQ ID NO 6) represents the amino-acid sequence of this protein.
- Sequence No. 7 represents the sequence encoding protease SpIB from
- Staphylococcus aureus along with its native signal peptide.
- Sequence No. 8 represents the amino-acid sequence of protease SpIB from
- Sequence No. 9 represents the sequence encoding a variant of protease SpIB from Staphylococcus aureus, in which the sequence encoding the native signal peptide has been replaced with a sequence encoding a signal peptide from Bacillus subtilis. Sequence No.
- SEQ ID NO 10 represents the amino-acid sequence of a variant of protease SpIB from Staphylococcus aureus, in which the native signal peptide sequence has been replaced with a signal peptide sequence from Bacillus subtilis (amino-acids from -29 to -l).
- Sequence No. 11 represents the sequence encoding a variant of protease SpIB from S. aureus with a signal peptide from B. subtilis containing the substitution S157A whereas sequence No. 12 (SEQ ID NO 12) represents the amino-acid sequence of this a variant.
- Sequence No. 13 represents the sequence encoding a variant of protease SpIB from S. aureus with a signal peptide from B. subtilis containing the substitution H39A whereas sequence No. 14 (SEQ ID NO 14) represents the amino-acid sequence of this variant.
- Sequence No. 15 represents the sequence encoding a variant of protease SpIB from 5. aureus with a signal peptide from B. subtilis containing the substitution D77A whereas sequence No. 16 (SEQ ID NO 16) represents the amino-acid sequence of this variant.
- Sequence No. 17 represents the sequence encoding a fusion protein containing the sequence of mature SpIB from S. aureus with an attached histidine tag and a sequence recognized by SpIB whereas sequence No. 18 (SEQ ED NO 18) represents the amino-acid sequence of this protein.
- Figure Ia contains a comparison of the amino-acid sequences of closely related proteases: protease SpIA, protease SpIC, V8 (protease V8 from Staphylococcus aureus also known as glutamylendopeptidase), ETA - epidermolytic toxin A from Staphylococcus aureus as well as a distantly related enzyme, trypsin (a model enzyme of the trypsin-like proteases), whereas Figure Ib contains a comparison of amino-acid sequences of closely related proteases: protease SpIB, protease SpIC, V8 (protease V8 from Staphylococcus aureus also known as glutamylendopeptidase), ETA - epidermolytic toxin A from Staphylococcus aureus as well as a distantly related enzyme, trypsin.
- protease SpIA protease SpIC
- V8 protetease V8
- Figure 2a represents a comparison of the amino-acid sequences of closely related proteases based on their tertiary structures as well as the tertiary structure determined for protease SpIA; (chymotryps - chymotrypsin; enterokina - enterokinase; czynnik - factor X (ten)); whereas Figure 2b represents a comparison of the amino-acid sequences of closely related proteases based on their tertiary structures as well as the tertiary structure determined for protease SpIB; in both cases residues particularly significant for structure retention and protease activity have been indicated.
- the initial experiment which made subsequent experiments possible was the determination of pH and temperature optima, and allowable other salt and reagent concentrations as well as enzyme stability. For this purpose, it was necessary to design a method of quantifying enzyme activity.
- known chymotrypsin-] ike elastase-like and trypsin-like protease substrates only one substrate has been identified as one even minimally digested by protease SpIA (N-Suc-Ala-Ala-Pro-Phe-pNA). Other multiple tested substrates were not hydrolysed.
- the enzymes in question retain activity in the presence of reagents including without limitation: imidazole, glutathione, DTT, mercaptoethanol, biotin, EDTA, ammonium sulphate, maltose, or sacharose.
- the enzymes in question can be stored frozen without noticeable activity loss, as well as being frozen and melted repeatedly. They can be lyophilised as well. They can also be stored at 4°C without significant activity loss. All of the above conditions constitute advantageous forms of storage of the enzymes, which is very important in everyday practice.
- coli ⁇ -galactosidase carbonic anhydrase, human serum alpha-2-macroglobulin, cytochrome c, goat IgG, RNAse, fibrinogen, whale myoglobin, a series of human and murine serpins) do not undergo proteolysis even during an extended incubation with an excess of enzyme.
- protease SpIA requires an amino-acid with a large, relatively hydrophobic side chain (Y, F) at Pl of the substrate (bold).
- Y, F relatively hydrophobic side chain
- protease SpIB requires glutamine (Q) residues at Pl of the substrate
- protease SpIB was contaminated with other proteolytic activity.
- protease SpIB can, like the closely related protease SpIC, be a protein lacking proteolytic activity. J. MoI.
- Example 3 A method of producing the proteases SpIA and SpIB SEQ ID NO: 1 and 2 respectively represent the nucleotide sequence of the gene encoding protease SpIA of Staphylococcus aureus as well as its corresponding amino-acid sequence.
- the nucleotide numeration begins from “a(l)” of the translation start triplet (atg) and ends at "a(708)" of the translation stop triplet (taa).
- the polypeptide chain of the protease arises via translation in conjunction with the signal peptide (amino-acid residues from M(-35) to A(-l)) which is cleaved off by a signal protease during secretion.
- protease SpIA The active, extracellular form of protease SpIA is produced, which can be harvested from the culture medium (amino-acid residues from El to K200).
- SEQ ID NO: 7 and 8 respectively represent the nucleotide sequence of the gene encoding protease SpIB from Staphylococcus aureus as well as its corresponding amino-acid sequence.
- the nucleotide numeration begins from "a(l)" of the translation start triplet (atg) and ends at "a(723)" of the translation stop triplet (taa).
- the polypeptide chain of the protease arises via translation in conjunction with the signal peptide (amino-acid residues from M(-36) to A(-l)) which is cleaved off by a signal protease during secretion.
- the active, extracellular form of protease SpIA is produced, which can be harvested from the culture medium (amino-acid residues from El to K204). From herewith in this description we use the numeration introduced in these two sequences.
- Sequences encoding the mature protease SpIA (El to K200) or SpIB (El to K204) have been cloned into appropriate expression plasmids, yielding a plasmid facilitating the production of the extracellular mature protease SpIA or SpIB in gram-positive bacteria.
- the sequence of the fusion protein consisting of the secretory signal sequence recognized by B. subtilis as well as the mature form of the SpIA or SpIB proteases, and a nucleotide sequence encoding the proteins has been shown in SEQ ID No. 4 and in SEQ ED No. 3 for SpIA and in SEQ ID No. 10 and SEQ ID No. 9 for SpIB.
- B. subtilis bacteria were transformed with an expression plasmid and transformant selection was performed on plates containing kanamycine (50 ⁇ g/ml). Selected clones were used to inoculate small volumes of liquid medium (TSB; Sigma) containing a selective antibiotic and incubated at 37°C with intensive mixing for 8 to 10 h. Such a start culture was then used to inoculate the main culture (4-16L of liquid medium with antibiotics) and incubated with intensive mixing at 37°C for 13 to 16 hours. All of the purification steps were carried out at 4°C. The bacteria were separated from the medium by centrifugation at 6000xg for 30min.
- the secreted protein in the medium separated from the bacteria was precipitated out using ammonium sulphate to 80% saturation (561g/L w 4 0 C).
- the precipitated proteins were separated from the medium using centrifugation (15000xg, Ih), dissolved in a small volume of acetate buffer, 50 mM pH 5.5, and dialysed overnight in a large excess volume of the same buffer.
- the dialysed sample was subjected to ion-exchange chromatography on SP Sepharose FF
- fractions containing the peak protein levels were collected. Collected fractions eluted in buffer with a conductivity of about 27 mS/cm for SpIA or 30 mS/cm for
- the method described in example 3 made it possible to efficiently produce the protein in question facilitating the further analysis of its structure, in particular the production of the crystalline form of the proteases SpIA and SpIB and the determination of their tertiary structure, which in effect resulted in the determination of the substrate specificity of both proteases.
- the determined coordinates of individual atoms of the mature SpIA and SpIB proteins were collected in Table 1 and Table 2 respectively.
- the analysis of the constructed model showed that protease SpIA and SpIB both exhibit a structure characteristic of Sl family proteases (trypsin-like/chymotrypsin-like) without any indication in the structure of the catalytic triad warranting the weak activity observed.
- P2 primarily Y170, H39 and D78,
- S157 ⁇ A completely lacks proteolytic activity.
- Pl primarily S175, H172, T152 to N156, A174
- P2 primarily F173, H39 and D77
- P4 primarily F173 and Y186
- protease SpIB A comparison of the tertiary structures of a fully active form (identical to the native protein) and poorly active form (containing two additional amino-acids at the N-terminus) of protease SpIB indicates the role of the precise positioning of the N-terminus of the protein as well as the initial glutamic acid residue (El).
- FIG 1 V28 to V40; D77 to 181; G120 to P122 as well as G155 to 1171. Furthermore, it is clear that individual residues are conserved: 150; S134; 1146; V188 and 1198.
- proteases SpIA and SpIB do not cleave other proteins other than ⁇ -casein, which also contain whole series of Phe and Tyr residues (for SpIA) as well as Asp, Asn and GIn (for SpIB), it was only at this stage of the research that it became obvious that contrary to the state of the art the trypsin-like proteases SpIA and SpIB likely possess a much higher substrate specificity in comparison with their closely related (protease V8) and distantly related (trypsin, chymotrypsin and others) homologues.
- the initial stage of selection selects fluorescent cells (where the protein of interest is expressed) whereafter these cells are put into contact with the tested protease and then, the cells which do not exhibit fluorescence are selected, meaning those for which the fluorescent portion has been cleaved off.
- the gene encoding the protein of interest is examined from the strains selected in this manner, allowing to determine the cleavage sequences.
- This method made it possible to sieve 64 million substrates and to gather information regarding amino- acids at positions P5 to Pl', not only Pl (as in the technique described above). Using this method, we selected the following sequences respectively recognized and cleaved by protease SpIA:
- LVPR*GS thrombin cleavage site in the above plasmid
- YLY*S a sequence encoding the determined as described above consensus sequence specific for SpIA
- WELQ*G the determined as described above consensus sequence specific for SpIB
- plasmids facilitating the expression of a GST-SpIA cleavage site-staphostatin A fusion protein or a GST-SpIB cleavage site-staphostatin A fusion protein respectively.
- Such proteins were produced in E. coli bacteria and purified using the affinity of the GST fusion protein for immobilised glutathione as described in MoI. Microbiol. (2003).
- protease SpIA and protease SpIB are not catalytically inefficient enzymes, as was suggested by the ⁇ -casein and carboxymethylated lysosyme digestions. To the contrary, this means that they are catalytically very efficient enzymes, but only with respect to substrates of an appropriate sequence, which is unexpectedly (concerning the state of the art) much moree ⁇ ! cd than in the case of known trypsin-like proteases.
- staphostatin A released each time from the fusion protein through SpIA or SpIB digestion and determined, using the Edman degradation method, its N-terminal sequence showing that protease SpIA and SpIB digest specifically and precisely within the recognised sequence only in the spot indicated by * (i.e. SpIA: YLY*S, and SpIB: WELQ*G).
- PCNA Proliferating Cell Nuclear Antigen
- the purified protein (35kDa) was subjected to SpIB digestion at 5:1 protein to
- Tris-HCl buffer pH 8.0 Tris-HCl buffer pH 8.0.
- the cleavage products were analysed by SDS-PAGE.
- the recombinant protein was efficiently cleaved by SpIB (98% at 24 hours) at the inserted site producing as expected a 32 kDa fragment which was not cleaved, degraded or otherwise further affected by prolonged incubation with SpIB.
- SpIB cleavage of fusion protein Histag-LVPR-CRP cAMP receptor protein (CRP; also known as catabolite gene activator protein [CAP]) was expressed with an N-terminal His-tag fusion (Histag-LVPR-CRP).
- the original construct contained thrombin cleavage site.
- the purified protein 25kDa was subjected to SpIB digestion at 9:1 protein to SpIB molar ratio at room temperature for 1, 7, and 24 hours in 5OmM Tris-HCl buffer pH 8.0.
- the cleavage products were analysed by SDS-PAGE. No recombinant protein cleavage or degradation was detected in any of the samples.
- thrombin cleavage site in the original construct was exchanged to a site recognized by SpIB protease (LVPR ⁇ WELQ).
- the purified Histag- WELQ-CRP was subjected to SpIB digestion at 25:1 and 50:1 protein to SpIB molar ratio at room temperature for 2, 4, 6, 9 and 24 hours in 5OmM Tris-HCl buffer pH 8.0.
- the cleavage products were analysed by SDS-PAGE.
- the recombinant protein was efficiently cleaved by SpIB (95% and 75% at 24hours respectively at 25:1 and 50:1 protein to SpIB molar ratio) at the inserted site producing as expected a 22 kDa fragment which was not cleaved, degraded or otherwise further affected by prolonged incubation with SpIB.
- SpIB cleavage of fusion protein GST-LVPR-CRP(W85L) cAMP receptor protein (CRP; also known as catabolite gene activator protein [CAP]) W85L mutant was expressed with an N-terminal GST (Glutathione S-transferase) fusion (GST-LVPR-CRP(W85L)).
- the original construct contained thrombin cleavage site.
- the purified protein (49kDa) was subjected to SpIB digestion at 3:1 protein to SpIB molar ratio at room temperature for 1, 7, and 24 hours in 5OmM Tris-HCl buffer pH 8.0.
- the cleavage products were analysed by SDS-PAGE. No recombinant protein cleavage was detected in any of the samples.
- Following the thrombin cleavage site in the original construct was exchanged to a site recognized by SpIB protease (LVPR ⁇ WELQ).
- the purified GST- WELQ-CRP(W85L) was subjected to SpIB digestion at 25:1 and 50:1 protein to SpIB molar ratio at room temperature for 2, 4, 6, 9 and 24 hours in 5OmM Tris-HCl buffer pH 8.0.
- the cleavage products were analysed by SDS-PAGE.
- the recombinant protein was efficiently cleaved by SpIB (95% at 9 and 24 hours respectively for 25:1 and 50:1 protein to SpIB molar rato) at the inserted site producing as expected a 26kDa and 22kDa fragments which were not cleaved, degraded or otherwise further affected by prolonged incubation with SpIB.
- CD GLU 1 9.251 40.165 10.946 93 CG2 ILE 12 21.983 40.226 30.542
- CD LYS 6 22.759 49.707 18.992 132 O THR 16 28.076 33.759 25.400
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Abstract
The invention discloses a method of obtaining proteases from Staphylococcus aureus, particularly the proteases SpIA or SpIB, their use in the specific hydrolysis of a polypeptide chain, amino-acid sequence recognized by them and their use.
Description
A protease from Staphylococcus aureus, particularly SpIA or SpIB, peptides it recognises and their use
The present invention relates to a method of obtaining proteases from Staphylococcus aureus, particularly the proteases SpIA or SpIB, their use in the specific hydrolysis of a polypeptide chain, amino-acid sequence recognized by them and their use. Proteases (proteinases, peptidases, proteolytic enzymes) exhibiting a high substrate specificity (recognizing and hydrolysing only particular peptide bonds) are used on a wide scale in laboratories and the biotechnological industry for specific polypeptide hydrolysis (primarily the following: enterokinase, factor X, thrombin, TEV protease, PreScission™ protease, as well as V8 and trypsin, with lesser specificity but also widely used). The enzymes described are used primarily, but not solely, for removing the so-called marker polypeptides (also known as tags, fusion tags, polypeptides containing marker polypeptides, affinity tags, polypeptides containing affinity tags; all descriptions are used interchangibly in the following text), recombinant polypeptide fragments useful at various stages of analysis or production (such as ones for detection or purification) but undesirable in the finished product. In theory, the high specificity of the enzyme used in conjunction with an efficiently recognized site introduced in between the tag and a portion of the polypeptide being the desired final product facilitates the precise removal of the tag, without risking the degradation of the desired product. However, the absence of entirely specific proteolytic enzymes means that in many cases their activity results in not only the desired removal of the fusion tag, but also the non-specific degradation of the polypeptide of interest, should it contain sites similar to the sequence specifically recognized by the enzyme. Furthermore, the most popular of the enzymes used today are not available as recombinant proteins or, due to production difficulties, are much more expensive than native proteins isolated from blood (thrombin, factor X) or intestines (enterokinase). The use of native proteins, however, carries the risk of contaminating the preparations with the undesirable activity of other enzymes or pathogens. These factors create a real need for novel enzymes which will fulfil specific tasks.
It is particularly preferential to obtain proteases with a narrow substrate specificity, which may find use as precise biotechnological tools (example patent descriptions: US 4 543 329, US 5 013 653, US 6 906 176, US 7 189 540 ).
The amino-acid sequences of certain proteases of Staphylococcus aureus, including proteases SpIB and SpIA, are known and have been published in J. MoI. Biol. (2006) 358, 270-279. This report also revealed a an inefficient, laboratory method of producing
recombinant SpIB protease in E. coli and examining the casein lytic activity of the above preparation using zymography. Neither the substrate specificity of SpIB nor its potential uses nor an efficient method of producing it were described. To date, no method has been revealed of producing recombinant SpIA protein, nor of purifying native or recombinant SpIA. The proteolytic activity of this enzyme has not been demonstrated directly, and neither has its substrate specificity. Thus, the state of the art concerning the SpIA protein consists its amino-acid sequence and makes it possible to confirm its homology to the amino-acid sequences of chymotrypsin-like serine proteases. However, no method is known of producing the protein, whether this protein really is a protease, its substrate specificity remains unknown and by the same token its potential uses. Furthermore, many chymotrypsin-like serine proteases are known (in the case of structural similarity the term "chymotrypsin-like" relates to the same group of proteases as the term "trypsin-like" and herein they are used interchangeably; only for descriptions of activity type are they differentiated, but as such they are not used in the description unless indicated otherwise), to which SpIA and SpIB also belong (SpIB -MΕROPS: SO 1.282). Amino-acid sequence similarity to other proteases of this group made it possible to include both proteases, SpIB and SpIA in the Sl family according to the generally accepted classification using the MΕROPS database (http://merops.sanger.ac.uk/; Rawlings, N.D., Morton, F.R. & Barrett, AJ. (2006) MEROPS: the peptidase database. Nucleic Acids Res 34, D270-D272). The widely held belief which is also expressed in the leading protease reference, the MΕROPS database, states that: "All characterised peptidases belonging to the chymotrypsin-like family (family Sl) are endopeptidases. There are also numerous homologues not being peptidases themselves, in which the catalytic residues have been substituted. There are three main types of activity: trypsin-like, in which the digestion of the amide substrate occurs following Arg or Lys residues at position Pl, chymotrypsin- like, in which digestion occurs following a hydrophobic amino-acid at Pl and elastase-like, in which digestion occurs behind an Ala residue at Pl. The substrate specificity of the Sl family depends solely on the amino-acid located at Pl. Most peptidases of this family undergo secretion and possess an N-terminal signal peptide. They are synthesized in the form of (mostly inactive) precursors with an additional N-terminal sequence, whose removal yields the active form of the enzyme. Activation does not always require the removal of the propeptide.". As is shown in the further sections of this description, general indications in the state of the art can lead only to false conclusions regarding the substrate
specificity of both proteases from Staphylococcus aureus, SpIB and SpIA, and to regard them as enzymes of little industrial value.
Given the above described state of the art, the goal of the present invention is to yield a highly specific protease and the method of its manufacture, and the characteristics of its activity facilitating its industrial use.
Unexpectedly, the authors of the present invention have ascertained that both the SpIB and
SpIA peptidases possess a much higher than expected substrate specificity. Basing on this discovery, the authors propose new, specific substrates for the SpIB or SpIA proteases and methods of hydrolysis and/or producing proteins making use of such peptides (sequence fragments) and new uses for the SpIB or SpIA proteases. The generation of these results was made possible by a novel efficient method of producing the SpIB and SpIA proteases, which constitutes the next aspect of the present invention.
The subject of the present invention is a polypeptide exhibiting affinity for the active centre of a protease of Staphylococcus aureus, e.g. recognized or recognized and hydrolized, particularly protease SpIA or SpIB, containing the amino-acid sequence Xaal-
Xaa2-Xaa3-Xaa4-Xaa5, where: for protease SpIA:
Xaal is an amino-acid selected from among: Trp, Tyr, Phe, VaI, He, Leu,
Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser,
Tyr, Phe, Asp or Pro,
Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He,
VaI, Thr,
Xaa4 is omitted or is any given amino-acid, preferentially selected from among: Ser, Thr,
GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr,
Xaa5 is omitted or is any given an amino-acid, whereas in the case of protease SpIB:
Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser,
Thr or GIy,
Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg,
Lys, Ser or Thr,
Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy,
Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn,
Xaa5 is omitted or is any given an amino-acid, preferentially selected form among: Thr,
Ser, VaI, GIy, Ala, GIu, Met, GIn, Asp, Asn.
Preferentially a polypeptide according to the present invention is characterised in that in the case of protease SpIA it contains a sequence selected from among:
Trp-Leu-Tyr, Trp-Leu-Tyr-Ser, Tyr-Glu-Tyr-Ala, Tyr-Glu-Tyr-Ser, Tyr-Glu-Tyr, Tyr-
Met-Tyr, Tyr-Met-Tyr-Ser, Tyr-Ala-Tyr-Ser, Tyr-Ala-Tyr, Tyr-Thr-Tyr-Ser, Tyr-Thr-Tyr,
Tyr-Leu-Tyr-Gly, Tyr-Leu-Tyr, Tyr-Leu-Tyr-Ser, Phe-Leu-Tyr-Ser, Phe-Leu-Tyr, VaI-
Leu-Tyr-Thr, Val-Leu-Tyr, Tφ-Leu-Ser-Thr, Trp-Leu-Ser, Trp-Met-Asn-Thr, Trp-Met-
Asn, Tφ-Tφ-Tyr-Thr, Tφ-Tφ-Tyr, Tyr-Tφ-Tφ-Tyr, Tyr-Tφ-Tφ, Tyr-Tφ-Met-Asn,
Tyr-Tφ-Met, Tyr-Tφ-Leu-Ser, Tyr-Tφ-Leu, Tyr-Leu-Phe, Phe-Leu-Phe, Tφ-Leu-Phe,
Tyr-Leu-Tφ, Phe-Leu-Tφ, Tφ-Leu-Tφ, whereas in the case of protease SpIB it preferentially contains sequence selected from among:
Tφ-Glu-Leu-Gln-Gly, Tφ-Glu-Leu-Gln-Ser, Tφ-Glu-Leu-Gln-Val, Tφ-Glu-Leu-Gln-
AIa, Tφ-Glu-Leu-Gln-Glu, Tφ-Glu-Leu-Gln-Met, Tφ-Glu-Leu-Gln-Gln, Tφ-Glu-Leu-
Gln-Asn, Tφ-Glu-Leu-Gln-Asp, Tφ-Glu-Leu-Gln, Tφ-Glu-Leu-Thr, Tφ-Glu-Val-Gln,
Val-Glu-Leu-Gln, Tφ-Gln-Leu-Asp, Tφ-Val-Leu-Gln, Phe-Glu-Val-Glu, Gly-Arg-Gly-
Val-Gly, Gly-Arg-Gly-Val, Val-Glu-Ile-Asp, Val-Val-Leu-Gln, Val-Val-Leu-Gln-Ser, He-
Glu-Ser-Gln, Ile-Glu-Ser-Gln-Ser.
The next subject of the present invention is a protein or peptide recognized by protease
SpIA or SpIB, containing an amino-acid sequence containing a polypeptide according to the present invention defined above.
The next subjects of the present invention are nucleotide sequences encoding a polypeptide according to the present invention defined above, as well as nucleotide sequences encoding a protein according to the present invention defined above.
The next subject of the present invention is a use of a polypeptide sequence according to the present invention defined above or its derivative in the production of a protein recognized by protease SpIA or SpIB or their derivatives.
The next subject of the present invention is a use of a nucleotide sequences according to the present invention defined above or its derivatives in the production of a protein recognized by protease SpIA or SpIB or their derivatives.
The next the subject of the present invention is a method of producing desired proteins characterised in that: a) a fusion protein is produced containing the sequence Zl-Xaal-Xaa2-Xaa3-Xaa4-Xaa5-
Z2, wherein in the case of protease SpIA:
Xaal is an amino-acid selected from among: Tφ, Tyr, Phe, VaI, He, Leu,
Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser, Tyr, Phe, Asp and Pro,
Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He, VaI, Thr,
Xaa4 is omitted or is any given an amino-acid, preferentially selected from among: Ser, Thr, GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr, Xaa5 is omitted or is any given an amino-acid whereas in the case of protease SpIB:
Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser, Thr or GIy,
Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg, Lys, Ser or Thr,
Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy, Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn. Xaa5 is omitted or is any given an amino-acid.
Both in the case of SpIB protease and SpIA protease Zl and Z2 are polypeptide(s) containing one or more amino-acids, where one of them denotes a polypeptide containing a desirable protein or peptide and the other a polypeptide containing a marker polypeptide, b) the fusion protein is isolated, preferentially using a chromatography technique using a medium exhibiting an affinity for the marker polypeptide, c) a hydrolysis reaction of the fusion protein is performed using a protease exhibiting the enzymatic activity of protease SpIA or protease SpIB and preferentially the desired protein is purified from the reaction mixture.
For the purposes of this description a polypeptide containing a marker polypeptide, also known as a tag, an affinity tag or marker polypeptide, should be understood as a sequence facilitating the isolation of a polypeptide containing it, particularly through affinity chromatography or other desirable methods. For the purpose of this description affinity chromatography describes a separation (fractionation) method of proteins or peptides containing marker polypeptide based on its affinity for any particular suitable media and therefore is used in a broader meening than the general use of the term. A specialist will be able to propose a series of sequences of this type based on common knowledge, which may be used to design an isolation system for the protein produced, particularly through affinity chromatography. For example, but without limitation, the introduction of a sequence recognized by an antibody facilitates the isolation of a protein containing it using said
antibody. Another example are amino-acid sequences containing an affinity for glutathione. The next example are techniques based on the well known formation of complexes of certain metal ions and some amino-acid residues. The best known example of this system is complex formation between nickel ions and the imidazole rings of histidines introduced into the polypeptide chain to be isolated. The next example are amino-acid sequences containing affinity for streptavidin or its derivatives. The next example are amino-acid sequences containing affinity for particular selected sugars. The next example are amino-acid sequences containing charged amino-acids having affinity for charged media. The next example are amino-acid sequences containing hydrophobic amino-acids having affinity for hydrophobic media. All such systems, without any limitation to the above examples, consist of a marker amino-acid sequence and a substance for which such a sequence has a strong enough affinity make it possible to design a purification system for proteins containing the marker sequence. Usually this will be an affinity chromatography technique on a medium containing said substance. A desired protein being a part of a fusion protein according to the present invention described above may be, though without limitation, any given known protein, whose amino-acid sequence or coding sequence is known. For example, this may be a therapeutic protein, whose production is desirable due to its therapeutic properties. Based on the instructions revealed in the present description and common knowledge, a specialist will be able to design a sequence encoding a fusion protein containing the sequence encoding a desired protein. Amino-acid sequences or sequences encoding known proteins may, for example but without limitation, be obtained from the GenBank database accessible at the URL http://www.ncbi.nlm.nih.gov/Genbank/index.html, which contains the sequences of known genes and amino-acid sequences of known proteins. To increase the level of expression of the fusion protein in a bacterial system or other system of choice, one may use known methods of increasing expression levels in bacterial cells or other cells of choice, which encompass by the way of example but without limitation the use of strong promoters, the use of transcription enhancing sequences or the use of preferentially used codons by a given bacterial cell or other cells of choice.
Preferentially, a method according to the present invention is characterised in that in the case of protease SpIA a fusion protein contains a sequence selected from among: Zl -Trp- Leu-Tyr-Z2, Z 1 -Trp-Leu-Tyr-Ser-Z2, Z 1 -Tyr-Glu-Tyr-Ala-Z2, Z 1 -Tyr-Glu-Tyr-Ser-Z2, Zl-Tyr-Glu-Tyr-Z2, Zl-Tyr-Met-Tyr-Z2, Zl-Tyr-Met-Tyr-Ser-Z2, Zl-Tyr-Ala-Tyr-Ser- Z2, Zl-Tyr-Ala-Tyr-Z2, Zl-Tyr-Thr-Tyr-Ser-Z2, Zl-Tyr-Thr-Tyr-Z2, Zl-Tyr-Leu-Tyr-
Gly-Z2, Zl-Tyr-Leu-Tyr-Z2, Zl-Tyr-Leu-Tyr-Ser-Z2, Zl-Phe-Leu-Tyr-Ser-Z2, Zl-Phe- Leu-Tyr-Z2, Zl-Val-Leu-Tyr-Thr-Z2, Zl-Val-Leu-Tyr-Z2, Zl-Tφ-Leu-Ser-Thr-Z2, Zl- Trp-Leu-Ser-Z2, Zl-Trp-Met-Asn-Thr-Z2, Zl-Trp-Met-Asn-Z2, Zl-Tφ-Tφ-Tyr-Thr-Z2, Zl-Tφ-Tφ-Tyr-Z2, Zl-Tyr-Tφ-Tφ-Tyr-Z2, Zl-Tyr-Tφ-Tφ-Z2, Zl-Tyr-Tφ-Met-Asn- Z2, Zl-Tyr-Tφ-Met-Z2, Zl-Tyr-Tφ-Leu-Ser-Z2, Zl-Tyr-Tφ-Leu-Z2, Zl-Tyr-Leu-Phe- Z2, Zl-Phe-Leu-Phe-Z2, Zl-Tφ-Leu-Phe-Z2, Zl-Tyr-Leu-Tφ-Z2, Zl-Phe-Leu-Tφ-Z2, Z 1-Tφ-Leu-Tφ-Z2, whereas in the case of protease SpIB a fusion protein preferentially contains a sequence selected from among: Zl-Tφ-Glu-Leu-Gln-Gly-Z2, Zl-Tφ-Glu-Leu- Gln-Z2, Zl-Tφ-Glu-Leu-Gln-Ser-Z2, Zl-Tφ-Glu-Leu-Gln-Val-Z2, Zl-Tφ-Glu-Leu-Gln- Ala-Z2, Zl-Tφ-Glu-Leu-Gln-Glu-Z2, Zl-Tφ-Glu-Leu-Gln-Met-Z2, Zl-Tφ-Glu-Leu- Gln-Gln-Z2, Zl-Tφ-Glu-Leu-Gln-Asn-Z2, Zl-Tφ-Glu-Leu-Gln-Asp-Z2, Zl-Tφ-Glu- Leu-Thr-Z2, Zl-Tφ-Glu-Val-Gln-Z2, Zl-Val-Glu-Leu-Gln-Z2, Zl-Tφ-Gln-Leu-Asp-Z2, Zl-Tφ-Val-Leu-Gln-Z2, Zl-Phe-Glu-Val-Glu-Z2, Zl-Gly-Arg-Gly-Val-Gly-Z2, Zl-GIy- Arg-Gly-Val-Z2, Zl-Val-Glu-Ile-Asp-Z2, Zl-Val-Val-Leu-Gln-Z2, Zl-Val-Val-Leu-Gln- Ser-Z2, Zl-Ile-Glu-Ser-Gln-Z2, Zl-Ile-Glu-Ser-Gln-Ser-Z2.
In a preferential embodiment of a method according to the present invention, the hydrolysis is performed at 0°C to 45°C, and at a pH from 5.0 to 8.0 in the case of protease SpIA or at a pH from 5.0 to 9.0 in the case of protease SpIB. Equally preferentially, the hydrolysis is performed in a buffer with a concentration of 1 to 50OmM, wherein in the case of protease SpIA the buffer is an N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, TAPS, CHES, MOPS, Bis-Tris, phosphate, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine or Tris buffer, whereas in the case of protease SpIB the buffer is a N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, MOPS, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine, TAPS, ethanolamine, CHES, phosphate, Bis-Tris, CAPS or Tris buffer. Equally preferentially, the hydrolysis is performed in a solution containing 0 to 50OmM NaCl.
The next the subject of the present invention is a variant of protease SpIA or SpIB, characterised in that it contains an amino-acid sequence containing at least one of the following modifications:
- substitution of histidine at position 39 of the SpIB sequence for another amino-acid,
- substitution of histidine at position 39 of the SpIA sequence for another amino-acid,
- substitution of aspartic acid at position 77 of the SpIB sequence for another amino-acid,
- substitution of aspartic acid at position 78 of the SpIA sequence for another amino-acid,
- substitution of serine at position 157 of the SpIB sequence for another amino-acid,
- substitution of serine at position 154 of the SpIA sequence for another amino-acid,
- attachment via a peptide bond to the amino-acid at the N-terminal or C-terminal end of the mature protease SpIA or SpIB of a polypeptide containing at least one of the following sequences: a known secretory sequence, a known bacterial secretory sequence, a known fungal secretory sequence, a sequence containing a methionine residue, a sequence of a polypeptide exhibiting affinity for the active centre of protease SpIA or SpIB according to the present invention as defined above, a sequence recognized by a proteolytic enzyme, a known marker polypeptide sequence, or a sequence of a polypeptide exhibiting the properties of a marker polypeptide.
- substitution of surface residues of SpIA or SpIB as respectively defined in Table 1 and Table 2 with residues allowing convenient attachment by chemical methods of entities allowing immobilization or specific tags
- incorporation into the sequence of protease SpIA or SpIB of a marker polypeptide sequence preferrentially in a manner not affecting the hydrolytic activity towards polypeptides according to the present invention as defined above.
Preferentially, but without limitation, a protease variant according to the present invention is characterised in that the secretory sequence is a bacterial secretory sequence recognized by Bacillus subtilis or other gram positive bacterium or other gram negative bacterium or a strain of fungi.
Equally preferentially, a protease variant according to the present invention is characterised in that it contains a sequence selected from among: SEQ ID No.: 4, SEQ ID NO: 6, SEQ
ID No.: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18.
The next the subject of the present invention is a nucleotide sequence encoding a protease variant according to the present invention as defined above.
Preferentially this nucleotide sequence contains a nucleotide sequence selected from among: SEQ ID No.: 3, SEQ ID NO: 5, SEQ ID No.: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15 or SEQ ID NO: 17.
The next the subject of the present invention is a method of producing protease SpIA or
SpIB or their variants characterised in that: a) the expression of a protein according to the present invention, as defined above, is carried out in the cells of a bacteria or other host, e.g. fungal host cells, preferentially the
protein is encoded by a nucleotide sequence according to the present invention defined above, and subsequently; b) the desired enzyme or a fraction containing it is isolated.
In a preferential embodiment of the method according to the present invention, the bacterial host is a strain of Bacillus subtilis or other gram positive bacterium or other gram negative bacterium or a strain of a fungi expressing a protein encoded by a nucleotide sequence designated SEQ ID No.: 3 or SEQ ID No.: 9.
In an equally preferential embodiment of the method according to the present invention, during stage b) the fermentation broth is separated from the bacterial mass through centrifugation, and the secretory proteins in the medium separated from the bacteria are precipitated out with ammonium sulphate or other suitable agent, the precipitated proteins are separated and dissolved in a small volume of buffer and dialysed against a buffer with pH 5.5 (±1.5).
In an equally preferential embodiment of the method according to the present invention, during stage b) the isolated protein is additionally purified using affinity chromatography, ion exchange chromatography, hydrophobic chromatography, reversed phase chormatography, and/or molecular sieving, and finally, the purified preparation is possibly concentrated and crystallised.
In one of the preferential embodiments of the method of producing an active protease according to the present invention, the catalytic ability of the same enzyme or of another enzyme may be used to precisely hydrolyse the polypeptide chain. In this method, an enzyme is produced with an N-terminal or C-terminal fusion tag selected preferentially from the large pool of tags described or a new peptide with desirable tag qualities. Such a tag can be constituted by a histidine tag (His-tag), but is not limited to it. The fusion tag and the sequence of the protease according to the present invention are separated by an inserted sequence recognized and cleaved by a protease according to the present invention or a sequence recognized and cleaved by another enzyme capable of precisely hydrolysing the polypeptide chain. Following the production of said fusion protein, it is isolated using the properties of the tag and then the tag is cleaved off using catalytic properties of the protease according to the present invention or another enzyme capable of precisely hydrolysing the polypeptide chain. In the case of cleavage using protease SpIA (for SpIA fusions), or SpIB (for SpIB fusions) the protease of the present invention liberated from the tag increases the pool of active enzyme increasing the rate of cleavage. Cleavage of the tag can be performed directly in or on the media (column) used to isolate the fusion protein or
following elution, where the former method facilitates the simultaneous purification of the protease from the fusion tag, whereas in the latter case, it is desirable to perform another purification step. The additional purification step is also desirable during the use of an enzyme capable of precisely hydrolysing the polypeptide chain other than the protease according to the present invention or when SpIB is prepared using SpIA or SpIA is prepared using SpIB to separate said enzyme from the protease according to the present invention.
The next the subject of the present invention is the use of the protease according to the present invention to specifically hydrolyse a polypeptide containing the amino-acid sequence Xaal-Xaa2-Xaa3-Xaa4-Xaa5 (ie. Yl-Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Y2, where
Yl and Y2 denotes a polypeptide containing one or more amino-acids), where: in the case of protease SpIA:
Xaal is an amino-acid selected from among: Trp, Tyr, Phe, VaI, He, Leu,
Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser,
Tyr, Phe, Asp, Pro,
Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He,
VaI, Thr,
Xaa4 is omitted or is any given an amino-acid, preferentially selected from among: Ser,
Thr, GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr
Xaa5 is omitted or is any given amino-acid, whereas in the case of protease SpIB:
Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser,
Thr or GIy,
Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg,
Lys, Ser or Thr,
Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy,
Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn, where, preferentially,
Xaa5 is omitted or is any given amino-acid preferentially selected form among: Thr, Ser,
VaI, GIy, Ala, GIu, Met, GIn, Asp, Asn. in the case of protease SpIA, the hydrolysed polypeptide preferentially contains a sequence selected from among: Trp-Leu-Tyr, Trp-Leu-Tyr-Ser, Tyr-Glu-Tyr-Ala, Tyr-Glu-Tyr-Ser,
Tyr-Glu-Tyr, Tyr-Met-Tyr, Tyr-Met-Tyr-Ser, Tyr-Ala-Tyr-Ser, Tyr-Ala-Tyr, Tyr-Thr-Tyr-
Ser, Tyr-Thr-Tyr, Tyr-Leu-Tyr-Gly, Tyr-Leu-Tyr, Tyr-Leu-Tyr-Ser, Phe-Leu-Tyr-Ser,
Phe-Leu-Tyr, Val-Leu-Tyr-Thr, Val-Leu-Tyr, Trp-Leu-Ser-Thr, Tφ-Leu-Ser, Trp-Met- Asn-Thr, Trp-Met-Asn, Tφ-Tφ-Tyr-Thr, Tφ-Tφ-Tyr, Tyr-Tφ-Tφ-Tyr, Tyr-Tφ-Tφ, Tyr-Tφ-Met-Asn, Tyr-Tφ-Met, Tyr-Tφ-Leu-Ser, Tyr-Tφ-Leu, Tyr-Leu-Phe, Phe-Leu- Phe, Tφ-Leu-Phe, Tyr-Leu-Tφ, Phe-Leu-Tφ, Tφ-Leu-Tφ, whereas in the case of protease SpIB the hydrolysed polypeptide preferentaially contains a sequence selected from among: Tφ-Glu-Leu-Gln-Gly, Tφ-Glu-Leu-Gln-Ser, Tφ-Glu- Leu-Gln-Val, Tφ-Glu-Leu-Gln-Ala, Tφ-Glu-Leu-Gln-Glu, Tφ-Glu-Leu-Gln-Met, Tφ- Glu-Leu-Gln-Gln, Tφ-Glu-Leu-Gln-Asn, Tφ-Glu-Leu-Gln-Asp, Tφ-Glu-Leu-Gln, Tφ- Glu-Leu-Thr, Tφ-Glu-Val-Gln, Val-Glu-Leu-Gln, Tφ-Gln-Leu-Asp, Tφ-Val-Leu-Gln, Phe-Glu-Val-Glu, Gly-Arg-Gly-Val-Gly, Gly-Arg-Gly-Val, Val-Glu-Ile-Asp, Val-Val- Leu-Gln, Val-Val-Leu-Gln-Ser, Ile-Glu-Ser-Gln, Ile-Glu-Ser-Gln-Ser. Equally preferentially, the hydrolysis is performed at a temperature ranging from 0°C to 45°C, pH from 4.0 to 10.0, preferentially from 5.0 to 8.0. Equally preferentially, the hydrolysis is performed in an N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl -malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, TAPS, CHES, MOPS, Bis-Tris, phosphate, CAPS triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine, ethanolamine or Tris buffer with a concentration of 1 to 25OmM. Equally preferentially, the hydrolysis is performed in a solution containing from 0 to 50OmM NaCl. The next the subject of the present invention is a protease exhibiting the activity of protease SpIA characterised in that it possesses an active centre encompassing a catalytic triad containing at least one from among the amino-acids: His, Asp and Ser, wherein the RMSD of Ca carbons of the main chain being a part of the amino-acids forming the catalytic triad is no greater than 2,2A, preferentially no greater than 1,8 A, in comparison with the Ca carbons of the main chain being a part of the amino-acids His39, Asp78 and Ser 154 contained in protease SpIA with a tertiary structure defined in Table 1. Preferentially, a protease according to the present invention is characterised in that the RMSD Ca carbons of the main chain within well defined secondary structures of the molecular core (thus not including loops, other mobile elements, fragments exposed to the outside of the molecule and its other features poorly defined in the state of the art) is no greater than 2A, preferentially no greater than 1,5 A, in combination with their corresponding structural Ca carbon atoms of the main chain contained in the protease SpIA with the tertiary structure defined in Table 1.
Equally preferentially, a protease according to the present invention is characterised in that the well defined secondary structure of the molecular core contains fragments corresponding to the structure of the protein SpIA selected from among the following sequences: Val4 to Glu6, Asnlό to Ala20, Gly24 to Val29, Thr33 to Asn37, Val51 to Ala53, Asn64 to Val67, Ile70 to Glu72, Leu79 to His85, Argl l2 to Ilel lό, Metl28 to Ilel35, Phel42 to Phel45, Serl54 to Leul59, Glyl67 to Alal71, Asnl81 to Tyrl85, Glul92 to Glnl95.
Equally preferentially, a protease according to the present invention is characterised in that it contains a fragment forming an α-helix corresponding to the structure of the fragment of the SpIA protein selected preferentially from among the following sequences: Lys38 to Ala41, Glul92 to Asnl96.
Equally preferentially, a protease according to the present invention is characterised in that it contains a fragment forming an β- strandcorresponding to the structure of the fragment of the SpIA protein selected preferentially from among the following sequences: Val4 to Lys5, VaI 18 to Ala20, Thr25 to Val28, Thr33 to Thr36, Val51 to Ala53, Asn64 to Val67, Asp69 to Glu72, Ala80 to Val84, Argl 12 to Del 16, Phel29 to Glyl33, Phel42 to Phel45, VaI 158 to Leul59, GIy 167 to Alal71, Asnl81 to VaI 184.
Equally preferentially, a protease according to the present invention is characterised in that it possesses a tertiary structure for which the RMSD of Ca carbons of the main chain is no larger than 2,5 A, preferentially no larger than 1 ,8 A, in combination with the Ca carbons of the main chain contained in protease SpIA with a tertiary structure defined in Table 1. Equally preferentially, a protease according to the present invention is characterised in that it contains structural elements preferentially selected from among:
- at the position corresponding to GIu 1 of the sequence of SpIA it contains an amino-acid selected from among: GIu, Asp, GIn, Asn;
- at the position corresponding to Val28 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val29 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile34 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val35 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Thr36 it contains an amino-acid selected from among: Ser, Thr;
- at the position corresponding to His39 it contains His;
- at the position corresponding to Val67 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile70 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Asp78 it contains Asp;
- at the position corresponding to Leu79 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile81 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Val82 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Val98 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Ser, Thr;
- at the position corresponding to GIy 117 it contains GIy;
- at the position corresponding to Tyrl lδ it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to Met 128 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Alal49 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Glnl50 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Prol51 it contains Pro;
- at the position corresponding to GIy 152 it contains GIy;
- at the position corresponding to Asnl53 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Serl54 it contains Ser;
- at the position corresponding to GIy 155 it contains GIy;
- at the position corresponding to Ser 156 it contains an amino-acid selected from among: VaI, Ala, Ser, Thr, GIy;
- at the position corresponding to Prol57 it contains Pro;
- at the position corresponding to GIy 167 it contains GIy;
- at the position corresponding to He 168 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Leul69 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Tyrl70 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to Alal71 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to GIy 172 it contains GIy;
- at the position corresponding to GIu 177 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Serl78 it contains an amino-acid selected from among: Ser, Thr, VaI, Leu, He, Ala;
- at the position corresponding to Asnlδl it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Phe 193 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to Ilel94 it contains an amino-acid selected from among: VaI, Leu, He, Ala.
The next the subject of the present invention is a protease exhibiting protease SpIB activity, characterised in that it contains an active centre formed by, among others, the catalytic triad His, Asp and Ser, wherein the RMSD of all atoms forming the amino-acids of the catalytic triad is no greater than 3.0A, preferentially no greater than 2 A, in combination with His 39, Asp 77 and Ser 157 contained in protease SpIB with a tertiary structure defined in Table 2.
Preferentially a protease exhibiting protease SpIB activity according to the present invention is characterised in that the RJVlSD of Ca carbons of the main chain within well defined secondary structures of the molecular core (thus not including loops, other mobile elements, fragments exposed to the outside of the molecule and its other features poorly defined in the state of the art) is no greater than 2 A, preferentially no greater than 1.5 A, in combination with their corresponding structural Ca carbon atoms of the main chain contained in the protease SpIB with the tertiary structure defined in Table 2.
Preferentially, a protease exhibiting protease SpIB activity according to the present invention is characterised in that the well-defined secondary structure of the molecular core contains fragments corresponding to the structure of the protein SpIB selected from among the following sequences: VaW to Lysό, Thrlό to Ala20, Ala24 to Val29, Thr33 to Val40, Ile50 to Ala52, Ile63 to Asn71, Val78 to Glu84, Argl l5 to Ilel l9, Leul31 to Vall38, Serl45 to Tyrl48, Thrl52 to Leul62, Glyl70 to Serl75, Alal85 to Tyrl89, Lysl96 to Alal99.
Preferentially, a protease exhibiting protease SpIB activity according to the present invention is characterised in that it contains a fragment forming an α-helix corresponding to the structure of the fragment of the SpIB protein selected preferentially from among the following sequences: Lys38 to Ser41, Lysl96 to Glu200.
Preferentially, a protease exhibiting protease SpIB activity according to the present invention is characterised in that it contains a fragment forming a β- strand corresponding to the structure of the fragment of the SpIB protein selected preferentially from among the following sequences: Val4 to Thr5, VaI 18 to Ala20, Thr25 to Val28, Thr33 to Thr36, Arg49 to Ala52, Ile63 to Asn71, Ser79 to Val83, Argl l5 to Ilel l9, Tyrl32 to Glyl36, Serl45 to Tyrl48, Vallόl to Leul62, Glyl70 to Serl75, Alal85 to Vall88. Preferentially, a protease exhibiting protease SpIB activity according to the present invention is characterised in that it possesses a tertiary structure for which the RMSD of Ca carbons of the main chain is no larger than 3A, preferentially no larger than 2.5A, in comparison with the Ca carbons of the main chain contained in protease SpIB with a tertiary structure defined in Table 2.
Preferentially, a protease exhibiting protease SpIB activity according to the present invention is characterised in that possesses the following structural elements:
- at the position corresponding to GIu 1 of the sequence of SpIB it contains an amino-acid selected from among: GIu, Asp, GIn, Asn;
- at the position corresponding to Val28 w of the SpIB sequence it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val29 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile34 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Leu35 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Thr36 it contains an amino-acid selected from among: Ser, Thr;
- at the position corresponding to His39 it contains His;
- at the position corresponding to Ile66 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile69 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Asp77 it contains Asp;
- at the position corresponding to Val78 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val80 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Ile81 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to VaIl 18 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Ser, Thr;
- at the position corresponding to GIy 120 it contains GIy;
- at the position corresponding to Tyrl21 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to Leul31 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to GIy 155 it contains GIy;
- at the position corresponding to Asnl56 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Ser 157 it contains Ser;
- at the position corresponding to GIy 158 it contains GIy;
- at the position corresponding to Ser 159 it contains an amino-acid selected from among: VaI, Ala, Ser, Thr, GIy;
- at the position corresponding to Pro 160 it contains Pro;
- at the position corresponding to GIy 170 it contains GIy;
- at the position corresponding to HeI 71 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Phe 197 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to He 198 it contains an amino-acid selected from among:
VaI, Leu, He, Ala.
The next the subject of the present invention is a method of storage of protease exhibiting protease SpIA activity according to the invention, as defined above, or of protease exhibiting protease SpIB activity according to the invention, as defined above, wherein the said enzymes are preferentially stored for more than 12 hours: a.) in solution preferentially at temperatures from +250C to O0C b.) in solution containing antifreezing agent at temperatures from +25°C to -850C c.) frozen at the temperatures form O0C to -2000C preferentially in a -2O0C freezer, -7O0C freezer, dry ice or liquid nitrogen d.) liophylized (freeze-dried) preferentially in moisture free containers or moisture free, vacuum sealed containers or moisture free, inert gas filled containers.
In the sequences, the following symbols denote: A, Ala, alanine; V, VaI, valine; L, Leu, leucine; I, He, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M,
Met, methionine; G, GIy, glycine; S, Ser, serine; T, Thr, threonine; C, Cys, cysteine; Y,
Tyr, tyrosine; N, Asn, asparagine; Q GIn, glutamine; D, Asp, aspartic acid; E, GIu, glutamic acid; K, Lys, lysine; R, Arg, arginine; H, His, histidine;
Xaa5 in the case of protease SpIB and Xaa4 and Xaa5 in the case of protease SpIA may be omitted or be any given amino-acid since, unexpectedly, SpIA and SpIB differ from other proteases characterised by high substrate specificity, which usually also exhibit specificity towards the amino-acid immediately following the hydrolysed bond (at the newly formed
N-terminus arising from the hydrolysis of the peptide bond, meaning the Pl' position according to the nomenclature scheme proposed by: Schechter, L, and Berger, A. (1967)
Biochem. Biophys. Res. Commun. 27, 157-162). In the case of protease SpIB we observe this preference only towards Pl ' amino-acids selected from among: Thr, Ser, VaI, GIy,
Ala, GIu, Met, GIn, Asp, Asn, Lys, Leu, Phe , whereas for protease SpIA we observe this preference only towards Pl' amino-acids selected from among: Ser, Thr, GIy, Ala, VaI,
GIn and GIu. However, the replacement of the preferred amino-acids with other residues does not lead to the complete loss of hydrolysis of the bonds in question. This characteristic is particularly preferential, because it facilitates the arbitrary design of the N- terminus of the released products.
Another aspect of the present invention relates to proteins exhibiting a defined protease activity according to the present invention due to the retention by these proteins of the tertiary structure of the protease defined in Table 1 or 2 respectively.
There is a commonly used parameter defining the similarity of tertiary structures, which makes it possible to define groups of proteins according to the present invention with desirable qualities. This parameter is root mean square distance (deviation), RMSD, or simply RMS (the notations should be treated as synonymous and were used as such in the description). The value of the RMSD parameter is calculated based on a comparison of the orientation of corresponding atoms following the superimposition of compared structures for best fit. The value of the parameter is expressed in Angstrøms (A) and was used as such throughout the text. In general, the lower the value of the parameter, the higher the similarity of the structures.
Thus, the subject of the present invention are proteins exhibiting proteolytic activity according to the present inventionbecause their tertiary structure is sufficiently similar to that of protease SpIA or SpIB. Said similarity is measured using the RMSD parameter for significant structural components of protease SpIA or SpIB in relation to corresponding structural components of the compared protein.
In particular, the subject of the present invention is an enzyme which preferentially fulfills at least one of the structural criteria defined in the Claims.
Analysis of the tertiary structure of the proteases described made it possible to localise regions and residues particularly significant in substrate recognition and catalysis. The subject of the present invention are thus proteins possessing residues corresponding with the following key amino-acid residues in the SpIA or SpIB sequences: a) residues of the so-called catalytic triad: S 154, H39 and D78 in SpIA, and S 157, H39 and D77 in SpIB. Substitution of these residues results in the complete loss of catalytic ability. For example, for the SpIB protease it has been shown that mutant S 157— >A completely lacks proteolytic activity. b) residues responsible for substrate recognition: for SpIA:
Pl: primarily A149, Q150, P151, N153, L169, A171, G172, E177, S178 and N181,
P2: primarily Y170, H39 and D78, and for SpIB:
Pl: primarily S175, H172, T152 to N156, A174,
P2: primarily F173, H39 and D77,
P3: primarily S 175
P4: primarily F173 and Y186,
c) glutamic acid residue at the N-terminal end of the polypeptide chain, which is responsible for the stabilisation of the N-terminus of the protein through hydrogen bonding and by the same token facilitates the expression of full proteolytic activity. This residue may be substituted by an aspartic acid residue with similar physico-chemical properties or by glutamine or by asparagine.
As shown in J. MoI. Biol. (2006) 358, 270-279, a comparison of the amino-acid sequences of homologous staphylococcal proteins (V8 protease as well as epidermolytic toxins) and trypsin has shown important regions essential for proper folding and/or functioning (Fig.
1): For SpIB these are: V28 to V40; D77 to 181; G120 to P122 as well as G155 to 1171, whereas for SpIA these are: V28 to 140; D78 to V82; Gl 17 to Pl 19 as well as G152 to
1168. Furthermore, the conservation of individual residues is clearly evident. For SpIB these are: 150; S134; 1146; V188 and 1198, whereas for SpIA these are: V51; S131; N181;
V184 and 1194. However, only the determination of the tertiary structure of the SpIA and
SpIB proteases makes it possible to compare their sequences via a structural comparison, and thereby to compare sequences of corresponding structural elements. First, the structure is compared, and where they are similar, the sequences are compared, even if they are not homologous in the classic sense. Such a solution carries much more information in comparison to an ordinary sequence alignment, because it indicates elements significant in the functioning of a protein. Such a comparison has been shown in Fig. 2, where appropriate fragments are grouped on the basis of structural similarity. This approach facilitates the differentiation of evidently conserved regions which are essential for protein functioning. Thus, a protein according to the present invention possessing the functional characteristics of SpIA should possess the following residues in locations corresponding to the following amino-acids in the SpIA sequence:
GIu 1 - this optimally consists of amino-acids selected from among: GIu, Asp, GIn, Asn
Val28 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Val29 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Ile34 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Val35 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Thr36 - this optimally consists of amino-acids selected from among Ser, Thr;
His39 - histidine of the catalytic triad; this optimally consists of His;
Val67 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Ile70 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Asp78 - aspartic acid of the catalytic triad; this optimally consists of Asp;
Leu79 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Ileδl - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala, Met;
Val82 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala,
Met;
Val98 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala, Ser,
Thr;
Glyl 17 - this optimally consists of GIy;
Tyrl lδ- this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
Metl28 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala,
Met;
Alal49 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala;
Glnl50 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
Prol51 - this optimally consists of Pro;
Glyl52 - this optimally consists of GIy;
Asnl53 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
Serl54 - serine of the catalytic triad described earlier, must be Ser;
Glyl55 - this optimally consists of GIy;
Serl56 - this optimally consists of amino-acids selected from among VaI, Ala, Ser, Thr,
GIy;
Prol57 - this optimally consists of Pro;
Glyl67 - this optimally consists of GIy;
Ilel68 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala;
Leu 169 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala;
Tyrl70 - this optimally consists of amino-acids selected from among Tyr, Phe, Trp;
Alal71 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala;
Glyl72 - this optimally consists of GIy;
Glul77 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
Serl78 - this optimally consists of amino-acids selected from among Ser, Thr, VaI, Leu,
He, Ala;
Asnlδl - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
Phel93 - this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
Ilel94 -this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala.
By analogy, a protein according to the present invention which possesses the functional characteristics of the SpIB possess the following residues in locations corresponding to the following amino-acids in the SpIB sequence:
Glul - this optimally consists of amino-acids selected from among: GIu, Asp, GIn, Asn
Val28 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Val29 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Ile34 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Leu35 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Thr36 - this optimally consists of amino-acids selected from among Ser, Thr;
His39 - histidine of the catalytic triad;
Ile66 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Ile69 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Asp77 - aspartic acid of the catalytic triad;
Val78 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala;
Val80 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala,
Met;
He81 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala, Met;
VaIl 18 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala,
Ser, Thr;
Glyl20 - this optimally consists of GIy;
Tyrl21- this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
Leul31 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala,
Met
Glyl55 - this optimally consists of GIy;
Asnl56 - this optimally consists of amino-acids selected from among Asn, GIn, Asp, GIu;
Serl57 - serine of the catalytic triad described earlier, must be Ser;
Glyl58 - this optimally consists of GIy;
Serl59 - this optimally consists of amino-acids selected from among VaI, Ala, Ser, Thr,
GIy;
ProlόO - this optimally consists of Pro;
Glyl70 - this optimally consists of GIy;
Ilel71 - this optimally consists of amino-acids selected from among VaI, Leu, He, Ala;
Phel97 - this optimally consists of amino-acids with a large, hydrophobic side-chain selected from among Tyr, Phe, Trp;
Ilel98 - this optimally consists of amino-acids with a small, hydrophobic side-chain selected from among VaI, Leu, He, Ala.
A particular embodiment of the present invention though without limitation is a protein possessing the structure of protease SpIA described in Table 1 or a protein possessing the structure of protease SpIB described in Table 2.
According to the present invention, both protease SpIA and protease SpIB recognise specific amino-acid sequences and hydrolyse the polypeptide chain immediately behind or within the recognised sequence. Due to the length of the recognised sequence (three or four sequential amino-acids for SpIA or SpIB respectively) the number of identical sequences in the human proteome and other proteomes is small, thus the enzyme is suitable for removing fusion tags during the production of the remaining majority of human proteins and proteins of other organisms. The present invention encompasses the amino-acid sequences of a polypeptide chain specifically recognised and hydrolysed by protease SpIA or SpIB, nucleotide sequences encoding said amino-acid sequences (thus facilitating the production of polypeptides containing them using recombinant protein technology) as well as a method of specifically hydrolysing polypeptides containing said amino-acid sequences using protease SpIA or SpIB. The present invention also encompasses proteases SpIA and
SpIB themselves and enzymes posessing SpIA and SpIB activity due to close structural
similarity as defined herein, as enzymes recognizing or recognizing and hydrolysing selected amino-acid sequences as well as methods of producing protease SpIA or SpIB in a recombinant system. Furthermore, the present invention encompasses synthetic substrates based on sequences specifically recognised and hydrolysed by protease SpIA or SpIB. In summary, the most preferential advantages of the present invention can be of use in the following processes: a) recognition of a specific amino-acid sequence in a polypeptide chain (particularly in the sequence of a recombinant protein) and its specific hydrolysis at a precisely determined site within the recognised sequence or a small distance away from it, b) the highly efficient production of the proteases SpIA and SpIB.
Successive subjects and individual aspects of the present invention have been defined in the patent Claims.
To better illustrate the nature of the present invention, the present description has been supplemented with listed sequences and figures.
Sequence No. 1 (SEQ ID NO 1) represents the sequence encoding protease SpIA from
Staphylococcus aureus along with its native signal peptide.
Sequence No. 2 (SEQ ID NO 2) represents the amino-acid sequence of protease SpIA from
Staphylococcus aureus (mature protein: amino-acids from 1 to 200) along with its native signal peptide (amino-acids from -35 to -1).
Sequence No. 3 (SEQ ID NO 3) represents the sequence encoding variant protease SpIA from Staphylococcus aureus, whose sequence encoding the native signal peptide has been replaced with a sequence encoding a signal peptide recognised by Bacillus subtilis.
Sequence No. 4 (SEQ ID NO 4) represents the amino-acid sequence of variant of protease
SpIA from Staphylococcus aureus, in which the native signal peptide sequence has been replaced with signal peptide recognised by Bacillus subtilis (amino-acids from -29 to -1).
Sequence No. 5 (SEQ ID NO 5) represents the sequence encoding a fusion protein containing the sequence of mature SpIA from S. aureus with a histidine tag and a sequence recognized by SpIA whereas sequence No. 6 (SEQ ID NO 6) represents the amino-acid sequence of this protein.
Sequence No. 7 (SEQ ID NO 7) represents the sequence encoding protease SpIB from
Staphylococcus aureus along with its native signal peptide.
Sequence No. 8 (SEQ ID NO 8) represents the amino-acid sequence of protease SpIB from
Staphylococcus aureus (mature protein: amino-acids from 1 to 204) along with its native signal peptide (amino-acids from -36 to -1).
Sequence No. 9 (SEQ ID NO 9) represents the sequence encoding a variant of protease SpIB from Staphylococcus aureus, in which the sequence encoding the native signal peptide has been replaced with a sequence encoding a signal peptide from Bacillus subtilis. Sequence No. 10 (SEQ ID NO 10) represents the amino-acid sequence of a variant of protease SpIB from Staphylococcus aureus, in which the native signal peptide sequence has been replaced with a signal peptide sequence from Bacillus subtilis (amino-acids from -29 to -l).
Sequence No. 11 (SEQ ID NO 11) represents the sequence encoding a variant of protease SpIB from S. aureus with a signal peptide from B. subtilis containing the substitution S157A whereas sequence No. 12 (SEQ ID NO 12) represents the amino-acid sequence of this a variant.
Sequence No. 13 (SEQ ID NO 13) represents the sequence encoding a variant of protease SpIB from S. aureus with a signal peptide from B. subtilis containing the substitution H39A whereas sequence No. 14 (SEQ ID NO 14) represents the amino-acid sequence of this variant.
Sequence No. 15 (SEQ ID NO 15) represents the sequence encoding a variant of protease SpIB from 5. aureus with a signal peptide from B. subtilis containing the substitution D77A whereas sequence No. 16 (SEQ ID NO 16) represents the amino-acid sequence of this variant.
Sequence No. 17 (SEQ ID NO 17) represents the sequence encoding a fusion protein containing the sequence of mature SpIB from S. aureus with an attached histidine tag and a sequence recognized by SpIB whereas sequence No. 18 (SEQ ED NO 18) represents the amino-acid sequence of this protein.
Figure Ia contains a comparison of the amino-acid sequences of closely related proteases: protease SpIA, protease SpIC, V8 (protease V8 from Staphylococcus aureus also known as glutamylendopeptidase), ETA - epidermolytic toxin A from Staphylococcus aureus as well as a distantly related enzyme, trypsin (a model enzyme of the trypsin-like proteases), whereas Figure Ib contains a comparison of amino-acid sequences of closely related proteases: protease SpIB, protease SpIC, V8 (protease V8 from Staphylococcus aureus also known as glutamylendopeptidase), ETA - epidermolytic toxin A from Staphylococcus aureus as well as a distantly related enzyme, trypsin. In both cases, sequence similarities have been indicated using a grey-scale, the darker the grey, the higher the similarity. Regions of obvious sequence homology as well as individual conserved residues have been indicated with frames.
Figure 2a represents a comparison of the amino-acid sequences of closely related proteases based on their tertiary structures as well as the tertiary structure determined for protease SpIA; (chymotryps - chymotrypsin; enterokina - enterokinase; czynnik - factor X (ten)); whereas Figure 2b represents a comparison of the amino-acid sequences of closely related proteases based on their tertiary structures as well as the tertiary structure determined for protease SpIB; in both cases residues particularly significant for structure retention and protease activity have been indicated.
The examples below have been given solely to better illustrate the individual aspects of the present invention and do not constitute a limit of its scope, which has been defined in the attached Claims.
Example 1. Initial characteristics of the proteases SpIA and SpIB.
The initial experiment which made subsequent experiments possible was the determination of pH and temperature optima, and allowable other salt and reagent concentrations as well as enzyme stability. For this purpose, it was necessary to design a method of quantifying enzyme activity. Among known chymotrypsin-] ike, elastase-like and trypsin-like protease substrates only one substrate has been identified as one even minimally digested by protease SpIA (N-Suc-Ala-Ala-Pro-Phe-pNA). Other multiple tested substrates were not hydrolysed. Among known chymotrypsin-like, elastase-like and trypsin-like protease substrates not even one was found to be digested even minimally by protease SpIB. However it was determined that SpIB inefficiently digests a modified protein substrate - FTC casein. Each time during the digestion of an identified substrates, it was necessary to use a molar excess of enzyme in relation to the substrate as well as long (an hour or more) incubation times. Nevertheless, using the identified substrates, it was possible to determine the initial characteristics of protease SpIA activity as well as SpIB activity. For SpIA, we determined an pH optimum of 6.5 (+/- 1.5), and for SpIB we determined an optimal pH of 8.25 (+/-1.5,). Both enzymes demonstrated a lack of a clear dependence on common salts up to 0.5M, a lack of influence of reducing agents up to several mM, and a wide temperature tolerance.
On this basis we determined the basic parameters of the hydrolysis reaction recommended for reactions carried out using protease SpIA or SpIB. In effect, each subsequent experiment using SpIA was carried out at a temperature of 40C to 40°C, in 10 to 20OmM acetate, lactic acid, Bis-Tris, Tris, Pyridin, ADA, MOPS, MES, PIPES, ACES, BES, TES, HEPES, Tricin, Bicin, TAPS, CHES, CAPS, dimethylamin, Piperidin, phosphate or citrate buffer at a pH of 5.5 to 8.0 and 0 to 25OmM NaCl, whereas all experiments on SpIB were
carried out at 4°C to 40°C, in 10 to 20OmM Tris, Pyridin, Bis-Tris, ADA, MOPS, MES, PIPES, ACES, BES, TES, HEPES, Tricin, Bicin, TAPS, CHES, CAPS, dimethylamin, Piperidin or phosphate buffer at a pH of 6.0 to 9.0 and 0 to 25OmM NaCl. Furthermore it was determined that the enzymes in question retain activity in the presence of reagents including without limitation: imidazole, glutathione, DTT, mercaptoethanol, biotin, EDTA, ammonium sulphate, maltose, or sacharose.
Furthermore, it was determined that the enzymes in question can be stored frozen without noticeable activity loss, as well as being frozen and melted repeatedly. They can be lyophilised as well. They can also be stored at 4°C without significant activity loss. All of the above conditions constitute advantageous forms of storage of the enzymes, which is very important in everyday practice.
Example 2. Initial attempts to establish the substrate specificity of protease SpIB and SpIA Digestion of β-casein
Usually, to establish the substrate specificity of a protease, it is put into contact with various proteins, hydrolysis sites are then established and, following an appropriate number of trials, statistical analysis is used to determine the most optimal cleavage sites. Such standard approaches were also initially used for protease SpIA as well as for SpIB, but did not yield the expected results. It was shown, that a series of tested proteins (for SpIA these were: chicken egg lysosyme, human cytochrome c, whale myoglobin, bovine fibrinogen, RNAse, soy-seed trypsin inhibitor, elastin, and goat IgG, whereas for SpIB these were: chicken egg lysosyme, soy-seed trypsin inhibitor, human serum trasnferrin, bovine serum albumin, chicken ovalbumin, E. coli β-galactosidase, carbonic anhydrase, human serum alpha-2-macroglobulin, cytochrome c, goat IgG, RNAse, fibrinogen, whale myoglobin, a series of human and murine serpins) do not undergo proteolysis even during an extended incubation with an excess of enzyme.
Detectable SpIA and SpIB activity was only demonstrated using β-casein zymography (J. MoI. Biol. (2006) 358, 270-279 for SpIB). Further experiments using alternative methods (with the protease and casein contacted in solution and proteolysis product analysis using SDS-PAGE) have confirmed this fact, but also showed that to carry out the hydrolysis one needed to use a molar excess of enzyme and very long incubation times of over a dozen hours. This means that the enzyme "very reluctantly" hydrolyses β-casein (usually this type of research makes use of catalytic amounts of enzyme, that is a 1/100 or lesser concentration compared to the substrate and very short incubation times, several minutes or so). Using mass spectrometry and chemical amino-acid sequencing we have established
three SpIA hydrolysis sites in β-casein, as well as one site within the carboxymethylated lysosyme (the carboxymethylated lysosyme cleavage is described below, a space denotes the cleavage site):
KIHPF AQTQS
PVEPF TESQS
AFLLY QEPVL
GSTDY GILGI
According to this small, and thus poorly representative, group and according to common knowledge that in these types of proteases (proteases of family Sl) the specificity is determined by the Pl residue, it may be stated that protease SpIA requires an amino-acid with a large, relatively hydrophobic side chain (Y, F) at Pl of the substrate (bold). Such an assumption does not at all indicate why other proteins containing many tyrosine and phenylalanine residues are not cleaved, and particularly β-casein itself which was digested only into a few fragments despite the fact that it contains a number of Y and F residues. It also does nothing to explain why protease SpIA is so inefficient.
It should also be noted, that in the remaining positions of sequences in casein digested by
SpIA, meaning P5 to Pl as well as Pl' to P5' there were no common elements or characteristic pattern, which would suggest any role of these positions for substrate specificity of the protease in question.
Similar mass spectrometry and chemical amino-acid sequencing methods were used to elucidate four SpIB cleavage sites within β-casein (spaces denote cleavage sites):
EEQQQ TEDEL
FAQTQ SLVYP
FTESQ SLTLT
LPLLQ SWMHQ
On the basis of this small, and thus poorly representative, group it could be assumed according to common knowledge, that in this type of protease specificity is determined by the residue at Pl, and protease SpIB requires glutamine (Q) residues at Pl of the substrate
(bold). Such an assumption does not explain why other proteins containing many glutamine (Q) residues are not cleaved, and in particular β-casein itself, which was digested into only a few fragments, despite the fact that it contains a number of Q residues.
Furthermore, this assumption does not clarify why protease SpIB is so inefficient.
It should also be noted, that in the remaining positions of sequences in casein digested by
SpIB, meaning P5 to Pl as well as Pl' to P5' there were no common elements or
characteristic pattern, which would suggest any role of these positions for substrate specificity of the protease in question. It seems, though, that at Pl' S or T residues are preferred.
Digestion of synthetic substrates, denatured proteins and synthetic peptides In the face of unsuccessful experiments described above, accordingly to the state of the art we adopted the working assumption that proteases SpIA and SpIB can digest only in particular, exposed regions of proteins, and due to the hidden orientation of other regions containing F and Y residues (SpIA) or Q residues (SpIB) inside of the molecular structure of proteins used as substrates, these are not recognized nor digested. In the research on SpIA we reanalysed selected proteins following denaturation (carboxymethylated lysosyme, denatured apomioglobin with the heme group removed), as well as selected peptides arising from the degradation of a haemoglobin molecule. We also tested synthetic substrates characterised by absence of secondary structure, possessing Pl residues with hydrophobic side chains, since the state of the art as well as experimental digestion of casein indicate that such substrate should be hydrolysed. We tested the following substrates (Pl residues are in bold): N-Ala-Phe-pNa N-Gly-Phe-pNA N-Suc-Phe-pNA N-Acetyl-L-Tyr-pNa N-Meo-Suc-Ala-Ala-Pro-Val-pNA N-Suc-L-Phe-pNA Nsuc-Ala-Ala-Ala-pNa Z-Leu-Leu-Leu-Amc Sue-Ala- Ala-Ala- Amc
In all cases, despite the use of a molar excess of SpIA as well as prolonged incubation times (up to 72h), we were unable to demonstrate the hydrolysis of the polypeptides in question. Only carboxylated lysosyme was weakly hydrolysed into large fragments (few cleavage sites). Only one of the cleavage sites in carboxymethylated lysosyme was determined.
In the face of unsuccessful experiments on SpIB described above, we adopted the working assumption according to the state of the art that protease SpIB can only hydrolyse substrates in particular, exposed regions of proteins, and due to the hidden orientation of other regions containing Q residues in the interior of the molecular structure of the proteins
used as substrates they are not recognized nor digested. For this reason we reanalysed proteins following denaturation (carboxymethylated lysosyme, carboxymethylated BPTI, denatured apocytochrome c and apomioglobin with the heme group removed), as well as the synthetic peptide KEGLTETTFEEDGVATGNHEYCVEV and fluorescent synthetic substrates characterised by their lack of secondary structures Abz-Glu-Ala-Leu-Gly-Thr-
Ser-Pro-Arg-Lys(Dnp)-Asp-OH and Abz-Gln-Gly-Ile-Gly-Thr-Ser-Arg-Pro-Lys(Dnp)-
Asp-OH. Furthermore, we chemically synthesized and tested peptides corresponding to regions flanking cleavage sites in the casein sequence: FTESQSLTLT as well as
EEQQQTEDEL.
In all cases, despite the use of a molar excess of SpIB as well as protracted incubation times (up to 72h), we were unable to demonstrate the hydrolysis of the polypeptides in question.
This result is surprising in the view of the state of the art and particularly surprising in the case of the latter two peptides with sequences identical to cleavage sites determied as described.
In summary, the above results show that the standard method of examining substrate specificity described above and performed according to common knowledge and the state of the art failed entirely in the case of proteases SpIA and SpIB.
Confirmation that protein SpIB is a protease
In light of the above results, the digestion of β-casein molecules in a molar excess of SpIB and a prolonged incubation time, a possible explanation was that the examined sample of
SpIB was contaminated with other proteolytic activity. In other words, protease SpIB can, like the closely related protease SpIC, be a protein lacking proteolytic activity. J. MoI.
Biol. (2006) 358, 270-279 reports a lack of proteolytic activity of protein SpIC, very closely related to protease SpIB. Using an excess of SpIB, small quantities of contaminants may have exhibited some activity.
Such an eventuality was eliminated by exchanging the catalytic serine residue of the enzyme for an alanine residue. In trypsin-like proteases, such a substitution always leads to the complete inhibition of activity. The purified mutant SpIB (S157→A) exhibited a complete inability to hydrolyse β-casein even at a threefold molar excess and 72 hours incubation. This experiment proved the role of the S 157 residue in the catalysis mechanism of SpIB as well as confirmed that it was this enzyme and not contaminants that was responsible for the hydrolysis of β-casein.
Example 3. A method of producing the proteases SpIA and SpIB
SEQ ID NO: 1 and 2 respectively represent the nucleotide sequence of the gene encoding protease SpIA of Staphylococcus aureus as well as its corresponding amino-acid sequence. The nucleotide numeration begins from "a(l)" of the translation start triplet (atg) and ends at "a(708)" of the translation stop triplet (taa). The polypeptide chain of the protease arises via translation in conjunction with the signal peptide (amino-acid residues from M(-35) to A(-l)) which is cleaved off by a signal protease during secretion. The active, extracellular form of protease SpIA is produced, which can be harvested from the culture medium (amino-acid residues from El to K200). Likewise, SEQ ID NO: 7 and 8 respectively represent the nucleotide sequence of the gene encoding protease SpIB from Staphylococcus aureus as well as its corresponding amino-acid sequence. The nucleotide numeration begins from "a(l)" of the translation start triplet (atg) and ends at "a(723)" of the translation stop triplet (taa). The polypeptide chain of the protease arises via translation in conjunction with the signal peptide (amino-acid residues from M(-36) to A(-l)) which is cleaved off by a signal protease during secretion. The active, extracellular form of protease SpIA is produced, which can be harvested from the culture medium (amino-acid residues from El to K204). From herewith in this description we use the numeration introduced in these two sequences.
Sequences encoding the mature protease SpIA (El to K200) or SpIB (El to K204) have been cloned into appropriate expression plasmids, yielding a plasmid facilitating the production of the extracellular mature protease SpIA or SpIB in gram-positive bacteria. The sequence of the fusion protein consisting of the secretory signal sequence recognized by B. subtilis as well as the mature form of the SpIA or SpIB proteases, and a nucleotide sequence encoding the proteins has been shown in SEQ ID No. 4 and in SEQ ED No. 3 for SpIA and in SEQ ID No. 10 and SEQ ID No. 9 for SpIB.
In order to produce the recombinant proteins, B. subtilis bacteria were transformed with an expression plasmid and transformant selection was performed on plates containing kanamycine (50μg/ml). Selected clones were used to inoculate small volumes of liquid medium (TSB; Sigma) containing a selective antibiotic and incubated at 37°C with intensive mixing for 8 to 10 h. Such a start culture was then used to inoculate the main culture (4-16L of liquid medium with antibiotics) and incubated with intensive mixing at 37°C for 13 to 16 hours. All of the purification steps were carried out at 4°C. The bacteria were separated from the medium by centrifugation at 6000xg for 30min. The secreted protein in the medium separated from the bacteria was precipitated out using ammonium
sulphate to 80% saturation (561g/L w 40C). The precipitated proteins were separated from the medium using centrifugation (15000xg, Ih), dissolved in a small volume of acetate buffer, 50 mM pH 5.5, and dialysed overnight in a large excess volume of the same buffer.
The dialysed sample was subjected to ion-exchange chromatography on SP Sepharose FF
(GE Healthcare) and fractions containing the peak protein levels were collected. Collected fractions eluted in buffer with a conductivity of about 27 mS/cm for SpIA or 30 mS/cm for
SpIB. In doubtful cases, the fractions were examined for proteolytic activity using
Zymography or for proteins of an appropriate molecular mass using SDS-PAGE or another appropriate means. The eluate was dialysed in 50 mM acetate buffer pH 5.0 for SpIA or pH
4.8 for SpIB and then ion-exchange chromatography was performed on a SOURCE 15S
(GE Healthcare). The fraction containing the main protein peak was collected and then a molecular sieve was used, Superdex S75 in PBS. The preparation obtained in this fashion was concentrated, aliquoted and frozen at -20°C.
Example 4. Determination of the tertiary structure and substrate specificity of protease
SpIA as well as protease SpIB.
The method described in example 3 made it possible to efficiently produce the protein in question facilitating the further analysis of its structure, in particular the production of the crystalline form of the proteases SpIA and SpIB and the determination of their tertiary structure, which in effect resulted in the determination of the substrate specificity of both proteases.
Analysis of the tertiary structure of the proteases SpIA and SpIB
In order to indicate possible structural determinants of the observed weak kinetics of hydrolysis of peptide bonds, as well as to possibly indicate more appropriate substrates we determined the tertiary structures of SpIA and SpIB using X-Ray crystallography.
The determined coordinates of individual atoms of the mature SpIA and SpIB proteins were collected in Table 1 and Table 2 respectively. The analysis of the constructed model showed that protease SpIA and SpIB both exhibit a structure characteristic of Sl family proteases (trypsin-like/chymotrypsin-like) without any indication in the structure of the catalytic triad warranting the weak activity observed.
For SpIA, though two of four models contained in the asymetric unit showed a small divergence of the His39 side chain from the position normally observed in serine proteases, this is more likely the result of internal interactions in the crystal than of an intrinsic property of SpIA. All the more so, since the remaining two models show no divergence from the generally accepted pattern for Sl serine proteases. Furthermore, the analysis
showed a well formed Pl site in the SpIA protein structure, capable of receiving amino- acids with relatively large, hydrophobic side chains of amino-acids such as Y and F, as well as possibly W.
In the case of SpIB, the analysis showed a well formed Pl site capable of accepting the amino-acids D, E, Q or N as well as a characteristic hydrophobic "patch" on the surface of the protein at P3/P4 indicating the possibility that protease SpIB recognizes other substrate residues outside of Pl, and the lack of such recognition (of an appropriate site on the substrate) can warrant the weak proteolytic activity observed in earlier experiments.
Moreover it was determined that both in case of SpIA and SpIB a structure of the so called
"oxyanion hole", a characteristic feature of the catalytic machinery of serine proteases of family Sl is not preformed. A common knowledge on serine proteases of family Sl would suggest that such enzymes should exibit week or no catalytic activity, though examples to the contrary are also described in the state of the art. Therefore at that point of studies on
SpIA and SpIB the effect of this structural feature described could not have been unambigously anticipated.
Key amino-acid residues in the sequence of protease SpIA
From the state of the art it is known that key residues for trypsin-like serine proteases are the residues of the so-called catalytic triad. For protease SpIA, the tertiary structure obtained confirms that these are S 154, H39 and D78. Substitution of these residues results in a total loss of catalytic ability.
On the basis of the tertiary structure of protease SpIA as well as the modelling of the substrate docking method, based, among others, on the knowledge of the structures of complexes of homologous proteins with their substrates and inhibitors, it is evident that the residues responsible for substrate recognition are:
Pl: primarily A149, Q150, P151, N153, L169, A171, G172, E177, S178 and N181,
P2: primarily Y170, H39 and D78,
A comparison of the amino-acid sequences as well as tertiary structures of homologous staphylococcal proteins (protease V8 as well as epidermolytic toxins) and trypsin indicates important regions in the protein sequence for the proper folding and/or functioning (see
Figure 1): V28 to 140; D78 to V82; Gl 17 to Pl 19 as well as G152 to 1168. Furthermore, clear conservation is also observed of residues : V51; S131; N181; V184 and 1194.
Key amino-acid residues in the sequence of protease SpIB
From the state of the art it is known that key residues for trypsin-like serine proteases are the residues of the so-called catalytic triad. For protease SpIB, the tertiary structure
obtained confirms that these areS157, H39 and D77. Substitution of these residues results in a total loss of catalytic ability. The appropriate amino-acid sequences of such mutants have been shown in SEQ ID No: 11-16. Using the sequences revealed as well as the method described in Example 3 it was possible to obtain appropriate mutants and subject them to further analysis. For example, it was experimentally confirmed that the mutant
S157→A completely lacks proteolytic activity.
On the basis of the determined tertiary structure of protease SpIB as well as the modelling of the substrate docking method, based, among others, on the knowledge of the structures of complexes of homologous proteins with their substrates and inhibitors, it is evident that the residues responsible for substrate recognition are:
Pl: primarily S175, H172, T152 to N156, A174
P2: primarily F173, H39 and D77
P3: primarily S 175
P4: primarily F173 and Y186
A comparison of the tertiary structures of a fully active form (identical to the native protein) and poorly active form (containing two additional amino-acids at the N-terminus) of protease SpIB indicates the role of the precise positioning of the N-terminus of the protein as well as the initial glutamic acid residue (El).
A comparison of the amino-acid sequences as well as the tertiary structure of homologous staphylococcal proteins (protease V8 as well as epidermolytic toxins) and trypsin indicates regions in the protein sequence essential for its proper folding and/or functioning (See
Figure 1): V28 to V40; D77 to 181; G120 to P122 as well as G155 to 1171. Furthermore, it is clear that individual residues are conserved: 150; S134; 1146; V188 and 1198.
Analysis of synthetic substrate libraries
Since the determined as described structures of both SpIA and SpIB do not indicate te reasons for "ineficient" catalysis observed in described experiments we searched for other possible determinants of "efficiency" not defined in the state of the art. We used a combinatory library of 104976 synthetic substrates during further stages of searching for optimal substrates for SpIA and SpIB. The library contains substrates which at positions P4 to Pl contain all possible permutations of 18 amino-acids (except methionine and cysteine) and position Pl' is occupied by 7-amide-4-fluoromethylcumarine, a fluorochrome which exhibits fluorescence following cleavage from the peptide portion by a protease which makes it possible to detect preferred substrates (detailed description in Biol. Chem. (2004).
385: 1093-1098). During the first stage of the experiments, according to assumption based
on the state of the art that the Pl residue determines the specificity of trypsin-like proteases, we concentrated on the preferred residues at Pl. A search of the library with each of the studied proteases showed that protease SpIA tolerates only the following amino acid side chains at position Pl out of 18 tested amino-acids: Phe and Tyr; whereas protease SpIB at Pl of 18 tested amino-acids tolerates only the following amino-acids: Asp, Asn, GIn (results in accordance with the results of β-casein digestion as well as with predictions based on protease structure analysis). The rate of digestion of the selected substrates was comparable with the rates of other proteases, showing that the tested proteases are not inefficient, as was suggested by the earlier results described in the state of the art and examples 1 and 2.
The library used makes it impossible to determine the results of selection at the P2 to P4 positions. However, looking for an answer as to why proteases SpIA and SpIB do not cleave other proteins other than β-casein, which also contain whole series of Phe and Tyr residues (for SpIA) as well as Asp, Asn and GIn (for SpIB), it was only at this stage of the research that it became obvious that contrary to the state of the art the trypsin-like proteases SpIA and SpIB likely possess a much higher substrate specificity in comparison with their closely related (protease V8) and distantly related (trypsin, chymotrypsin and others) homologues.
Analysis of the CLiPS combinatorial library
The high substrate specificity made it necessary to search a much larger number of substrates to find the preferentially selected ones, and thus forced the use of the much more advanced CLiPS method (PNAS, (2006). 130: 7583-7588). Very briefly, in a library compiled in this fashion, a protein on the exterior of a bacterial cell membrane is synthesized in such a way, that it contains all possible permutations of the linear sequence of several amino-acids (each strain of bacteria in the library contains proteins of a particular sequence, but different than the other strains within the same library). At the end of the variable sequence, there is a fluorescent sequence. The initial stage of selection (flow cytometry) selects fluorescent cells (where the protein of interest is expressed) whereafter these cells are put into contact with the tested protease and then, the cells which do not exhibit fluorescence are selected, meaning those for which the fluorescent portion has been cleaved off. Next, the gene encoding the protein of interest is examined from the strains selected in this manner, allowing to determine the cleavage sequences. This method made it possible to sieve 64 million substrates and to gather information regarding amino- acids at positions P5 to Pl', not only Pl (as in the technique described above). Using this
method, we selected the following sequences respectively recognized and cleaved by protease SpIA:
P3P2Pi*Pi'
RWL Y * - - WL Y * S
GWLY * - I Y E_Y * A L Y E_Y * -
V Y E_Y * -
V Y M_Y * -
V Y A_Y * S F Y T_Y * S L Y L Y * G A F L Y * S T F L Y * - I V_L Y * T
V V_L Y * T
V W L S_* T YWMN * T
V W W_Y * T AWL Y * -
(Y, W, F)LY* (T, S) C o n s e n s u s s e q u e n c e and by protease SpIB:
P4P3P2Pi*Pi' W E L Q * S W E L Q * G W E L T_* G W E L T_* V W E V_Q * E V_E L Q * S W E L Q * V W E L Q * S W E L Q * E W E L Q * M W E L Q * S W E L Q * A W Q_L D_* A W V_L Q * A W E L Q * C o n s e n s u s s e q u e n c e
Bold type indicates the amino-acids corresponding to a carefully selected consensus sequence, amino-acids divergent from the consensus sequence are underlined, an asterisk indicates the cleavage site.
Now that both SpIA and SpIB were unambigously shown to exhibit catalytic activity towards a very limited number of the substrates (unexpected, very high substrate specificity) the above described feature of unperformed oxy anion hole was suggested a previously unobserved among serine proteases of family Sl mechanism of improving (limiting) the substrate specificity of the enzyme.
In the light of the earlier experiments and the state of the art, the results obtained are certainly not obvious. To date, the knowledge of dozens of trypsin-like proteases almost exclusively indicates position Pl as the determinant of substrate specificity in this type of proteins. Likewise, protease V8, highly homologous with SpIA and SpIB, and also from Staphylococcus aureus exhibits specificity exclusively for Pl residues. This is the reason, why we initially anticipated, according to the state of the art, that this will also be the case for the SpIA and SpIB proteases. Because proteases specific solely for Pl are not particularly specific when measured using the CLiPS method, this method is not indicated for the establishment of specificity for these types of enzymes. Only a series of unsuccessful attempts to fit SpIA and SpIB into the information from the state of the art forced the authors to test another, less likely hypothesis regarding the specificity of the protease examined, which hypothesis unexpectedly turned out to be true. Example 5. The use of proteases SpIA and SpIB in the specific hydrolysis of proteins containing amino-acid sequences according to the present invention The accuracy of the choice of consensus sequence as well as the utility of protease SpIA and SpIB were confirmed in subsequent experiments. We made use of a plasmid facilitating the expression of staphostatin A as a fusion protein with GST cleaved off with thrombin (described in MoI. Microbiol. (2003). 49: 1051-1066)). The incubation of the fusion protein GST-thrombin cleavage site-staphostatin A with protease SpIA or SpIB respectively, even using protracted incubation times with an excess of enzyme did not lead to the hydrolysis of the peptide chain which was visualized using SDS-PAGE. Using genetic engineering methods (point mutagenesis) we substituted the thrombin cleavage site in the above plasmid (LVPR*GS) for a sequence encoding the determined as described above consensus sequence specific for SpIA (YLY*S) or the determined as described above consensus sequence specific for SpIB (WELQ*G) yielding plasmids facilitating the expression of a GST-SpIA cleavage site-staphostatin A fusion protein or a GST-SpIB
cleavage site-staphostatin A fusion protein respectively. Such proteins were produced in E. coli bacteria and purified using the affinity of the GST fusion protein for immobilised glutathione as described in MoI. Microbiol. (2003). 49: 1051-1066 for the GST-thrombin cleavage site-staphostatin A fusion protein. When such a protein was put into contact with protease SpIA or SpIB respectively, very rapid hydrolysis of the polypeptide chain was observed at several minutes of incubation and a hundredfold molar excess of substrate over the enzyme. This means that protease SpIA and protease SpIB are not catalytically inefficient enzymes, as was suggested by the β-casein and carboxymethylated lysosyme digestions. To the contrary, this means that they are catalytically very efficient enzymes, but only with respect to substrates of an appropriate sequence, which is unexpectedly (concerning the state of the art) much moree \ !cd than in the case of known trypsin-like proteases.
Furthermore, we isolated the staphostatin A released each time from the fusion protein through SpIA or SpIB digestion and determined, using the Edman degradation method, its N-terminal sequence showing that protease SpIA and SpIB digest specifically and precisely within the recognised sequence only in the spot indicated by * (i.e. SpIA: YLY*S, and SpIB: WELQ*G).
A similar result should be obtained through an experiment, in which a sequence recognized by protease SpIA or SpIB according to the description, particularly the appropriate consensus sequences YLYS or WELQG, are placed between a histidine tag (His-Tag), or any other tag and a protein of interest, or between any given protein of interest and any tag, so as to yield the precise cleavage of the tag from the protein of interest. Example 6. The role of the Pl' position in substrate recognition by SpIB For many specific proteases, a significant role in substrate recognition is played not only by the P positions, but also by Pl' (i.e. for thrombin this must be a small amino-acid (usually GIy)). This is rather disadvantageous, since it does makes it impossible to arbitrarily shape the N-terminus of the protein after tag removal. The analysis of substrates selected using CLiPS shows that in the case of SpIB, this residue is of no great consequence (at the Pl' position we find S, G, V, A but also amino-acids with large side- chains such as E or M).
A similar result should be obtained in an experiment in which in a GST-protease SpIB cleavage site-staphostatin A fusion protein, at the Pl' position substitutions of various amino acids are made (e.g. substitution of the sequence of WELQ*G for WELQ*Q or
WELQ*N). To confirm the lack of influence of the introduced change on the rate of hydrolysis of the fusion protein following experiments were performed.
The following amino acids were introduced at the Pl' position of the GST-protease SpIB cleavage site-staphostatin A fusion protein: E, K, N, Q, L, F, M. In effect we obtained the following constructs, containing in the fusion protein described in Example 5, the following sequence fragments in place of WELQ G:
WELQ E
WELQK
WELQ N
WELQ Q
WELQ L
WELQ F
WELQ M
All recombinant proteins produced with these constructs were cleaved efficiently by protease SpIB, confirming little influence of the Pl' position on substrate recognition and hydrolysis. The cleavage site immediately following the recognized consensus sequence
(WELQ) was confirmed in each case by the sequencing of the newly released N-terminus of the protein using Edman degradation.
SpIB cleavage of fusion protein Histag-LVPR-PCNA
Proliferating Cell Nuclear Antigen (PCNA) was expressed with an N-terminal His-tag fusion (Histag-LVPR-PCNA). The original construct contained thrombin cleavage site
(LVPR). The purified protein (35kDa) was subjected to SpIB digestion at 5:1 protein to
SpIB molar ratio at room temperature for 1, 7, and 24 hours in 5OmM Tris-HCl buffer pH
8.0. The cleavage products were analysed by SDS-PAGE. No recombinant protein cleavage or degradation was detected in any of the samples. Following the thrombin cleavage site in the original construct was exchanged to a site recognized by SpIB protease
(LVPR →WELQ). The purified Histag- WELQ-PCNA was subjected to SpIB digestion at
100:1 protein to SpIB molar ratio at room temperature for 2, 4, 6, 9 and 24 hours in 5OmM
Tris-HCl buffer pH 8.0. The cleavage products were analysed by SDS-PAGE. The recombinant protein was efficiently cleaved by SpIB (98% at 24 hours) at the inserted site producing as expected a 32 kDa fragment which was not cleaved, degraded or otherwise further affected by prolonged incubation with SpIB.
SpIB cleavage of fusion protein Histag-LVPR-CRP
cAMP receptor protein (CRP; also known as catabolite gene activator protein [CAP]) was expressed with an N-terminal His-tag fusion (Histag-LVPR-CRP). The original construct contained thrombin cleavage site. The purified protein (25kDa) was subjected to SpIB digestion at 9:1 protein to SpIB molar ratio at room temperature for 1, 7, and 24 hours in 5OmM Tris-HCl buffer pH 8.0. The cleavage products were analysed by SDS-PAGE. No recombinant protein cleavage or degradation was detected in any of the samples. Following the thrombin cleavage site in the original construct was exchanged to a site recognized by SpIB protease (LVPR →WELQ). The purified Histag- WELQ-CRP was subjected to SpIB digestion at 25:1 and 50:1 protein to SpIB molar ratio at room temperature for 2, 4, 6, 9 and 24 hours in 5OmM Tris-HCl buffer pH 8.0. The cleavage products were analysed by SDS-PAGE. The recombinant protein was efficiently cleaved by SpIB (95% and 75% at 24hours respectively at 25:1 and 50:1 protein to SpIB molar ratio) at the inserted site producing as expected a 22 kDa fragment which was not cleaved, degraded or otherwise further affected by prolonged incubation with SpIB. SpIB cleavage of fusion protein GST-LVPR-CRP(W85L) cAMP receptor protein (CRP; also known as catabolite gene activator protein [CAP]) W85L mutant was expressed with an N-terminal GST (Glutathione S-transferase) fusion (GST-LVPR-CRP(W85L)). The original construct contained thrombin cleavage site. The purified protein (49kDa) was subjected to SpIB digestion at 3:1 protein to SpIB molar ratio at room temperature for 1, 7, and 24 hours in 5OmM Tris-HCl buffer pH 8.0. The cleavage products were analysed by SDS-PAGE. No recombinant protein cleavage was detected in any of the samples. Following the thrombin cleavage site in the original construct was exchanged to a site recognized by SpIB protease (LVPR → WELQ). The purified GST- WELQ-CRP(W85L) was subjected to SpIB digestion at 25:1 and 50:1 protein to SpIB molar ratio at room temperature for 2, 4, 6, 9 and 24 hours in 5OmM Tris-HCl buffer pH 8.0. The cleavage products were analysed by SDS-PAGE. The recombinant protein was efficiently cleaved by SpIB (95% at 9 and 24 hours respectively for 25:1 and 50:1 protein to SpIB molar rato) at the inserted site producing as expected a 26kDa and 22kDa fragments which were not cleaved, degraded or otherwise further affected by prolonged incubation with SpIB.
Table 1. Coordinates of the tertiary structure of SpIA from Staphylococcus aureus, legend: NA- atomic order number, A - type of atom, AK- amino-acid, NAK - amino-acid order number in the primary structure, X, Y, I - atomic coordinates
NA A AK NAK X Y Z
1126 O SER 156 28 473 -6. 826 38 325
1127 N PRO 157 30 484 -7. 047 39 429
1128 CA PRO 157 31 108 -8. 228 38 729
1129 CB PRO 157 32 467 -8. 353 39 398
1130 CG PRO 157 32 341 -7. 612 40 676
1131 CD PRO 157 31 417 -6. 455 40 385
1132 C PRO 157 30 385 -9. 533 38 957
1133 O PRO 157 29 899 —9 773 40 037
1134 N VAL 158 30 333 -10 .387 37 954
1135 CA VAL 158 29 686 -11 .682 38 077
1136 CB VAL 158 28 450 -11 .903 37 089
1137 CGl VAL 158 27 923 -13 .307 37 217
1138 CG2 VAL 158 27 357 -10 .885 37 297
1139 C VAL 158 30 807 -12 .642 37 722
1140 O VAL 158 31 368 -12 .640 36 559
1141 N LEU 159 31 129 -13 .457 38 729
1142 CA LEU 159 32. 313 -14 .288 38 694
1143 CB LEU 159 33. 189 -13 .959 39 905
1144 CG LEU 159 33. 683 -12 .548 40. 104
1145 CDl LEU 159 34. 560 -12 .460 41. 378
1146 CD2 LEU 159 34. 502 -12 .182 38. 921
1147 C LEU 159 31. 988 -15 .767 38. 679
1148 O LEU 159 31. 149 -16 .213 39. 484
1149 N ASN 160 32. 663 -16 .551 37. 833
1150 CA ASN 160 32. 575 -18 .007 38. 024
1151 CB ASN 160 32. 926 -18 .777 36. 742
1152 CG ASN 160 34. 386 -18 .634 36. 318
1153 ODl ASN 160 34. 712 -18 .797 35. 116
1154 ND2 ASN 160 35. 277 -18 .320 37. 286
1155 C ASN 160 33. 324 -18 .536 39. 285
1156 O ASN 160 33. 899 -17 .775 40. 041
1157 N SER 161 33. 266 -19 .840 39. 538
1158 CA SER 161 34. 041 -20 .531 40. 604
1159 CB SER 161 33. 816 -22 .040 40. 498
1160 OG SER 161 34. 367 -22 .538 39. 255
1161 C SER 161 35. 584 -20 .210 40. 592
1162 O SER 161 36. 160 -19 .965 41. 698
1163 N LYS 162 36. 211 -20 .178 39. 381
1164 CA LYS 162 37. 627 -19 .774 39. 202
1165 CB LYS 162 38. 189 -20 .051 37. 796
1166 CG LYS 162 37. 980 -21 .392 37. 144
1167 CD LYS 162 38. 917 -21 .460 35. 920
1168 CE LYS 162 38. 303 -22 .324 34. 814
1169 NZ LYS 162 37. 433 -23 .419 35. 425
1170 C LYS 162 37. 906 -18 .286 39. 362
1171 O LYS 162 38. 968 -17 .847 38. 894
1172 N HIS 163 36. 979 -17 .511 39. 958
1173 CA HIS 163 37. 040 -16 .044 39. 998
1174 CB HIS 163 37. 999 -15 .589 41. 093
1175 CG HIS 163 37. 741 -16 .257 42. 412
1176 NDl HIS 163 38. 734 -16 .883 43. 141
1177 CEl HIS 163 38. 204 -17 .403 44. 231
1178 NE2 HIS 163 36. 905 -17 .152 44. 230
1179 CD2 HIS 163 36. 589 -16 .438 43. 102
1180 C HIS 163 37. 335 -15 .311 38. 702
1181 O HIS 163 37. 997 -14 .303 38. 733
1182 N GLU 164 36. 859 -15 .785 37. 553
1183 CA GLU 164 36. 954 -14 .963 36. 325
1184 CB GLU 164 37. 348 -15 .850 35. 172
1185 CG GLU 164 38. 408 -16 .857 35. 535
1186 CD GLU 164 38. 447 -18 .008 34. 497
1187 OEl GLU 164 37. 416 -18 .719 34. 278
1188 OE2 GLU 164 39. 499 -18 .169 33. 863
1189 C GLU 164 35. 596 -14 .256 36. 026
1190 O GLU 164 34. 570 -14 .773 36. 492
1191 N VAL 165 35. 614 -13 .087 35. 320
1192 CA VAL 165 34. 403 -12 .257 35. 007
1193 CB VAL 165 34. 514 -10 .625 34. 703
1194 CGl VAL 165 33. 819 -9. 648 35. 765
1195 CG2 VAL 165 35. 789 -10 .215 34. 309
1196 C VAL 165 33. 657 -12 .831 33. 842
1197 O VAL 165 34. 178 -12 .894 32. 741
1198 N ILE 166 32. 379 -13 .161 34. 076
Table 2. Coordinates of the tertiary structure of SpIB from Staphylococcus aureus, legend: NA- atomic order number, A - type of atom, AK- amino-acid, NAK - amino-acid order number in the primary structure, X, Y, I - atomic coordinates
AK NAK X NA A AK NAK
N GLU 1 10.688 39.517 6.273 89 CA ILE 12 23.045 38.511 29.008
CA GLU 1 10.569 40.577 7.288 90 CB ILE 12 21.702 39.218 29.455
CB GLU 1 10.333 39.952 8.665 91 CGl ILE 12 21.029 39.865 28.261
CG GLU 1 9.837 40.901 9.754 92 CDl ILE 12 19.625 40.425 28.547
CD GLU 1 9.251 40.165 10.946 93 CG2 ILE 12 21.983 40.226 30.542
OEl GLU 1 8.374 39.289 10.757 94 C ILE 12 22.717 37.420 28.001
OE2 GLU 1 9.667 40.471 12.083 95 O ILE 12 22.994 37.538 26.795
C GLU 1 11.858 41.360 7.274 96 N PHE 13 22.123 36.351 28.496
O GLU 1 12.925 40.827 6.940 97 CA PHE 13 21.724 35.268 27.610
N ASN 2 11.763 42.633 7.613 98 CB PHE 13 21.355 34.021 28.430
CA ASN 2 12.949 43.448 7.819 99 CG PHE 13 20.971 32.844 27.592
CB ASN 2 13.143 44.442 6.674 100 CDl PHE 13 19.632 32.556 27.322
CG ASN 2 14.510 45.115 6.693 101 CEl PHE 13 19.283 31.462 26.521
ODl ASN 2 15.192 45.178 7.728 102 CZ PHE 13 20.281 30.646 25.987
ND2 ASN 2 14.926 45.631 5.526 103 CE2 PHE 13 21.614 30.923 26.241
C ASN 2 12.789 44.141 9.162 104 CD2 PHE 13 21.958 32.029 27.048
O ASN 2 12.416 45.328 9.242 105 C PHE 13 20.542 35.792 26.787
N ASN 3 13.033 43.376 10.222 106 O PHE 13 19.660 36.488 27.352
CA ASN 3 12.929 43.887 11.577 107 N PRO 14 20.437 35.414 25.489
CB ASN 3 11.732 43.269 12.308 108 CA PRO 14 21.259 34.446 24.695
CG ASN 3 11.568 43.811 13.722 109 CB PRO 14 20.313 34.045 23.559
ODl ASN 3 11.828 44.991 13.985 110 CG PRO 14 19.526 35.319 23.294
ND2 ASN 3 11.128 42.953 14.637 111 CD PRO 14 19.357 35.997 24.666
C ASN 3 14.232 43.633 12.315 112 C PRO 14 22.523 35.038 24.111
O ASN 3 14.395 42.625 12.997 113 O PRO 14 23.323 34.295 23.506
N VAL 4 15.154 44.569 12.147 114 N TYR 15 22.736 36.338 24.304
CA VAL 4 16.495 44.448 12.647 115 CA TYR 15 23.813 37.051 23.597
CB VAL 4 17.501 44.721 11.514 116 CB TYR 15 23.613 38.555 23.694
CGl VAL 4 18.926 44.579 12.001 117 CG TYR 15 22.173 38.891 23.362
CG2 VAL 4 17.218 43.810 10.339 118 CDl TYR 15 21.688 38.763 22.058
C VAL 4 16.653 45.484 13.730 119 CEl TYR 15 20.350 39.033 21.753
O VAL 4 16.344 46.660 13.508 120 CZ TYR 15 19.497 39.423 22.773
N THR 5 17.111 45.065 14.908 121 OH TYR 15 18.188 39.684 22.470
CA THR 5 17.231 45.982 16.043 122 CE2 TYR 15 19.947 39.551 24.082
CB THR 5 16.038 45.834 17.033 123 CD2 TYR 15 21.284 39.273 24.370
OGl THR 5 16.019 44.513 17.581 124 C TYR 15 25.205 36.659 24.060
CG2 THR 5 14.704 46.067 16.330 125 O TYR 15 26.195 36.864 23.342
C THR 5 18.547 45.772 16.767 126 N THR 16 25.271 36.112 25.270
O THR 5 19.031 44.642 16.866 127 CA THR 16 26.512 35.543 25.808
N LYS 6 19.123 46.856 17.260 128 CB THR 16 26.452 35.379 27.349
CA LYS 6 20.371 46.797 18.006 129 OGl THR 16 25.250 34.677 27.717
CB LYS 6 20.966 48.198 18.161 130 CG2 THR 16 26.478 36.779 27.980
CG LYS 6 22.207 48.286 19.024 131 C THR 16 26.973 34.254 25.110
CD LYS 6 22.759 49.707 18.992 132 O THR 16 28.076 33.759 25.400
C LYS 6 20.157 46.136 19.366 133 N GLY 17 26.166 33.746 24.173
O LYS 6 19.220 46.465 20.090 134 CA GLY 17 26.599 32.636 23.303
N VAL 7 21.038 45.202 19.711 135 C GLY 17 27.200 33.085 21.980
CA VAL 7 20.924 44.523 20.986 136 O GLY 17 27.612 32.242 21.177
CB VAL 7 21.754 43.195 21.045 137 N VAL 18 27.246 34.405 21.738
CGl VAL 7 21.655 42.613 22.448 138 CA VAL 18 27.700 34.908 20.465
CG2 VAL 7 21.196 42.170 20.048 139 CB VAL 18 26.709 36.016 19.894
C VAL 7 21.300 45.455 22.126 140 CGl VAL 18 27.257 36.589 18.580
O VAL 7 22.444 45.904 22.233 141 CG2 VAL 18 25.341 35.415 19.687
N LYS 8 20.319 45.738 22.983 142 C VAL 18 29.122 35.457 20.571
CA LYS 8 20.528 46.643 24.119 143 O VAL 18 29.542 36.050 21.599
CB LYS 8 19.201 46.882 24.875 144 N VAL 19 29.894 35.203 19.503
C LYS 8 21.612 46.093 25.057 145 CA VAL 19 31.254 35.703 19.397
O LYS 8 22.629 46.755 25.305 146 CB VAL 19 32.323 34.534 19.460
N ASP 9 21.429 44.863 25.546 147 CGl VAL 19 32.147 33.697 20.733
CA ASP 9 22.397 44.307 26.498 148 CG2 VAL 19 32.201 33.660 18.213
CB ASP 9 21.740 44.015 27.844 149 C VAL 19 31.455 36.420 18.050
CG ASP 9 22.755 43.777 28.947 150 O VAL 19 30.702 36.196 17.068
ODl ASP 9 23.926 43.454 28.637 151 N ALA 20 32.464 37.286 18.043
OD2 ASP 9 22.391 43.920 30.139 152 CA ALA 20 32.792 38.120 16.885
C ASP 9 23.048 43.037 25.940 153 CB ALA 20 32.876 39.588 17.315
O ASP 9 22.404 42.015 25.800 154 C ALA 20 34.128 37.741 16.245
N THR 10 24.327 43.136 25.629 155 O ALA 20 35.153 37.687 16.904
CA THR 10 25.040 42.046 24.939 156 N PHE 21 34.090 37.514 14.929
CB THR 10 26.085 42.628 23.969 157 CA PHE 21 35.281 37.599 14.075
OGl THR 10 26.877 43.598 24.665 158 CB PHE 21 35.088 36.631 12.911
CG2 THR 10 25.395 43.294 22.773 159 CG PHE 21 34.989 35.193 13.339
C THR 10 25.702 41.075 25.918 160 CDl PHE 21 36.146 34.385 13.393
O THR 10 26.357 40.112 25.504 161 CEl PHE 21 36.047 33.035 13.801
N ASN 11 25.513 41.333 27.218 162 CZ PHE 21 34.784 32.506 14.130
CA ASN 11 26.149 40.583 28.306 163 CE2 PHE 21 33.649 33.312 14.091
CB ASN 11 26.927 41.530 29.238 164 CD2 PHE 21 33.760 34.651 13.668
C ASN 11 25.149 39.726 29.088 165 C PHE 21 35.394 39.055 13.562
O ASN 11 25.394 39.312 30.241 166 O PHE 21 34.553 39.887 13.907
N ILE 12 24.021 39.431 28.456 167 N LYS 22 36.410 39.359 12.733
NA AK NAK NA AK NAK
168 CA LYS 22 36.596 40.704 12.116 251 N ILE 34 33. .306 26, .826 19.163
169 CB LYS 22 37.718 40.669 11.056 252 CA ILE 34 32. .368 27, .717 18.459
174 C LYS 22 35.322 41.294 11.502 253 CB ILE 34 32. .910 29, .186 18.405
175 O LYS 22 34.921 42.431 11.839 254 CGl ILE 34 34, .134 29, .314 17.530
176 N SER 23 34.666 40.535 10.623 255 CDl ILE 34 34. ,581 30. .783 17.235
177 CA SER 23 33.413 41.017 10.031 256 CG2 ILE 34 33. ,212 29. .759 19.811
178 CB SER 23 33.696 41.760 8.729 257 C ILE 34 32. .068 27. .161 17.058
179 OG SER 23 34.054 40.822 7.740 258 O ILE 34 32. .816 26. .300 16.543
180 C SER 23 32.444 39.866 9.807 259 N LEU 35 30. ,923 27. .551 16.495
181 O SER 23 31.776 39.770 8.769 260 CA LEU 35 30. ,576 27. ,230 15.119
182 N ALA 24 32.366 38.978 10.801 261 CB LEU 35 29. ,186 26. ,583 15.051
183 CA ALA 24 31.463 37.853 10.726 262 CG LEU 35 28. 954 25. 460 14.090
184 CB ALA 24 32.085 36.712 9.948 263 CDl LEU 35 29. 960 24. ,312 14.419
185 C ALA 24 31.154 37.435 12.172 264 CD2 LEU 35 27. 527 24. ,985 14.255
186 O ALA 24 31.710 38.007 13.127 265 C LEU 35 30. 567 28. ,513 14.304
187 N THR 25 30.278 36.442 12.288 266 O LEU 35 30. 199 29. 589 14.788
188 CA THR 25 29.734 35.999 13.559 267 N THR 36 31. 024 28. 417 13.055
189 CB THR 25 28.196 36.155 13.529 268 CA THR 36 30. 840 29. 586 12.134
190 OGl THR 25 27.868 37.496 13.076 269 CB THR 36 31. 992 30. 616 12.197
191 CG2 THR 25 27.561 35.922 14.929 270 OGl THR 36 31. 663 31. 784 11.405
192 C THR 25 30.112 34.523 13.777 271 CG2 THR 36 33. 356 30. 000 11.694
193 O THR 25 30.394 33.808 12.825 272 C THR 36 30. 710 28. 942 10.740
194 N GLY 26 30.074 34.062 15.028 273 O THR 36 30. 522 27. 710 10.640
195 CA GLY 26 29.965 32.620 15.268 274 N ASN 37 30. 773 29. 750 9.672
196 C GLY 26 29.278 32.455 16.608 275 CA ASN 37 30. 744 29. 156 8.314
197 O GLY 26 28.869 33.445 17.219 276 CB ASN 37 30. 149 30. 148 7.288
198 N PHE 27 29.199 31.237 17.118 277 CG ASN 37 28. 740 30. 615 7.617
199 CA PHE 27 28.500 31.078 18.399 278 ODl ASN 37 28. 412 31. 794 7.373
200 CB PHE 27 26.960 31.025 18.211 279 ND2 ASN 37 27. 914 29. 740 8.118
201 CG PHE 27 26.472 29.939 17.310 280 C ASN 37 32. 150 28. 849 7.808
202 CDl PHE 27 26.108 28.697 17.824 281 O ASN 37 33. 112 29. 512 8.220
203 CEl PHE 27 25.644 27.720 17.015 282 N LYS 38 32. 263 27. 881 6.870
204 CZ PHE 27 25.519 27.938 15.626 283 CA LYS 38 33. 563 27. 630 6.236
205 CE2 PHE 27 25.867 29.187 15.106 284 CB LYS 38 33. 476 26. 501 5.216
206 CD2 PHE 27 26.350 30.157 15.936 285 CG LYS 38 33. 184 25. 163 5.809
207 C PHE 27 28.983 29.849 19.128 286 CD LYS 38 32. 697 24. 253 4.713
208 O PHE 27 29.545 28.944 18.512 287 CE LYS 38 32. 468 22. 874 5.223
209 N VAL 28 28.744 29.814 20.432 288 NZ LYS 38 31. 857 22. 015 4.218
210 CA VAL 28 29.341 28.760 21.282 289 C LYS 38 34. 041 28. 869 5.532
211 CB VAL 28 29.472 29.266 22.756 290 O LYS 38 35. 229 29. 144 5.543
212 CGl VAL 28 30.061 28.192 23.651 291 N HIS 39 33. 119 29. 655 4.952
213 CG2 VAL 28 30.362 30.506 22.807 292 CA HIS 39 33. 552 30. 815 4.168
214 C VAL 28 28.475 27.517 21.247 293 CB HIS 39 32. 458 31. 299 3.193
215 O VAL 28 27.270 27.598 21.431 294 CG HIS 39 31. 335 32. 046 3.828
216 N VAL 29 29.097 26.334 21.068 295 NDl HIS 39 30. 077 31. 502 3.979
217 CA VAL 29 28.313 25.092 21.066 296 CEl HIS 39 29. 278 32. 413 4.517
218 CB VAL 29 28.368 24.405 19.679 297 NE2 HIS 39 29. 976 33. 512 4.718
219 CGl VAL 29 27.482 25.221 18.678 298 CD2 HIS 39 31. 254 33. 318 4.267
220 CG2 VAL 29 29.831 24.296 19.160 299 C HIS 39 34. 072 31. 895 5.058
221 C VAL 29 28.763 24.113 22.161 300 O HIS 39 34. 767 32. 803 4.607
222 O VAL 29 28.094 23.123 22.453 301 N VAL 40 33. 761 31. 805 6.359
223 N GLY 30 29.885 24.411 22.770 302 CA VAL 40 34. 418 32. 647 7.358
224 CA GLY 30 30.387 23.518 23.805 303 CB VAL 40 33. 502 32. 875 8.592
225 C GLY 30 31.656 24.018 24.411 304 CGl VAL 40 34. 228 33. 641 9.746
226 O GLY 30 32.107 25.123 24.129 305 CG2 VAL 40 32. 257 33. 586 8.143
227 N LYS 31 32.306 23.147 25.185 306 C VAL 40 35. 776 32. 096 7.799
228 CA LYS 31 33.552 23.532 25.852 307 O VAL 40 36. 795 32. 808 7.763
229 CB LYS 31 34.118 22.312 26.593 308 N SER 41 35. 807 30. 843 8.231
230 CG LYS 31 35.392 22.633 27.358 309 CA SER 41 37. 044 30. 291 8.823
231 CD LYS 31 35.949 21.312 27.929 310 CB SER 41 36. 802 28. 919 9.443
232 CE LYS 31 37.189 21.599 28.747 311 OG SER 41 36. 231 28. 010 8.527
233 NZ LYS 31 37.531 20.378 29.569 312 C SER 41 38. 189 30. 201 7.806
234 C LYS 31 34.596 24.013 24.852 313 O SER 41 39. 353 30. 135 8.194
235 O LYS 31 34.949 23.254 23.927 314 N LYS 42 37. 849 30. 206 6.513
236 N ASN 32 35.107 25.237 25.042 315 CA LYS 42 38. 910 30. 108 5.501
237 CA ASN 32 36.114 25.845 24.185 316 CB LYS 42 38. 330 29. 867 4.117
238 CB ASN 32 37.472 25.158 24.377 317 CG LYS 42 37. 654 31. 042 3.506
239 CG ASN 32 37.910 25.177 25.824 318 CD LYS 42 37. 411 30. 717 2.008
240 ODl ASN 32 37.621 26.139 26.561 319 CE LYS 42 36. 334 31. 603 1.422
241 ND2 ASN 32 38.544 24.109 26.255 320 NZ LYS 42 36. 729 33. 035 1.463
242 C ASN 32 35.797 25.818 22.705 321 C LYS 42 39. 837 31. 309 5.517
243 O ASN 32 36.730 25.901 21.895 322 O LYS 42 40. 996 31. 226 5.083
244 N THR 33 34.527 25.694 22.355 323 N ASN 43 39. 351 32. 415 6.064
245 CA THR 33 34.167 25.434 20.942 324 CA ASN 43 40. 163 33. 613 6.256
246 CB THR 33 33.800 23.964 20.738 325 CB ASN 43 39. 267 34. 840 6.081
247 OGl THR 33 34.860 23.140 21.266 326 CG ASN 43 38. 699 34. 922 4.700
248 CG2 THR 33 33.632 23.650 19.226 327 ODl ASN 43 39. 337 34. 500 3.739
249 C THR 33 33.049 26.295 20.372 328 ND2 ASN 43 37. 484 35. 440 4.588
250 O THR 33 31.983 26.432 20.986 329 C ASN 43 40. 931 33. 700 7.561
NA A AK NAK NA A AK NAK
330 O ASN 43 41.640 34.685 7. 800 409 CD2 HIS 53 29 .900 39.050 26.624
331 N TYR 44 40.766 32.680 8. 403 410 C HIS 53 30 .503 40.410 23.375
332 CA TYR 44 41.380 32.640 9. 715 411 O HIS 53 30 .140 41.003 24.407
333 CB TYR 44 40.282 32.499 10 .799 412 N PRO 54 31 .208 41.014 22.392
334 CG TYR 44 39.581 33.816 10 .912 413 CA PRO 54 31 .604 42.425 22.535
335 CDl TYR 44 40.054 34.799 11 .782 414 CB PRO 54 32 .485 42.680 21.294
336 CEl TYR 44 39.450 36.043 11 .842 415 CG PRO 54 31 .968 41.691 20.246
337 CZ TYR 44 38.381 36.321 11 .010 416 CD PRO 54 31 .655 40.446 21.100
338 OH TYR 44 37.781 37.580 11 .082 417 C PRO 54 30 .382 43.355 22.502
339 CE2 TYR 44 37.896 35.369 10 .144 418 O PRO 54 29 .368 43.046 21.832
340 CD2 TYR 44 38.500 34.124 10 .093 419 N ASN 55 30 .493 44.460 23.249
341 C TYR 44 42.385 31.519 9. 786 420 CA ASN 55 29 .509 45.545 23.220
342 O TYR 44 42.372 30.645 8. 942 421 CB ASN 55 29 .217 46.031 24.642
343 N LYS 45 43.222 31.545 10 .815 425 C ASN 55 30 .063 46.689 22.356
344 CA LYS 45 44.121 30.439 11 .052 426 O ASN 55 31 .219 46.647 21.935
345 CB LYS 45 45.518 30.800 10 .531 427 N SER 56 29 .245 47.710 22.085
346 CG LYS 45 46.108 32.001 11 .242 428 CA SER 56 29 .630 48.765 21.126
347 CD LYS 45 47.533 32.302 10 .745 429 CB SER 56 28 .466 49.744 20.890
348 CE LYS 45 48.226 33.306 11 .667 430 OG SER 56 27 .245 49.058 20.621
349 NZ LYS 45 47.618 34.698 11 .675 431 C SER 56 30 .897 49.547 21.530
350 C LYS 45 44.212 30.222 12 .557 432 O SER 56 31 .576 50.134 20.673
351 O LYS 45 43.819 31.077 13 .346 433 N ASP 57 31 .218 49.544 22.827
352 N VAL 46 44.742 29.069 12 .932 434 CA ASP 57 32 .317 50.355 23.352
353 CA VAL 46 45.187 28.823 14 .308 435 CB ASP 57 31 .796 51.720 23.832
354 CB VAL 46 45.883 27.426 14 .411 439 C ASP 57 33 .095 49.647 24.468
355 CGl VAL 46 46.455 27.200 15 .766 440 O ASP 57 32 .705 49.693 25.656
356 CG2 VAL 46 44.904 26.320 14 .070 441 N LYS 58 34 .173 48.973 24.058
357 C VAL 46 46.117 29.974 14 .748 442 CA LYS 58 35 .207 48.449 24.966
358 O VAL 46 47.065 30.366 14 .024 443 CB LYS 58 35 .630 49.521 26.001
359 N GLY 47 45.841 30.504 15 .943 448 C LYS 58 34 .902 47.098 25.647
360 CA GLY 47 46.545 31.646 16 .469 449 O LYS 58 35 .796 46.248 25.732
361 C GLY 47 45.863 32.989 16 .291 450 N GLY 59 33 .667 46.883 26.113
362 O GLY 47 46.262 33.964 16 .930 451 CA GLY 59 33 .378 45.702 26.960
363 N ASP 48 44.887 33.077 15 .389 452 C GLY 59 33 .155 44.394 26.196
364 CA ASP 48 44.088 34.307 15 .275 453 O GLY 59 32 .487 44.404 25.149
365 CB ASP 48 43.230 34.309 14 .035 454 N ASN 60 33 .681 43.276 26.729
366 CG ASP 48 44.028 34.571 12 .783 455 CA ASN 60 33 .534 41.948 26.086
367 ODl ASP 48 45.175 35.085 12 .879 456 CB ASN 60 34 .532 41.846 24.904
368 OD2 ASP 48 43.498 34.235 11 .718 457 CG ASN 60 35 .984 41.917 25.350
369 C ASP 48 43.152 34.421 16 .456 458 ODl ASN 60 36 .392 41.201 26.263
370 O ASP 48 42.951 33.447 17 .144 459 ND2 ASN 60 36 .780 42.770 24.706
371 N ARG 49 42.527 35.590 16 .613 460 C ASN 60 33 .708 40.791 27.110
372 CA ARG 49 41.737 35.848 17 .809 461 O ASN 60 34 .027 41.045 28.283
373 CB ARG 49 42.343 37.017 18 .582 462 N GLY 61 33 .514 39.540 26.677
374 CG ARG 49 43.592 36.533 19 .290 463 CA GLY 61 33 .667 38.360 27.557
375 CD ARG 49 44.783 37.415 19 .028 464 C GLY 61 35 .061 37.740 27.451
376 NE ARG 49 46.003 36.865 19 .623 465 O GLY 61 35 .315 36.643 27.924
377 CZ ARG 49 46.839 37.540 20 .411 466 N GLY 62 35 .964 38.484 26.842
378 NHl ARG 49 46.616 38.818 20 .717 467 CA GLY 62 37 .338 38.043 26.679
379 NH2 ARG 49 47.917 36.926 20 .883 468 C GLY 62 37 .713 37.938 25.218
380 C ARG 49 40.251 36.036 17 .523 469 O GLY 62 36 .846 37.865 24.335
381 O ARG 49 39.848 36.404 16 .409 470 N ILE 63 39 .019 37.886 24.995
382 N ILE 50 39.424 35.681 18 .503 471 CA ILE 63 39 .616 37.666 23.672
383 CA ILE 50 37.985 36.000 18 .452 472 CB ILE 63 40 .591 38.803 23.289
384 CB ILE 50 37.060 34.752 18 .196 473 CGl ILE 63 39 .807 40.115 23.141
385 CGl ILE 50 37.132 33.663 19 .296 474 CDl ILE 63 40 .666 41.323 22.959
386 CDl ILE 50 36.338 33.925 20 .613 475 CG2 ILE 63 41 .272 38.474 21.965
387 CG2 ILE 50 37.447 34.048 16 .894 476 C ILE 63 40 .339 36.334 23.746
388 C ILE 50 37.635 36.694 19 .760 477 O ILE 63 41 .083 36.092 24.680
389 O ILE 50 38.339 36.522 20 .761 478 N TYR 64 40 .059 35.449 22.790
390 N THR 51 36.557 37.473 19 .743 479 CA TYR 64 40. .546 34.068 22.817
391 CA THR 51 36.133 38.234 20 .926 480 CB TYR 64 39. .369 33.105 23.041
392 CB THR 51 36.113 39.742 20 .659 481 CG TYR 64 38. .678 33.439 24.340
393 OGl THR 51 37.451 40.114 20 .326 482 CDl TYR 64 39 .104 32.874 25.544
394 CG2 THR 51 35.766 40.479 21 .965 483 CEl TYR 64 38, .489 33.207 26.740
395 C THR 51 34.764 37.742 21 .330 484 CZ TYR 64 37, .451 34.114 26.734
396 O THR 51 33.894 37.598 20 .506 485 OH TYR 64 36, .836 34.508 27.915
397 N ALA 52 34.601 37.439 22 .616 486 CE2 TYR 64 37. .021 34.701 25.569
398 CA ALA 52 33.336 36.903 23 .087 487 CD2 TYR 64 37, .620 34.358 24.365
399 CB ALA 52 33.535 36.050 24 .309 488 C TYR 64 41, .280 33.697 21.540
400 C ALA 52 32.398 38.076 23 .406 489 O TYR 64 40, .938 34.178 20.468
401 O ALA 52 32.791 39.018 24 .087 490 N SER 65 42. .279 32.832 21.690
402 N HIS 53 31.180 37.969 22 .900 491 CA SER 65 43. .130 32.365 20.575
403 CA HIS 53 30.089 38.950 23 .146 492 CB SER 65 44. .504 31.982 21.184
404 CB HIS 53 29.099 38.446 24 .218 493 OG SER 65 45. .431 31.657 20.174
405 CG HIS 53 29.686 38.203 25 .588 494 C SER 65 42. .504 31.137 19.919
406 NDl HIS 53 30.017 36.942 26 .054 495 O SER 65 42. .099 30.202 20.607
407 CEl HIS 53 30.444 37.036 27 .304 496 N ILE 66 42. .453 31.088 18.583
408 NE2 HIS 53 30.381 38.302 27 .671 497 CA ILE 66 42. .003 29.915 17.903
NA AK NAK NA A AK NAK
498 CB ILE 66 41.714 30.237 16.411 577 O LYS 75 28.367 22.833 1.760
499 CGl ILE 66 40.496 31.207 16.327 578 N GLU 76 27.528 21.421 3.284
500 CDl ILE 66 40.191 31.668 14.874 579 CA GLU 76 27.055 22.480 4.189
501 CG2 ILE 66 41.396 28.923 15.662 580 CB GLU 76 26.204 21.864 5.318
502 C ILE 66 43.021 28.765 18.024 581 CG GLU 76 24.892 21.208 4.762
503 O ILE 66 44.196 28.976 17.758 582 CD GLU 76 25.054 19.758 4.319
504 N LYS 67 42.578 27.571 18.424 583 OEl GLU 76 26.192 19.216 4.324
505 CA LYS 67 43.503 26.389 18.506 584 OE2 GLU 76 24.005 19.173 3.967
506 CB LYS 67 43.577 25.815 19.948 585 C GLU 76 28.145 23.372 4.735
507 CG LYS 67 42.368 25.092 20.370 586 O GLU 76 29.257 22.909 5.068
508 CD LYS 67 42.601 24.493 21.754 587 N ASP 77 27.832 24.670 4.831
509 CE LYS 67 41.373 23.768 22.323 588 CA ASP 77 28.819 25.725 5.103
510 NZ LYS 67 41.805 23.005 23.560 589 CB ASP 77 28.306 27.048 4.480
511 C LYS 67 43.257 25.284 17.494 590 CG ASP 77 29.384 28.086 4.280
512 O LYS 67 44.131 24.401 17.257 591 ODl ASP 77 30.521 27.925 4.763
513 N LYS 68 42.088 25.280 16.878 592 OD2 ASP 77 29.112 29.101 3.582
514 CA LYS 68 41.769 24.287 15.868 593 C ASP 77 28.989 25.853 6.647
515 CB LYS 68 41.333 22.973 16.525 594 O ASP 77 28.567 26.839 7.261
516 CG LYS 68 41.596 21.719 15.698 595 N VAL 78 29.616 24.848 7.251
517 CD LYS 68 41.330 20.400 16.474 596 CA VAL 78 29.703 24.730 8.718
518 CE LYS 68 41.533 19.209 15.514 597 CB VAL 78 28.817 23.564 9.223
519 NZ LYS 68 41.215 17.878 16.112 598 CGl VAL 78 29.094 23.278 10.776
520 C LYS 68 40.670 24.797 14.994 599 CG2 VAL 78 27.344 23.867 8.992
521 O LYS 68 39.768 25.465 15.487 600 C VAL 78 31.141 24.423 9.070
522 N ILE 69 40.742 24.448 13.702 601 O VAL 78 31.736 23.493 8.503
523 CA ILE 69 39.675 24.748 12.715 602 N SER 79 31.706 25.184 10.003
524 CB ILE 69 40.140 25.815 11.653 603 CA SER 79 33.070 24.944 10.454
525 CGl ILE 69 40.541 27.110 12.360 604 CB SER 79 33.941 26.044 9.817
526 CDl ILE 69 41.188 28.224 11.503 605 OG SER 79 35.276 25.966 10.239
527 CG2 ILE 69 39.067 26.038 10.599 606 C SER 79 33.117 24.973 11.980
528 C ILE 69 39.348 23.433 12.048 607 O SER 79 32.500 25.852 12.585
529 O ILE 69 40.252 22.748 11.495 608 N VAL 80 33.858 24.040 12.588
530 N ILE 70 38.088 23.031 12.130 609 CA VAL 80 34.044 24.010 14.064
531 CA ILE 70 37.681 21.741 11.601 610 CB VAL 80 34.051 22.539 14.584
532 CB ILE 70 37.272 20.726 12.689 611 CGl VAL 80 34.466 22.489 16.035
533 CGl ILE 70 38.328 20.588 13.787 612 CG2 VAL 80 32.665 21.910 14.427
534 CDl ILE 70 37.861 21.152 15.071 613 C VAL 80 35.363 24.698 14.389
535 CG2 ILE 70 36.992 19.368 12.081 "614 O VAL 80 36.389 24.406 13.775
536 C ILE 70 36.477 21.963 10.738 615 N ILE 81 35.331 25.621 15.342
537 O ILE 70 35.402 22.199 11.250 616 CA ILE 81 36.494 26.372 15.745
538 N ASN 71 36.641 21.883 9.423 617 CB ILE 81 36.259 27.903 15.544
539 CA ASN 71 35.499 22.025 8.513 618 CGl ILE 81 35.722 28.206 14.145
540 CB ASN 71 35.987 22.385 7.099 619 CDl ILE 81 36.676 27.853 13.094
541 CG ASN 71 36.525 23.821 6.991 620 CG2 ILE 81 37.525 28.681 15.887
542 ODl ASN 71 36.554 24.583 7.961 621 C ILE 81 36.693 26.153 17.243
543 ND2 ASN 71 36.939 24.196 5.770 622 O ILE 81 35.797 26.490 18.051
544 C ASN 71 34.692 20.748 8.429 623 N GLN 82 37.827 25.580 17.626
545 O ASN 71 35.265 19.632 8.437 624 CA GLN 82 38.170 25.472 19.073
546 N TYR 72 33.377 20.891 8.302 625 CB GLN 82 38.844 24.134 19.384
547 CA TYR 72 32.469 19.760 8.114 626 CG GLN 82 38.051 22.928 18.981
548 CB TYR 72 31.015 20.236 8.218 627 CD GLN 82 38.764 21.646 19.374
549 CG TYR 72 29.993 19.149 7.932 628 OEl GLN 82 39.895 21.689 19.850
550 CDl TYR 72 29.979 17.976 8.686 629 NE2 GLN 82 38.136 20.515 19.120
551 CEl TYR 72 29.038 16.971 8.457 630 C GLN 82 39.097 26.598 19.412
552 CZ TYR 72 28.114 17.112 7.455 631 O GLN 82 39.902 27.013 18.562
553 OH TYR 72 27.203 16.093 7.235 632 N VAL 83 39.004 27.082 20.659
554 CE2 TYR 72 28.087 18.261 6.675 633 CA VAL 83 39.842 28.144 21.172
555 CD2 TYR 72 29.030 19.290 6.924 634 CB VAL 83 39.021 29.425 21.551
556 C TYR 72 32.680 19.126 6.719 635 CGl VAL 83 38.455 30.087 20.263
557 O TYR 72 32.611 19.826 5.729 636 CG2 VAL 83 37.951 29.084 22.610
558 N PRO 73 32.965 17.810 6.657 637 C VAL 83 40.617 27.654 22.399
559 CA PRO 73 33.037 17.180 5.341 638 O VAL 83 40.176 26.722 23.070
560 CB PRO 73 33.818 15.868 5.618 639 N GLU 84 41.771 28.253 22.650
561 CG PRO 73 34.387 16.043 7.065 640 CA GLU 84 42.471 28.040 23.921
562 CD PRO 73 33.268 16.826 7.722 641 CB GLU 84 43.780 28.837 23.987
563 C PRO 73 31.629 16.959 4.776 642 CG GLD 84 44.947 28.145 23.251
564 O PRO 73 30.889 16.057 5.180 643 CD GLU 84 45.246 26.717 23.778
565 N GLY 74 31.237 17.833 3.857 644 OEl GLU 84 45.090 26.422 25.000
566 CA GLY 74 29.910 17.767 3.268 645 OE2 GLU 84 45.622 25.868 22.950
567 C GLY 74 29.673 19.118 2.611 646 C GLU 84 41.544 28.435 25.056
568 O GLY 74 30.491 20.025 2.764 647 O GLU 84 40.893 29.491 24.993
569 N LYS 75 28.574 19.252 1.884 648 N GLU 85 41.417 27.547 26.029
570 CA LYS 75 28.286 20.514 1.163 649 CA GLU 85 40.569 27.766 27.210
571 CB LYS 75 27.101 20.355 0.175 650 CB GLU 85 40.651 26.538 28.130
572 CG LYS 75 25.766 20.104 0.824 651 CG GLU 85 39.573 26.486 29.232
573 CD LYS 75 24.594 19.932 -0.116 652 CD GLU 85 39.283 25.087 29.780
574 CE LYS 75 23.270 20.077 0.652 653 OEl GLU 85 39.476 24.053 29.086
575 NZ LYS 75 22.050 19.684 -0.133 654 OE2 GLU 85 38.788 25.033 30.912
576 C LYS 75 28.052 21.691 2.098 655 C GLU 85 40.928 29.032 27.987
NA A AK NAK NA AK NAK
656 O GLϋ 85 40.033 29.712 28.541 735 O PHE 95 40.075 32.535 30.170
657 N ARG 86 42.222 29.351 28.034 736 N ASN 96 38.944 31.938 32.024
658 CA ARG 86 42.717 30.548 28.716 737 CA ASN 96 38.388 30.743 31.386
659 CB ARG 86 44.106 30.345 29.352 738 CB ASN 96 37.901 29.741 32.432
660 CG ARG 86 44.641 31.626 30.051 739 CG ASN 96 37.748 28.353 31.870
661 CD ARG 86 46.065 31.476 30.632 740 ODl ASN 96 36.998 28.131 30.929
662 NE ARG 86 46.463 32.746 31.257 741 ND2 ASN 96 38.445 27.401 32.454
663 CZ ARG 86 46.162 33.093 32.513 742 C ASN 96 37.260 31.071 30.427
664 NHl ARG 86 45.478 32.264 33.296 743 O ASN 96 36.230 31.614 30.830
665 NH2 ARG 86 46.546 34.267 32.995 744 N PHE 97 37.446 30.701 29.160
666 C ARG 86 42.737 31.742 27.781 745 CA PHE 97 36.436 30.880 28.116
667 O ARG 86 43.477 31.757 26.766 746 CB PHE 97 36.835 29.997 26.906
668 N ALA 87 41.915 32.750 28.123 747 CG PHE 97 35.867 30.057 25.749
669 CA ALA 87 41.866 34.012 27.371 748 CDl PHE 97 35.813 31.184 24.915
670 CB ALA 87 40.861 35.017 27.996 749 CEl PHE 97 34.895 31.204 23.817
671 C ALA 87 43.221 34.666 27.204 750 CZ PHE 97 34.077 30.109 23.576
672 O ALA 87 44.070 34.631 28.112 751 CE2 PHE 97 34.127 28.991 24.427
673 N ILE 88 43.406 35.270 26.030 752 CD2 PHE 97 35.032 28.980 25.482
674 CA ILE 88 44.512 36.184 25.791 753 C PHE 97 35.081 30.439 28.596
675 CB ILE 88 44.633 36.544 24.261 754 O PHE 97 34.087 31.153 28.428
676 CGl ILE 88 44.833 35.272 23.431 755 N ASN 98 35.047 29.256 29.221
677 CDl ILE 88 45.976 34.312 23.950 756 CA ASN 98 33.797 28.620 29.557
678 CG2 ILE 88 45.743 37.582 23.992 757 CB ASN 98 34.040 27.160 29.832
679 C ILE 88 44.251 37.425 26.652 758 CG ASN 98 34.743 26.504 28.662
680 O ILE 88 45.148 37.919 27.342 759 ODl ASN 98 34.188 26.453 27.546
681 N GLO 89 43.014 37.916 26.612 760 ND2 ASN 98 35.996 26.119 28.868
682 CA GLU 89 42.575 39.003 27.490 761 C ASN 98 33.027 29.272 30.689
683 CB GLO 89 42.258 40.261 26.695 762 O ASN 98 31.840 28.995 30.863
684 CG GLO 89 43.507 40.865 26.047 763 N ASP 99 33.733 30.072 31.466
685 CD GLϋ 89 43.225 42.087 25.211 764 CA ASP 99 33.089 30.857 32.555
686 OEl GLϋ 89 42.225 42.803 25.503 765 CB ASP 99 34.121 31.186 33.616
687 OE2 GLϋ 89 44.018 42.331 24.262 766 CG ASP 99 34.529 29.980 34.431
688 C GLϋ 89 41.358 38.527 28.267 767 ODl ASP 99 33.704 29.035 34.655
689 O GLϋ 89 40.396 38.081 27.669 768 OD2 ASP 99 35.690 29.971 34.879
690 N ARG 90 41.433 38.584 29.598 769 C ASP 99 32.475 32.136 32.006
691 CA ARG 90 40.378 38.046 30.473 770 O ASP 99 31.648 32.768 32.679
692 CB ARG 90 40.782 38.115 31.967 771 N ASN 100 32.877 32.530 30.789
693 CG ARG 90 40.658 39.490 32.648 772 CA ASN 100 32.458 33.820 30.232
694 CD ARG 90 41.204 39.387 34.097 773 CB ASN 100 33.656 34.538 29.629
695 NE ARG 90 40.267 38.800 35.070 774 CG ASN 100 34.508 35.176 30.653
696 CZ ARG 90 40.597 38.086 36.166 775 ODl ASN 100 34.178 36.245 31.178
697 NHl ARG 90 39.640 37.642 36.988 776 ND2 ASN 100 35.644 34.547 30.943
698 NH2 ARG 90 41.862 37.787 36.457 777 C ASN 100 31.378 33.720 29.171
699 C ARG 90 39.023 38.694 30.257 778 O ASN 100 30.838 34.738 28.762
700 O ARG 90 38.914 39.845 29.789 779 N VAL 101 31.090 32.509 28.697
701 N GLY 91 37.983 37.930 30.547 780 CA VAL 101 30.135 32.307 27.635
702 CA GLY 91 36.635 38.474 30.570 781 CB VAL 101 30.863 31.866 26.311
703 C GLY 91 36.291 38.863 32.003 782 CGl VAL 101 31.962 32.812 25.927
704 O GLY 91 37.138 38.745 32.926 783 CG2 VAL 101 31.424 30.381 26.485
705 N PRO 92 35.068 39.360 32.205 784 C VAL 101 29.104 31.263 27.998
706 CA PRO 92 34.661 39.755 33.564 785 O VAL 101 29.296 30.471 28.953
707 CB PRO 92 33.282 40.419 33.344 786 N THR 102 28.013 31.227 27.224
708 CG PRO 92 33.349 40.885 31.940 787 CA THR 102 26.924 30.307 27.444
709 CD PRO 92 34.022 39.707 31.228 788 CB THR 102 25.645 31.045 27.917
710 C PRO 92 34.582 38.592 34.535 789 OGl THR 102 25.304 32.054 26.945
711 O PRO 92 34.621 38.810 35.760 790 CG2 THR 102 25.877 31.735 29.262
712 N LYS 93 34.484 37.368 34.014 791 C THR 102 26.614 29.600 26.123
713 CA LYS 93 34.425 36.179 34.844 792 O THR 102 26.143 30.233 25.195
714 CB LYS 93 33.242 35.294 34.444 793 N PRO 103 26.872 28.294 26.036
715 CG LYS 93 31.881 35.983 34.587 794 CA PRO 103 26.547 27.494 24.844
716 CD LYS 93 30.748 34.991 34.310 795 CB PRO 103 27.016 26.064 25.235
717 CE LYS 93 30.572 33.989 35.450 796 CG PRO 103 28.020 26.277 26.324
718 NZ LYS 93 30.525 32.535 35.018 797 CD PRO 103 27.528 27.495 27.092
719 C LYS 93 35.766 35.407 34.788 798 C PRO 103 25.046 27.440 24.560
720 O LYS 93 35.824 34.225 35.098 799 O PRO 103 24.232 27.489 25.505
721 N GLY 94 36.827 36.122 34.446 800 N PHE 104 24.669 27.307 23.295
722 CA GLY 94 38.175 35.601 34.534 801 CA PHE 104 23.277 27.081 22.945
723 C GLY 94 38.703 35.093 33.195 802 CB PHE 104 22.998 27.633 21.536
724 O GLY 94 38.155 35.398 32.145 803 CG PHE 104 23.078 29.139 21.463
725 N PHE 95 39.784 34.333 33.271 804 CDl PHE 104 22.131 29.929 22.112
726 CA PHE 95 40.474 33.849 32.078 805 CEl PHE 104 22.222 31.333 22.058
727 CB PHE 95 41.941 33.523 32.397 806 CZ PHE 104 23.262 31.960 21.383
728 CG PHE 95 42.804 34.727 32.401 807 CE2 PHE 104 24.208 31.192 20.757
729 CDl PHE 95 43.291 35.235 31.204 808 CD2 PHE 104 24.110 29.768 20.816
730 CEl PHE 95 44.080 36.400 31.173 809 C PHE 104 22.863 25.618 23.017
731 CZ PHE 95 44.365 37.063 32.352 810 O PHE 104 23.667 24.735 22.817
732 CE2 PHE 95 43.874 36.579 33.559 811 N LYS 105 21.583 25.403 23.263
733 CD2 PHE 95 43.085 35.409 33.591 812 CA LYS 105 20.941 24.106 23.229
734 C PHE 95 39.805 32.709 31.354 813 CB LYS 105 19.981 24.015 24.426
NA A AK NAK X Y Z 898 CA LYS 117 15.878 35.522 15.289
814 CG LYS 105 18.987 22.837 24 .458 899 CB LYS 117 15 .929 36 .175 16 .682
815 CD LYS 105 17.790 23.152 25 .344 900 CG LYS 117 16 .217 35 .196 17 .815
818 C LYS 105 20.155 24.015 21 .925 901 CD LYS 117 15 .916 35 .800 19 .208
819 O LYS 105 19.518 24.994 21 .501 902 CE LYS 117 14 .580 35 .300 19 .740
820 N TYR 106 20.150 22.834 21 .319 903 NZ LYS 117 14 .530 35 .544 21 .203
821 CA TYR 106 19.346 22.613 20 .121 904 C LYS 117 17 .262 35 .114 14 .842
822 CB TYR 106 19.639 21.241 19 .498 905 O LYS 117 17 .752 34 .019 15 .201
823 CG TYR 106 21.025 21.069 18 .931 906 N VAL 118 17 .868 36 .004 14 .067
824 CDl TYR 106 21.525 21.922 17 .942 907 CA VAL 118 19 .235 35 .874 13 .589
825 CEl TYR 106 22.822 21.743 17 .403 908 CB VAL 118 19 .339 36 .087 12 .045
826 CZ TYR 106 23.584 20.683 17 .840 909 CGl VAL 118 20, .792 35 .799 11 .555
827 OH TYR 106 24.825 20.480 17 .326 910 CG2 VAL 118 18 .305 35 .193 11 .344
828 CE2 TYR 106 23.096 19.824 18 .818 911 C VAL 118 20 .028 36 .926 14 .311
829 CD2 TYR 106 21.841 20.021 19 .358 912 O VAL 118 19. .752 38, .136 14 .179
830 C TYR 106 17.871 22.629 20 .438 913 N ILE 119 21. .001 36, .481 15 .106
831 O TYR 106 17.433 21.995 21 .409 914 CA ILE 119 21, .781 37. .397 15 .937
832 N ALA 107 17.110 23.303 19 .577 915 CB ILE 119 21. .754 36, .982 17 .453
833 CA ALA 107 15.670 23.130 19 .492 916 CGl ILE 119 20. .324 36. .862 17 .944
834 CB ALA 107 15.092 24.112 18 .468 917 CDl ILE 119 20. .186 35. .972 19, .208
835 C ALA 107 15.300 21.685 19 .120 918 CG2 ILE 119 22. .613 37. .924 18 .310
836 O ALA 107 16.093 20.963 18 .498 919 C ILE 119 23. .233 37. ,444 15, .467
837 N ALA 108 14.095 21.261 19 .497 920 O ILE 119 23. .887 36. ,408 15, .363
838 CA ALA 108 13.584 19.950 19 .090 921 N GLY 120 23. ,734 38. .647 15. .213
839 CB ALA 108 12.249 19.684 19 .733 922 CA GLY 120 25. ,102 38. ,805 14. .742
840 C ALA 108 13.455 19.881 17 .574 923 C GLY 120 25. 459 40. 236 14. .444
841 O ALA 108 13.612 18.814 16 .975 924 O GLY 120 24. 846 41. 165 14. .986
842 N GLY 109 13.186 21.023 16 .961 925 N TYR 121 26. 428 40. ,407 13. .548
843 CA GLY 109 13.022 21.102 15. .519 926 CA TYR 121 27. 017 41. 708 13. .281
844 C GLY 109 12.616 22.494 15. .132 927 CB TYR 121 28. 494 41. 642 13. .629
845 O GLY 109 12.702 23.432 15 .921 928 CG TYR 121 28. 729 41. 169 15. ,027
846 N ALA 110 12.167 22.643 13 .896 929 CDl TYR 121 28. 623 42. 054 16. ,108
847 CA ALA 110 11.661 23.928 13, .459 930 CEl TYR 121 28. 830 41. 633 17. 418
848 CB ALA 110 12.799 24.854 13, .008 931 CZ TYR 121 29. 116 40. 293 17. 660
849 C ALA 110 10.657 23.684 12. .351 932 OH TYR 121 29. 261 39. 895 18. 959
850 O ALA 110 10.773 22.715 11. .624 933 CE2 TYR 121 29. 211 39. 392 16. 619
851 N LYS 111 9.658 24.556 12. ,250 934 CD2 TYR 121 29. 011 39. 821 15. 298
852 CA LYS 111 8.631 24.387 11. .233 935 C TYR 121 26. 827 42. 161 11. 821
853 CB LYS 111 7.388 23.659 11. .807 936 O TYR 121 27. 760 42. 099 11. 033
854 CG LYS 111 6.526 24.519 12. ,735 937 N PRO 122 25. 615 42. 587 11. 449
858 C LYS 111 8.303 25.731 10. ,585 938 CA PRO 122 25. 402 42. 888 10. 020
859 O LYS 111 8.431 26.793 11. ,216 939 CB PRO 122 23. 881 43. 001 9.908
860 N ALA 112 7.954 25.663 9.297 940 CG PRO 122 23. 432 43. 427 11. 273
861 CA ALA 112 7.393 26.783 8.546 941 CD PRO 122 24. 387 42. 763 12. 243
862 CB ALA 112 6.771 26.274 7.256 942 C PRO 122 26. 111 44. 148 9.463
863 C ALA 112 6.363 27.533 9.376 943 O PRO 122 26. 053 44. 380 8.249
864 O ALA 112 5.526 26.918 10. 058 944 N HIS 123 26. 783 44. 930 10. 319
865 N GLY 113 6.453 28.852 9.366 945 CA HIS 123 27. 455 46. 185 9.909
866 CA GLY 113 5.519 29.675 10. 148 946 CB HIS 123 26. 616 47. 383 10. 345
867 C GLY 113 6.086 30.209 11. 446 947 CG HIS 123 25. 171 47. 299 9.957
868 O GLY 113 5.841 31.357 11. 809 948 NDl HIS 123 24. 151 47. 386 10. 880
869 N GLU 114' 6.876 29.401 12. 159 949 CEl HIS 123 22. 986 47. 290 10. 260
870 CA GLU 114 7.423 29.893 13. 425 950 NE2 HIS 123 23. 213 47. 154 8.965
871 CB GLU 114 8.022 28.748 14. 262 951 CD2 HIS 123 24. 572 47. 160 8.748
872 CG GLU 114 9.495 28.425 14. 000 952 C HIS 123 28. 880 46. 327 10. 496
873 CD GLU 114 9.979 27.343 14. 932 953 O HIS 123 29. 033 46. 460 11. 715
874 OEl GLU 114 9.840 26.155 14. 592 954 N PRO 124 29. 927 46. 348 9.639
875 OE2 GLU 114 10.430 27.683 16. 037 955 CA PRO 124 31. 306 46. 112 10. 118
876 C GLU 114 8.391 31.051 13. 231 956 CB PRO 124 31. 955 45. 432 8.906
877 O GLU 114 9.001 31.199 12. 157 957 CG PRO 124 31. 245 46. 082 7.690
878 N ARG 115 8.494 31.908 14. 248 958 CD PRO 124 29. 901 46. 609 8.181
879 CA ARG 115 9.347 33.094 14. 179 959 C PRO 124 32. 135 47. 351 10. 503
880 CB ARG 115 8.696 34.288 14. 886 960 O PRO 124 33. 344 47. 379 10. 237
881 CG ARG 115 7.450 34.815 14. 145 961 N TYR 125 31. 501 48. 335 11. 143
882 CD ARG 115 6.817 36.023 14. 851 962 CA TYR 125 32. 085 49. 672 11. 400
883 NE ARG 115 7.690 37.203 14. 953 963 CB TYR 125 31. 018 50. 580 12. 037
884 CZ ARG 115 8.099 37.925 13. 918 964 CG TYR 125 29. 973 49. 824 12. 827
885 NHl ARG 115 7.744 37.579 12. 687 971 C TYR 125 33. 426 49. 756 12. 180
886 NH2 ARG 115 8.886 38.975 14. 107 972 O TYR 125 34. 074 48. 736 12. 440
887 C ARG 115 10.743 32.833 14. 760 973 N LYS 126 33. 829 50. 989 12. 523
888 O ARG 115 10.874 32.237 15. 825 974 CA LYS 126 35. 069 51. 307 13. 273
889 N ILE 116 11.772 33.282 14. 052 975 CB LYS 126 34. 767 51. 671 14. 740
890 CA ILE 116 13.137 33.055 14. 530 976 CG LYS 126 33. 652 52. 710 14. 944
891 CB ILE 116 13.883 32.051 13. 597 980 C LYS 126 36. 265 50. 314 13. 076
892 CGl ILE 116 13.753 32.453 12. 105 981 O LYS 126 36. 804 50. 273 11. 960
893 CDl ILE 116 14.584 33.682 11. 737 982 N ASN 127 36. 727 49. 542 14. 077
894 CG2 ILE 116 13.320 30.660 13. 775 983 CA ASN 127 36. 367 49. 613 15. 497
895 C ILE 116 13.845 34.392 14. 707 988 C ASN 127 35. 156 48. 785 15. 943
896 O ILE 116 13.292 35.463 14. 400 989 O ASN 127 35. 008 47. 603 15. 581
897 N LYS 117 15.050 34.356 15. 247 990 N LYS 128 34. 316 49. 450 16. 741
NA AK NAK NA A AK NAK
991 CA LYS 128 33.058 48.947 17.324 1074 CGl VAL 138 11.202 27.816 9.037
992 CB LYS 128 32.048 50.102 17.408 1075 CG2 VAL 138 11.669 29.305 10.953
997 C LYS 128 32.389 47.721 16.678 1076 C VAL 138 9.760 30.007 7.661
998 O LYS 128 32.064 47.715 15.486 1077 O VAL 138 8.534 29.850 1.111
999 N TYR 129 32.213 46.678 17.484 1078 N MET 139 10.399 29.948 6.489
1000 CA TYR 129 31.340 45.566 17.135 1079 CA MET 139 9.693 29.682 5.229
1001 CB TYR 129 31.810 44.278 17.807 1080 CB MET 139 10.335 30.458 4.079
1002 CG TYR 129 33.169 43.756 17.400 1081 CG MET 139 10.555 31.920 4.382
1003 CDl TYR 129 33.367 43.195 16.140 1082 SD MET 139 9.036 32.905 4.502
1004 CEl TYR 129 34.600 42.698 15.755 1083 CE MET 139 8.073 32.220 5.800
1005 CZ TYR 129 35.658 42.735 16.642 1084 C MET 139 9.613 28.216 4.902
1006 OH TYR 129 36.876 42.229 16.245 1085 O MET 139 8.574 27.741 4.441
1007 CE2 TYR 129 35.495 43.281 17.916 1086 N SER 140 10.699 27.470 5.158
1008 CD2 TYR 129 34.240 43.785 18.282 1087 CA SER 140 10.716 26.030 4.940
1009 C TYR 129 29.974 45.918 17.704 1088 CB SER 140 10.887 25.721 3.456
1010 O TYR 129 29.856 46.215 18.894 1089 OG SER 140 12.008 26.439 2.963
1011 N VAL 130 28.944 45.896 16.874 1090 C SER 140 11.873 25.367 5.669
1012 CA VAL 130 27.606 46.192 17.342 1091 O SER 140 12.867 26.019 6.001
1013 CB VAL 130 27.049 47.522 16.732 1092 N VAL 141 11.718 24.068 5.900
1014 CGl VAL 130 25.711 47.847 17.309 1093 CA VAL 141 12.789 23.216 6.428
1015 CG2 VAL 130 28.030 48.660 16.927 1094 CB VAL 141 12.564 22.873 7.916
1016 C VAL 130 26.685 45.023 17.013 1095 CGl VAL 141 13.592 21.816 8.396
1017 O VAL 130 26.436 44.704 15.845 1096 CG2 VAL 141 12.618 24.141 8.761
1018 N LEU 131 26.168 44.380 18.052 1097 C VAL 141 12.857 21.949 5.594
1019 CA LEO 131 25.333 43.212 17.857 1098 O VAL 141 11.881 21.189 5.526
1020 CB LEU 131 25.330 42.363 19.134 1099 N GLU 142 13.998 21.714 4.960
1021 CG LEU 131 24.819 40.941 18.972 1100 CA GLU 142 14.167 20.557 4.124
1022 CDl LEU 131 25.813 40.167 18.095 1101 CB GLU 142 13.900 20.949 2.674
1023 CD2 LEU 131 24.684 40.303 20.372 1102 CG GLU 142 13.913 19.764 1.703
1024 C LEU 131 23.921 43.601 17.491 1106 C GLU 142 15.595 20.026 4.290
1025 O LEU 131 23.338 44.497 18.121 1107 O GLU 142 16.565 20.731 3.984
1026 N TYR 132 23.359 42.943 16.485 1108 N GLY 143 15.712 18.814 4.826
1027 CA TYR 132 21.970 43.153 16.126 1109 CA GLY 143 17.023 18.253 5.185
1028 CB TYR 132 21.834 43.698 14.694 1110 C GLY 143 17.884 19.251 5.952
1029 CG TYR 132 22.235 45.145 14.560 1111 O GLY 143 17.493 19.744 7.000
1030 CDl TYR 132 21.265 46.141 14.381 1112 N SER 144 19.060 19.549 5.421
1031 CEl TYR 132 21.618 47.463 14.246 1113 CA SER 144 19.983 20.450 6.108
1032 CZ TYR 132 22.943 47.809 14.289 1114 CB SER 144 21.434 20.038 5.834
1033 OH TYR 132 23.300 49.130 14.155 1115 OG SER 144 21.732 20.215 4.451
1034 CE2 TYR 132 23.927 46.848 14.466 1116 C SER 144 19.740 21.919 5.742
1035 CD2 TYR 132 23.559 45.520 14.588 1117 O SER 144 20.564 22.778 6.026
1036 C TYR 132 21.209 41.873 16.221 1118 N SER 145 18.606 22.214 5.101
1037 O TYR 132 21.808 40.794 16.225 1119 CA SER 145 18.340 23.592 4.718
1038 N GLU 133 19.892 42.000 16.345 1120 CB SER 145 18.037 23.721 3.213
1039 CA GLU 133 18.961 40.894 16.270 1121 OG SER 145 17.777 25.103 2.951
1040 CB GLU 133 18.218 40.743 17.599 1122 C SER 145 17.191 24.173 5.500
1041 CG GLU 133 17.224 39.610 17.642 1123 O SER 145 16.106 23.618 5.496
1042 CD GLU 133 16.815 39.267 19.075 1124 N ILE 146 17.443 25.292 6.169
1043 OEl GLU 133 17.673 39.351 19.981 1125 CA ILE 146 16.363 26.067 6.789
1044 OE2 GLU 133 15.633 38.897 19.284 1126 CB ILE 146 16.432 26.142 8.374
1045 C GLU 133 17.981 41.167 15.139 1127 CGl ILE 146 15.193 26.924 8.912
1046 O GLU 133 17.387 42.271 15.065 1128 CDl ILE 146 15.025 26.913 10.472
1047 N SER 134 17.813 40.183 14.260 1129 CG2 ILE 146 17.746 26.788 8.854
1048 CA SER 134 16.905 40.303 13.102 1130 C ILE 146 16.336 27.427 6.141
1049 CB SER 134 17.682 40.295 11.767 1131 O ILE 146 17.375 28.118 6.017
1050 OG SER 134 16.783 40.127 10.678 1132 N VAL 147 15.131 27.806 5.702
1051 C SER 134 15.870 39.212 13.121 1133 CA VAL 147 14.926 29.071 5.026
1052 O SER 134 16.215 38.026 13.207 1134 CB VAL 147 14.337 28.846 3.588
1053 N THR 135 14.579 39.587 13.051 1135 CGl VAL 147 14.176 30.187 2.873
1054 CA THR 135 13.504 38.612 13.225 1136 CG2 VAL 147 15.248 27.910 2.767
1055 CB THR 135 12.513 39.042 14.334 1137 C VAL 147 14.015 29.987 5.855
1056 OGl THR 135 11.845 40.239 13.916 1138 O VAL 147 13.019 29.541 6.380
1057 CG2 THR 135 13.241 39.299 15.630 1139 N TYR 148 14.390 31.250 5.979
1058 C THR 135 12.717 38.307 11.952 1140 CA TYR 148 13.637 32.178 6.807
1059 O THR 135 12.720 39.095 10.997 1141 CB TYR 148 14.198 32.227 8.248
1060 N GLY 136 12.071 37.151 11.928 1142 CG TYR 148 15.717 32.119 8.302
1061 CA GLY 136 11.275 36.746 10.772 1143 CDl TYR 148 16.526 33.249 8.243
1062 C GLY 136 10.772 35.335 10.800 1144 CEl TYR 148 17.904 33.150 8.259
1063 O GLY 136 11.254 34.524 11.606 1145 CZ TYR 148 18.487 31.892 8.357
1064 N PRO 137 9.804 35.005 9.915 1146 OH TYR 148 19.850 31.775 8.355
1065 CA PRO 137 9.256 33.674 9.905 1147 CE2 TYR 148 17.732 30.765 8.415
1066 CB PRO 137 7.911 33.845 9.180 1148 CD2 TYR 148 16.337 30.873 8.383
1067 CG PRO 137 8.131 34.944 8.277 1149 C TYR 148 13.654 33.552 6.188
1068 CD PRO 137 9.178 35.854 8.885 1150 O TYR 148 14.661 34.014 5.618
1069 C PRO 137 10.115 32.678 9.151 1151 N SER 149 12.539 34.244 6.333
1070 O PRO 137 10.774 33.032 8.162 1152 CA SER 149 12.504 35.599 5.819
1071 N VAL 138 10.093 31.439 9.634 1153 CB SER 149 11.152 35.886 5.164
1072 CA VAL 138 10.627 30.285 8.900 1154 OG SER 149 11.188 37.197 4.645
1073 CB VAL 138 10.726 29.026 9.812 1155 C SER 149 12.839 36.625 6.901
NA AK NAK NA AK NAK
1156 O SER 149 11.955 37.251 7. 498 1235 O VAL 161 18.740 29.382 15.510
1157 N ALA 150 14.130 36.815 7. 123 1236 N LEU 162 17.833 31.371 16.113
1158 CA ALA 150 14.617 37.739 8. 110 1237 CA LEU 162 17.222 30.865 17.345
1159 CB ALA 150 15.109 36.972 9. 345 1238 CB LEU 162 17.651 31.731 18.539
1160 C ALA 150 15.755 38.487 7. 456 1239 CG LEU 162 19.122 31.862 18.951
1161 O ALA 150 16.581 37.880 6. 789 1240 CDl LEU 162 19.254 32.588 20.288
1162 N HIS 151 15.813 39.799 7. 626 1241 CD2 LEU 162 19.841 30.505 19.026
1163 CA HIS 151 16.847 40.568 6. 957 1242 C LEU 162 15.708 30.885 17.242
1164 CB HIS 151 16.521 42.068 6. 966 1243 O LEU 162 15.147 31.746 16.533
1165 CG HIS 151 17.523 42.890 6. 224 1244 N ASN 163 15.050 29.959 17.923
1166 NDl HIS 151 17.444 43.104 4. 862 1245 CA ASN 163 13.573 30.031 18.032
1167 CEl HIS 151 18.456 43.857 4. 480 1246 CB ASN 163 12.959 28.656 18.161
1168 NE2 HIS 151 19.196 44.130 5. 541 1247 CG ASN 163 13.286 27.966 19.490
1169 CD2 HIS 151 18.630 43.540 6. 644 1248 ODl ASN 163 13.723 28.611 20.438
1170 C HIS 151 18.253 40.351 7. 508 1249 ND2 ASN 163 13.059 26.645 19.556
1171 O HIS 151 18.481 40.478 8. 706 1250 C ASN 163 13.123 30.988 19.141
1172 N THR 152 19.197 40.052 6. 608 1251 O ASN 163 13.945 31.699 19.721
1173 CA THR 152 20.604 39.836 6. 970 1252 N SER 164 11.805 31.046 19.397
1174 CB THR 152 20.963 38.321 6. 935 1253 CA SER 164 11.283 32.018 20.375
1175 OGl THR 152 20.607 37.770 5. 656 1254 CB SER 164 9.766 32.066 20.325
1176 CG2 THR 152 20.165 37.575 7. 994 1255 OG SER 164 9.249 30.768 20.363
1177 C THR 152 21.575 40.574 6. 056 1256 C SER 164 11.760 31.729 21.799
1178 O THR 152 21.224 40.897 4. 926 1257 O SER 164 11.821 32.631 22.644
1179 N GLU 153 22.784 40.833 6. 563 1258 N ASN 165 12.142 30.481 22.043
1180 CA GLU 153 23.890 41.345 5. 780 1259 CA ASN 165 12.711 30.123 23.318
1181 CB GLU 153 24.333 42.703 6. 306 1260 CB ASN 165 12.139 28.801 23.812
1182 CG GLU 153 23.265 43.830 6. 178 1261 CG ASN 165 12.441 28.589 25.281
1183 CD GLU 153 22.814 44.137 4. 740 1262 ODl ASN 165 12.424 29.547 26.057
1184 OEl GLU 153 23.563 43.838 3. 779 1263 ND2 ASN 165 12.783 27.371 25.653
1185 OE2 GLU 153 21.696 44.700 4. 573 1264 C ASN 165 14.254 30.125 23.351
1186 C GLU 153 25.063 40.353 5. 827 1265 O ASN 165 14.884 29.565 24.260
1187 O GLU 153 24.998 39.358 6. 545 1266 N ASN 166 14.852 30.775 22.359
1188 N SER 154 26.134 40.633 5. 089 1267 CA ASN 166 16.300 30.974 22.293
1189 CA SER 154 27.242 39.681 4. 990 1268 CB ASN 166 16.821 31.814 23.482
1190 CB SER 154 28.302 40.158 4. 001 1269 CG ASN 166 16.438 33.291 23.356
1191 OG SER 154 29.000 41.261 4. 517 1270 ODl ASN 166 16.334 33.815 22.246
1192 C SER 154 27.873 39.367 6. 356 1271 ND2 ASN 166 16.199 33.956 24.492
1193 O SER 154 28.288 38.230 6. 583 1272 C ASN 166 17.076 29.676 22.137
1194 N GLY 155 27.941 40.376 7. 237 1273 O ASN 166 18.220 29.603 22.548
1195 CA GLY 155 28.390 40.207 8. 618 1274 N GLU 167 16.423 28.663 21.576
1196 C GLY 155 27.626 39.171 9. 442 1275 CA GLU 167 17.071 27.387 21.263
1197 O GLY 155 28.139 38.732 10 .481 1276 CB GLU 167 16.129 26.211 21.445
1198 N ASN 156 26.416 38.805 9. 020 1277 CG GLU 167 15.478 26.204 22.849
1199 CA ASN 156 25.638 37.752 9. 696 1278 CD GLU 167 14.256 25.339 22.941
1200 CB ASN 156 24.146 37.786 9. 333 1279 OEl GLU 167 13.603 25.063 21.901
1201 CG ASN 156 23.376 38.872 10 .099 1280 OE2 GLU 167 13.936 24.935 24.080
1202 ODl ASN 156 22.871 39.825 9. 486 1281 C GLU 167 17.531 27.458 19.816
1203 ND2 ASN 156 23.345 38.776 11 .454 1282 O GLU 167 16.913 28.136 18.963
1204 C ASN 156 26.156 36.327 9. 426 1283 N LEU 168 18.622 26.749 19.538
1205 O ASN 156 25.627 35.372 10 .010 1284 CA LEU 168 19.244 26.785 18.230
1206 N SER 157 27.071 36.181 8. 461 1285 CB LEU 168 20.674 26.197 18.403
1207 CA SER 157 27.749 31.892 8. 231 1286 CG LEU 168 21.662 26.327 17.291
1208 CB SER 157 29.046 35.134 7. 467 1287 CDl LEU 168 21.999 27.785 16.934
1209 OG SER 157 28.723 35.358 6. 083 1288 CD2 LEU 168 22.903 25.546 17.775
1210 C SER 157 28.089 34.311 9. 594 1289 C LEU 168 18.486 25.998 17.160
1211 O SER 157 28.762 34.958 10 .387 1290 O LEU 168 18.188 24.792 17.339
1212 N GLY 158 27.626 33.102 9. 844 1291 N VAL 169 18.148 26.672 16.055
1213 CA GLY 158 27.978 32.389 11 .095 1292 CA VAL 169 17.502 26.001 14.922
1214 C GLY 158 27.033 32.676 12 .246 1293 CB VAL 169 16.092 26.546 14.537
1215 O GLY 158 27.190 32.106 13 .324 1294 CGl VAL 169 15.096 26.348 15.712
1216 N SER 159 26.003 33.517 12 .028 1295 CG2 VAL 169 16.141 27.965 14.084
1217 CA SER 159 25.011 33.799 13 .089 1296 C VAL 169 18.339 25.938 13.653
1218 CB SER 159 24.045 34.924 12 .638 1297 O VAL 169 17.998 25.167 12.747
1219 OG SER 159 24.765 36.155 12 .500 1298 N GLY 170 19.366 26.749 13.561
1220 C SER 159 24.168 32.600 13 .538 1299 CA GLY 170 20.295 26.641 12.427
1221 O SER 159 23.716 31.826 12 .738 1300 C GLY 170 21.426 27.617 12.557
1222 N PRO 160 23.891 32.487 14 .866 1301 O GLY 170 21.529 28.315 13.566
1223 CA PRO 160 22.909 31.501 15 .288 1302 N ILE 171 22.346 27.610 11.574
1224 CB PRO 160 22.979 31.553 16 .839 1303 CA ILE 171 23.411 28.623 11.500
1225 CG PRO 160 23.500 33.009 17 .143 1304 CB ILE 171 24.854 28.023 11.647
1226 CD PRO 160 24.420 33.313 15 .973 1305 CGl ILE 171 25.938 29.079 11.407
1227 C PRO 160 21.571 32.006 14 .833 1306 CDl ILE 171 27.370 28.574 11.711
1228 O PRO 160 21.340 33.229 14 .841 1307 CG2 ILE 171 25.019 26.723 10.836
1229 N VAL 161 20.713 31.067 14 .484 1308 C ILE 171 23.305 29.324 10.147
1230 CA VAL 161 19.320 31.310 14 .211 1309 O ILE 171 23.217 28.653 9.118
1231 CB VAL 161 18.935 30.688 12 .892 1310 N HIS 172 23.336 30.651 10.185
1232 CGl VAL 161 17.425 30.917 12 .599 1311 CA HIS 172 23.134 31.457 8.976
1233 CG2 VAL 161 19.855 31.244 11 .790 1312 CB HIS 172 23.024 32.925 9.379
1234 C VAL 161 18.601 30.598 15 .344 1313 CG HIS 172 22.829 33.843 8.209
NA A AK NAK NA AK NAK
1314 NDl HIS 172 21.652 33.874 7.493 1397 N TYR 186 19.229 31.994 4.258
1315 CEl HIS 172 21.773 34.739 6.500 1398 CA TYR 186 19.437 30.564 4.223
1316 NE2 HIS 172 22.988 35.260 6.541 1399 CB TYR 186 20.055 30.119 2.866
1317 CD2 HIS 172 23.669 34.718 7.607 1400 CG TYR 186 19.163 30.586 1.764
1318 C HIS 172 24.331 31.291 8.071 1401 CDl TYR 186 19.430 31.788 1.128
1319 O HIS 172 25.477 31.320 8.568 1402 CEl TYR 186 18.549 32.286 0.147
1320 N PHE 173 24.117 31.176 6.748 1403 CZ TYR 186 17.403 31.599 -0.140
1321 CA PHE 173 25.280 31.236 5.840 1404 OH TYR 186 16.577 32.140 -1.101
1322 CB PHE 173 25.783 29.820 5.453 1405 CE2 TYR 186 17.082 30.416 0.489
1323 CG PHE 173 24.981 29.182 4.385 1406 CD2 TYR 186 17.967 29.924 1.4841
1324 CDl PHE 173 25.431 29.228 3.056 1407 C TYR 186 20.349 30.187 5.371
1325 CEl PHE 173 24.657 28.684 2.045 1408 O TYR 186 21.369 30.829 5.611
1326 CZ PHE 173 23.433 28.082 2.347 1409 N GLY 187 19.926 29.168 6.094
1327 CE2 PHE 173 22.987 28.031 3.666 1410 CA GLY 187 20.779 28.609 7.172
1328 CD2 PHE 173 23.761 28.583 4.665 1411 C GLY 187 20.946 27.103 6.998
1329 C PHE 173 25.124 32.151 4.578 1412 O GLY 187 20.189 26.437 6.281
1330 O PHE 173 26.124 32.620 4.019 1413 N VAL 188 21.935 26.556 7.704
1331 N ALA 174 23.892 32.482 4.206 1414 CA VAL 188 22.100 25.091 7.793
1332 CA ALA 174 23.675 33.243 2.948 1415 CB VAL 188 23.574 24.705 7.963
1333 CB ALA 174 23.694 32.283 1.751 1416 CGl VAL 188 23.715 23.179 8.023
1334 C ALA 174 22.359 34.016 2.942 1417 CG2 VAL 188 24.402 25.266 6.815
1335 O ALA 174 21.404 33.626 3.590 1418 C VAL 188 21.307 24.632 8.988
1336 N SER 175 22.289 35.063 2.128 1419 O VAL 188 21.470 25.160 10.071
1337 CA SER 175 20.974 35.636 1.793 1420 N TYR 189 20.437 23.663 8.760
1338 CB SER 175 20.695 36.924 2.538 1421 CA TYR 189 19.627 23.075 9.815
1339 OG SER 175 21.905 37.573 2.866 1422 CB TYR 189 18.379 22.555 9.156
1340 C SER 175 20.826 35.876 0.298 1423 CG TYR 189 17.332 22.049 10.094
1341 O SER 175 21.752 35.635 -0.478 1424 CDl TYR 189 16.522 22.942 10.811
1342 N ASP 176 19.641 36.312 -0.097 1425 CEl TYR 189 15.527 22.474 11.668
1343 CA ASP 176 19.443 36.799 -1.448 1426 CZ TYR 189 15.334 21.115 11.786
1344 CB ASP 176 18.048 36.413 -1.944 1427 OH TYR 189 14.328 20.627 12.607
1345 CG ASP 176 17.887 34.894 -2.123 1428 CE2 TYR 189 16.118 20.205 11.080
1346 ODl ASP 176 18.887 34.190 -2.399 1429 CD2 TYR 189 17.105 20.678 10.226
1347 OD2 ASP 176 16.764 34.399 -1.980 1430 C TYR 189 20.407 21.892 10.394
1348 C ASP 176 19.634 38.297 -1.445 1431 O TYR 189 21.094 21.197 9.664
1349 O ASP 176 19.380 38.965 -0.449 1432 N PHE 190 20.297 21.668 11.699
1350 N VAL 177 20.157 38.839 -2.529 1433 CA PHE 190 21.160 20.674 12.319
1351 CA VAL 177 20.052 40.286 -2.728 1434 CB PHE 190 21.542 21.083 13.743
1352 CB VAL 177 21.376 40.943 -3.134 1435 CG PHE 190 22.365 22.356 13.785
1353 CGl VAL 177 21.967 40.268 -4.379 1436 CDl PHE 190 21.807 23.545 14.239
1354 CG2 VAL 177 21.141 42.447 -3.321 1437 CEl PHE 190 22.559 24.706 14.261
1355 C VAL 177 18.918 40.666 -3.692 1438 CZ PHE 190 23.868 24.711 13.812
1356 O VAL 177 17.744 40.801 -3.282 1439 CE2 PHE 190 24.429 23.537 13.337
1357 N ASP 181 11.008 44.174 3.566 1440 CD2 PHE 190 23.674 22.377 13.316
1358 CA ASP 181 11.761 43.230 2.730 1441 C PHE 190 20.567 19.287 12.212
1363 C ASP 181 11.331 41.773 2.954 1442 O PHE 190 19.791 18.832 13.053
1364 O ASP 181 11.519 41.213 4.047 1443 N THR 191 20.944 18.617 11.140
1365 N ASN 182 10.750 41.157 1.930 1444 CA THR 191 20.490 17.258 10.895
1366 CA ASN 182 10.327 39.764 2.054 1445 CB THR 191 20.782 16.848 9.457
1367 CB ASN 182 8.831 39.611 1.754 1446 OGl THR 191 22.188 16.965 9.231
1368 CG ASN 182 7.958 39.849 3.011 1447 CG2 THR 191 20.014 17.714 8.459
1369 ODl ASN 182 8.107 40.858 3.726 1448 C THR 191 21.306 16.346 11.809
1370 ND2 ASN 182 7.074 38.900 3.297 1449 O THR 191 22.366 16.763 12.323
1371 C ASN 182 11.251 38.760 1.321 1450 N PRO 192 20.823 15.115 12.031
1372 O ASN 182 10.836 37.681 0.878 1451 CA PRO 192 21.590 14.167 12.872
1373 N ARG 183 12.521 39.140 1.245 1452 CB PRO 192 20.846 12.830 12.656
1374 CA ARG 183 13.560 38.299 0.679 1453 CG PRO 192 19.471 13.225 12.331
1375 CB ARG 183 14.795 39.135 0.313 1454 CD PRO 192 19.560 14.519 11.550
1376 CG ARG 183 15.655 39.641 1.474 1455 C PRO 192 23.060 14.029 12.472
1377 CD ARG 183 16.914 40.297 0.942 1456 O PRO 192 23.940 13.997 13.324
1378 NE ARG 183 17.890 40.594 1.994 1457 N GLU 193 23.327 13.944 11.172
1379 CZ ARG 183 19.011 41.290 1.807 1458 CA GLO 193 24.674 13.789 10.676
1380 NHl ARG 183 19.303 41.782 0.604 1459 CB GLU 193 24.610 13.584 9.145
1381 NH2 ARG 183 19.842 41.514 2.820 1460 CG GLU 193 25.936 13.348 8.487
1382 C ARG 183 13.892 37.179 1.661 1461 CD GLU 193 25.834 13.028 6.987
1383 O ARG 183 13.674 37.312 2.872 1462 OEl GLU 193 26.823 12.480 6.410
1384 N ASN 184 14.397 36.071 1.130 1463 OE2 GLU 193 24.763 13.311 6.387
1385 CA ASN 184 14.733 34.925 1.968 1464 C GLU 193 25.559 14.988 11.050
1386 CB ASN 184 14.219 33.634 1.328 1465 O GLU 193 26.728 14.845 11.447
1387 CG ASN 184 12.718 33.501 1.417 1466 N ILE 194 25.007 16.198 10.928
1388 ODl ASN 184 12.065 34.165 2.235 1467 CA ILE 194 25.787 17.369 11.285
1389 ND2 ASN 184 12.155 32.634 0.593 1468 CB ILE 194 25.178 18.663 10.651
1390 C ASN 184 16.223 34.851 2.317 1469 CGl ILE 194 25.213 18.553 9.108
1391 O ASN 184 17.103 35.343 1.586 1470 CDl ILE 194 24.346 19.662 8.390
1392 N ALA 185 16.495 34.252 3.474 1471 CG2 ILE 194 25.957 19.845 11.092
1393 CA ALA 185 17.863 33.995 3.916 1472 C ILE 194 26.002 17.497 12.827
1394 CB ALA 185 18.198 34.827 5.153 1473 O ILE 194 27.085 17.816 13.304
1395 C ALA 185 17.988 32.493 4.194 1474 N LYS 195 24.969 17.212 13.595
1396 O ALA 185 16.984 31.780 4.323 1475 CA LYS 195 25.097 17.144 15.056
NA AK NAK X NA AK NAK
1476 CB LYS 195 23.779 16.672 15.648 1517 CA GLU 200 32.864 14.611 17.685
1477 CG LYS 195 22.734 17.783 15.597 1518 CB GLU 200 32.984 13.848 16.366
1478 CD LYS 195 21.483 17.402 16.428 1519 CG GLU 200 32.132 12.660 16.244
1479 CE LYS 195 20.397 18.478 16.341 1520 CD GLU 200 32.032 12.152 14.804
1480 NZ LYS 195 19.188 17.946 17.115 1521 OEl GLU 200 31.413 11.073 14.627
1481 C LYS 195 26.185 16.217 15.541 1522 OE2 GLU 200 32.528 12.852 13.863
1482 O LYS 195 26.924 16.543 16.459 1523 C GLU 200 33.982 15.639 17.804
1483 N LYS 196 26.239 15.038 14.951 1524 O GLU 200 35.087 15.321 18.252
1484 CA LYS 196 27.281 14.046 15.242 1525 N ASN 201 33.688 16.903 17.483
1485 CB LYS 196 27.032 12.778 14.434 1526 CA ASN 201 34.714 17.900 17.377
1486 CG LYS 196 28.078 11.668 14.671 1527 CB ASN 201 34.726 18.421 15.943
1487 CD LYS 196 27.969 10.638 13.546 1528 CG ASN 201 35.313 17.430 15.015
1488 CE LYS 196 26.505 10.198 13.407 1529 ODl ASN 201 36.526 17.193 15.050
1489 NZ LYS 196 25.958 10.253 12.014 1530 ND2 ASN 201 34.467 16.756 14.265
1490 C LYS 196 28.681 14.583 14.989 1531 C ASN 201 34.672 19.048 18.388
1491 O LYS 196 29.556 14.481 15.845 1532 O ASN 201 35.502 19.972 18.358
1492 N PHE 197 28.903 15.184 13.815 1533 N ILE 202 33.733 18.961 19.312
1493 CA PHE 197 30.177 15.790 13.531 1534 CA ILE 202 33.783 19.830 20.495
1494 CB PHE 197 30.160 16.384 12.096 1535 CB ILE 202 32.417 19.831 21.230
1495 CG PHE 197 31.242 17.380 11.870 1536 CGl ILE 202 31.464 20.780 20.486
1496 CDl PHE 197 30.974 18.760 11.981 1537 CDl ILE 202 30.012 20.508 20.697
1497 CEl PHE 197 31.991 19.686 11.792 1538 CG2 ILE 202 32.559 20.334 22.693
1498 CZ PHE 197 33.288 19.244 11.501 1539 C ILE 202 34.976 19.440 21.405
1499 CE2 PHE 197 33.539 17.890 11.383 1540 O ILE 202 35.248 18.232 21.606
1500 CD2 PHE 197 32.503 16.966 11.557 1541 N ASP 203 35.705 20.433 21.918
1501 C PHE 197 30.536 16.865 14.555 1542 CA ASP 203 36.840 20.174 22.802
1502 O PHE 197 31.656 16.937 15.033 1543 CB ASP 203 37.550 21.468 23.221
1503 N ILE 198 29.570 17.727 14.916 1544 CG ASP 203 38.949 21.209 23.777
1504 CA ILE 198 29.854 18.745 15.880 1545 ODl ASP 203 39.523 20.127 23.477
1505 CB ILE 198 28.665 19.718 16.021 1546 OD2 ASP 203 39.474 22.068 24.504
1506 CGl ILE 198 28.511 20.491 14.715 1547 C ASP 203 36.400 19.387 24.032
1507 CDl ILE 198 27.152 21.285 14.651 1548 O ASP 203 35.332 19.632 24.593
1508 CG2 ILE 198 28.924 20.695 17.181 1549 N LYS 204 37.247 18.444 24.459
1509 C ILE 198 30.220 18.107 17.228 1550 CA LYS 204 36.866 17.556 25.556
1510 O ILE 198 31.190 18.479 17.829 1551 CB LYS 204 36.428 16.176 25.034
1511 N ALA 199 29.488 17.093 17.627 1552 CG LYS 204 35.002 16.097 24.529
1512 CA ALA 199 29.763 16.450 18.925 1553 CD LYS 204 34.783 14.864 23.684
1513 CB ALA 199 28.649 15.466 19.288 1554 CE LYS 204 33.638 14.997 22.643
1514 C ALA 199 31.120 15.778 18.939 1555 NZ LYS 204 34.161 15.549 21.407
1515 O ALA 199 31.828 15.856 19.930 1556 C LYS 204 37.990 17.387 26.558
1516 N GLU 200 31.533 15.217 17.795 1557 O LYS 204 39.177 17.448 26.148
1558 OXT LYS 204 37.695 17.196 21.116
Claims
1. A polypeptide exhibiting affinity towards the active centre of Staphylococcus aureus protease particularly the proteases SpIA or SpIB, possessing the amino-acid sequence Xaal-Xaa2-Xaa3-Xaa4-Xaa5, where: in the case of protease SpIA:
Xaal is an amino-acid selected from among: Trp, Tyr, Phe, VaI, He, Leu, Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser, Tyr, Phe, Asp, Pro,
Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He, VaI, Thr,
Xaa4 is omitted or is any given amino-acid, preferentially selected from among: Ser, Thr, GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr, Xaa5 is omitted or is any given amino-acid, whereas in the case of SpIB:
Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser, Thr or GIy,
Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg, Lys, Ser or Thr,
Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy, Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn, Xaa5 is omitted or is any given amino-acid, preferentially selected form among: Thr, Ser, VaI, GIy, Ala, GIu, Met, GIn, Asp, Asn.
2. Polypeptide according to Claim 1, characterised in that in the case of protease SpIA it contains a sequence selected from among: Trp-Leu-Tyr, Trp-Leu-Tyr-Ser, Tyr-Glu-Tyr-Ala, Tyr-Glu-Tyr-Ser, Tyr-Glu-Tyr, Tyr- Met-Tyr, Tyr-Met-Tyr-Ser, Tyr-Ala-Tyr-Ser, Tyr-Ala-Tyr, Tyr-Thr-Tyr-Ser, Tyr-Thr-Tyr, Tyr-Leu-Tyr-Gly, Tyr-Leu-Tyr, Tyr-Leu-Tyr-Ser, Phe-Leu-Tyr-Ser, Phe-Leu-Tyr, VaI- Leu-Tyr-Thr, Val-Leu-Tyr, Trp-Leu-Ser-Thr, Trp-Leu-Ser, Trp-Met-Asn-Thr, Trp-Met- Asn, Trp-Trp-Tyr-Thr, Trp-Trp-Tyr, Tyr-Trp-Trp-Tyr, Tyr-Trp-Trp, Tyr-Trp-Met-Asn, Tyr-Trp-Met, Tyr-Trp-Leu-Ser, Tyr-Trp-Leu, Tyr-Leu-Phe, Phe-Leu-Phe, Trp-Leu-Phe, Tyr-Leu-Trp, Phe-Leu-Trp, Trp-Leu-Trp, whereas in the case of protease SpIB it contains a sequence selected from among: Trp-Glu-Leu-Gln-Gly, Trp-Glu-Leu-Gln-Ser, Trp-Glu-Leu-Gln-Val, Trp-Glu-Leu-Gln- AIa, Trp-Glu-Leu-Gln-Glu, Trp-Glu-Leu-Gln-Met, Trp-Glu-Leu-Gln-Gln, Trp-Glu-Leu- Gln-Asn, Trp-Glu-Leu-Gln-Asp, Trp-Glu-Leu-Gln, Trp-Glu-Leu-Thr, Trp-Glu-Val-Gln, Val-Glu-Leu-Gln, Trp-Gln-Leu-Asp, Trp-Val-Leu-Gln, Phe-Glu-Val-Glu, Gly-Arg-Gly- Val-Gly, Gly-Arg-Gly-Val, Val-Glu-Ile-Asp, Val-Val-Leu-Gln, Val-Val-Leu-Gln-Ser, He- Glu-Ser-Gln, Ile-Glu-Ser-Gln-Ser.
3. A protein recognized by protease SpIB or SpIA containing an amino-acid sequence of a polypeptide exhibiting affinity towards the active centre of protease SpIA or SpIB according to Claims 1-2.
4. A nucleotide sequence encoding a polypeptide according to Claims 1-2.
5. A nucleotide sequence encoding a protein according to Claim 3
6. Use of a polypeptide sequence according to Claims 1-2 or its derivative during the manufacturing of a protein recognized by protease SpIB or SpIA or their derivatives.
7. Use of a nucleotide sequence encoding a polypeptide according to Claims 1-2 or its derivative during the manufacturing of a protein recognized by protease SpIB or SpIA or their derivatives.
8. A method of producing a desired protein, characterised in that: a) a fusion protein is produced containing the sequence Zl-Xaal-Xaa2-Xaa3-Xaa4-Xaa5- Z2, where Zl and Z2 denotes a polypeptide containing one or more amino-acids, where one of them denotes a polypeptide containing a desired protein and the other containing a marker polypeptide, b) the fusion protein is isolated, preferentially by a chromatographic technique using a column exhibiting affinity for the marker polypeptide, c) a hydrolysis reaction is performed on the fusion protein using a protease exhibiting the enzymatic activity of protease SpIA or protease SpIB and preferentially, the desired protein is isolated from the reaction mixture, where in the case of protease SpIA use:
Xaal is an amino-acid selected from among: Trp, Tyr, Phe, VaI, He, Leu,
Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser,
Tyr, Phe, Asp, Pro,
Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He,
VaI, Thr,
Xaa4 is omitted or is any given amino-acid, preferentially selected from among: Ser, Thr,
GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr,
Xaa5 is omitted or is any given amino-acid, whereas in the case of protease SpIB use: Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser,
Thr or GIy,
Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg,
Lys, Ser or Thr,
Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy,
Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn.
Xaa5 is omitted or is any given amino-acid preferentially selected form among: Thr, Ser,
VaI, GIy, Ala, GIu, Met, GIn, Asp, Asn.
9. A method according to Claim 8, characterised in that in the case of protease SpIA use, the fusion protein contains a sequence selected from among: Zl-Trp-Leu-Tyr-Z2, Zl-Trp- Leu-Tyr-Ser-Z2, Zl-Tyr-Glu-Tyr-Ala-Z2, Zl-Tyr-Glu-Tyr-Ser-Z2, Zl-Tyr-Glu-Tyr-Z2, Zl-Tyr-Met-Tyr-Z2, Zl-Tyr-Met-Tyr-Ser-Z2, Zl-Tyr-Ala-Tyr-Ser-Z2, Zl-Tyr-Ala-Tyr- Z2, Zl-Tyr-Thr-Tyr-Ser-Z2, Zl-Tyr-Thr-Tyr-Z2, Zl-Tyr-Leu-Tyr-Gly-Z2, Zl-Tyr-Leu- Tyr-Z2, Zl-Tyr-Leu-Tyr-Ser-Z2, Zl-Phe-Leu-Tyr-Ser-Z2, Zl-Phe-Leu-Tyr-Z2, Zl-VaI- Leu-Tyr-Thr-Z2, Zl-Val-Leu-Tyr-Z2, Zl-Trp-Leu-Ser-Thr-Z2, Zl-Trp-Leu-Ser-Z2, Zl- Trp-Met-Asn-Thr-Z2, Zl-Trp-Met-Asn-Z2, Zl-Tφ-Tφ-Tyr-Thr-Z2, Zl-Tφ-Trp-Tyr-Z2, Zl-Tyr-Tφ-Tφ-Tyr-Z2, Zl-Tyr-Tφ-Tφ-Z2, Zl-Tyr-Tφ-Met-Asn-Z2, Zl-Tyr-Tφ-Met- Z2, Zl-Tyr-Tφ-Leu-Ser-Z2, Zl-Tyr-Tφ-Leu-Z2, Zl-Tyr-Leu-Phe-Z2, Zl-Phe-Leu-Phe- Z2, Zl-Tφ-Leu-Phe-Z2, Zl-Tyr-Leu-Tφ-Z2, Zl-Phe-Leu-Tφ-Z2, Zl-Tφ-Leu-Tφ-Z2, whereas in the case of protease SpIB use, the fusion protein contains a sequence selected from among: Zl-Tφ-Glu-Leu-Gln-Gly-Z2, Zl-Tφ-Glu-Leu-Gln-Z2, Zl-Tφ-Glu-Leu- Gln-Ser-Z2, Zl-Tφ-Glu-Leu-Gln-Val-Z2, Zl-Tφ-Glu-Leu-Gln-Ala-Z2, Zl-Tφ-Glu-Leu- Gln-Glu-Z2, Zl-Tφ-Glu-Leu-Gln-Met-Z2, Zl-Tφ-Glu-Leu-Gln-Gln-Z2, Zl-Tφ-Glu- Leu-Gln- Asn-Z2, Z 1 -Tφ-Glu-Leu-Gln- Asp-Z2, Z 1 -Tφ-Glu-Leu-Thr-Z2, Z 1 -Trp-Glu- Val-Gln-Z2, Zl-Val-Glu-Leu-Gln-Z2, Zl-Tφ-Gln-Leu-Asp-Z2, Zl-Tφ-Val-Leu-Gln-Z2, Zl-Phe-Glu-Val-Glu-Z2, Zl-Gly-Arg-Gly-Val-Gly-Z2, Zl-Gly-Arg-Gly-Val-Z2, Zl-VaI- Glu-Ile-Asp-Z2, Zl-Val-Val-Leu-Gln-Z2, Zl-Val-Val-Leu-Gln-Ser-Z2, Zl-Ile-Glu-Ser- Gln-Z2, Zl-Ile-Glu-Ser-Gln-Ser-Z2.
10. A method according to Claim 8, characterised in that the hydrolysis is performed at a temperature from 0°C to 45°C and at pH 5.0 to 8.0 in the case of SpIA or at pH 5.0 to 9.0 in the case of SpIB.
11. A method according to Claim 8, characterised in that the hydrolysis is performed in a buffer with a concentration of 1 to 50OmM, where in the case of protease SpIA the reaction is performed in N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, IAPS, CHES, MOPS, Bis-Tris, phosphate, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine, ethanolamine or Tris buffer, whereas in the case of protease SpIB the reaction is performed in N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, MOPS, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine, TAPS, ethanolamine, CHES, phosphate, Bis-Tris, CAPS or Tris buffer.
12. A method according to Claim 8, characterised in that the hydrolysis is performed in a solution containing 0 to 50OmM NaCl.
13. A variant of protease SpIA or SpIB, characterised in that it contains an amino-acid sequence containing at least one of the following modifications:
- substitution of histidine at position 39 of the SpIB sequence for another amino-acid,
- substitution of histidine at position 39 of the SpIA sequence for another amino-acid,
- substitution of aspartic acid at position 77 of the SpIB sequence for another amino-acid,
- substitution of aspartic acid at position 78 of the SpIA sequence for another amino-acid,
- substitution of serine at position 157 of the SpIB sequence for another amino-acid,
- substitution of serine at position 154 of the SpIA sequence for another amino-acid,
- attachment via a peptide bond to the amino-acid at the N-terminal or C-terminal end of the mature protease SpIA or SpIB of a polypeptide containing at least one of the following sequences: a known secretory sequence, a known bacterial secretory sequence, a known fungal secretory sequence, a sequence containing a methionine residue, a sequence of a polypeptide exhibiting affinity for the active centre of protease SpIA or SpIB according to Claims 1-2, a sequence recognized by a proteolytic enzyme, a known marker polypeptide sequence, or a sequence of a polypeptide exhibiting the properties of a marker polypeptide.
- substitution of surface residues of SpIA or SpIB as respectively defined in Table 1 and Table 2 with residues allowing convenient attachment by chemical methods of entities allowing immobilization or specific tags
- incorporation into the sequence of protease SpIA or SpIB of a marker polypeptide sequence preferrentially in a manner not affecting the hydrolytic activity towards polypeptides defined in Claims 1 and 2.
14. A variant of a protease according to Claim 13, characterised in that the secretory sequence is a bacterial secretory sequence recognized by Bacillus subtilis or other gram positive bacterium or other gram negative bacterium or a strain of fungi.
15. A variant of a protease according to Claim 13, characterised in that it contains a sequence selected from among: SEQ ID No.: 4, SEQ ID NO: 6, SEQ ID No.: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18.
16. A nucleotide sequence encoding a protease variant according to Claims 13-15.
17. A nucleotide sequence according to Claim 16, characterised in that it contains a nucleotide sequence selected from among: SEQ ID No.: 3, SEQ ID NO: 5, SEQ ID No.: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 lub SEQ ID NO: 17.
18. A method of producing protease SpIA or SpIB or their variants, characterised in that : a) the expression of the protein according to Claims 13-15 occurs in bacterial or fungal host cells, preferentially encoded by a nucleotide sequence according to Claims 16-17, and then; b) the desired enzyme or a fraction containing it is isolated.
19. A method according to Claim 18, characterised in that the bacterial host is a strain of Bacillus subtilis or other gram positive bacterium or other gram negative bacterium or a strain of a fungi expressing a protein encoded by the nucleotide sequence presented in SEQ ID No.: 3 or SEQ ID No.: 9.
20. A method according to Claim 18, characterised in that during stage b) the fermentation mixture is separated from the bacterial mass through centrifugation, secretory proteins found in the medium with bacteria removed are precipitated out with ammonium sulphate, and the precipitated proteins are separated and dissolved in a small volume of buffer and dialysed in a buffer with a pH of about 5.5.
21. A method according to Claim 20, characterised in that during stage b), the isolated protein is additionally purified using affinity chromatography and/or ion exchange chromatography and/or molecular sieving, and, finally, the purified preparation is possibly concentrated and crystallised.
22. Use of protease SpIA or SpIB for the specific hydrolysis of a polypeptide containing the amino-acid sequence Xaal-Xaa2-Xaa3-Xaa4-Xaa5 (ie. Yl-Xaal-Xaa2-Xaa3-Xaa4- Xaa5-Y2, where Yl and Y2 denotes a polypeptide containing one or more amino-acids), where: in the case of protease SpIA:
Xaal is an amino-acid selected from among: Tip, Tyr, Phe, VaI, He, Leu,
Xaa2 is an amino-acid selected from among: Leu, GIu, Met, Ala, Thr, Trp, He, VaI, Ser,
Tyr, Phe, Asp, Pro,
Xaa3 is an amino-acid selected from among: Tyr, Phe, Trp, Leu, Asn, GIn, Ser, Met, He,
VaI, Thr, Xaa4 is omitted or is any given amino-acid, preferentially selected from among: Ser, Thr,
GIy, Ala, VaI, Asn, Asp, GIn, GIu, Tyr
Xaa5 is omitted or is any given an amino-acid, whereas in the case of protease SpIB:
Xaal is an amino-acid selected from among: Trp, Ala, He, Leu, Met, Phe, Tyr, VaI, Ser,
Thr or GIy,
Xaa2 is an amino-acid selected from among GIu, GIn, Asp, Asn, VaI, Leu, He, GIy, Arg,
Lys, Ser or Thr,
Xaa3 is an amino-acid selected from among Leu, He, VaI, Thr, Ser, Pro or GIy,
Xaa4 is an amino-acid selected from among: GIn, GIu, Thr, Ser, Asp or Asn,
Xaa5 is omitted or is any given amino-acid, preferentially selected form among: Thr, Ser,
VaI, GIy, Ala, GIu, Met, GIn, Asp, Asn. where preferentially in the case of protease SpIA, the hydrolysed polypeptide contains a sequence selected from among: Trp-Leu-Tyr, Trp-Leu-Tyr-Ser, Tyr-Glu-Tyr-Ala, Tyr-Glu-Tyr-Ser, Tyr-Glu-Tyr,
Tyr-Met-Tyr, Tyr-Ala-Tyr-Ser, Tyr-Met-Tyr-Ser, Tyr-Ala-Tyr, Tyr-Thr-Tyr-Ser, Tyr-Thr-
Tyr, Tyr-Leu-Tyr-Gly, Tyr-Leu-Tyr, Tyr-Leu-Tyr-Ser, Phe-Leu-Tyr-Ser, Phe-Leu-Tyr,
Val-Leu-Tyr-Thr, Val-Leu-Tyr, Trp-Leu-Ser-Thr, Trp-Leu-Ser, Trp-Met-Asn-Thr, Trp-
Met-Asn, Trp-Trp-Tyr-Thr, Trp-Trp-Tyr, Tyr-Tφ-Trp-Tyr, Tyr-Trp-Trp, Tyr-Trp-Met-
Asn, Tyr-Trp-Met, Tyr-Tφ-Leu-Ser, Tyr-Trp-Leu, Tyr-Leu-Phe, Phe-Leu-Phe, Trp-Leu-
Phe, Tyr-Leu-Trp, Phe-Leu-Trp, Trp-Leu-Trp, whereas in the case of protease SpIB, the hydrolysed polypeptide contains a sequence preferentially selected from among: Trp-Glu-Leu-Gln-Gly, Trp-Glu-Leu-Gln-Ser, Trp-
Glu-Leu-Gln-Val, Trp-Glu-Leu-Gln-Ala, Trp-Glu-Leu-Gln-Glu, Trp-Glu-Leu-Gln-Met,
Trp-Glu-Leu-Gln-Gln, Trp-Glu-Leu-Gln-Asn, Tφ-Glu-Leu-Gln-Asp, Trp-Glu-Leu-Gln,
Trp-Glu-Leu-Thr, Tφ-Glu-Val-Gln, Val-Glu-Leu-Gln, Tφ-Gln-Leu-Asp, Tφ-Val-Leu-
GIn, Phe-Glu-Val-Glu, Gly-Arg-Gly-Val-Gly, Gly-Arg-Gly-Val, Val-Glu-Ile-Asp, VaI-
Val-Leu-Gln, Val-Val-Leu-Gln-Ser, Ile-Glu-Ser-Gln, Ile-Glu-Ser-Gln-Ser.
23. A method according to Claim 22, characterised in that the hydrolysis is performed at a temperature from 0°C to 45°C and at pH 5.0 to 8.0 in the case of SpIA or at pH 5.0 to 9.0 in the case of SpIB.
24. A method according to Claim 22, characterised in that the hydrolysis is performed in a buffer with a concentration of 1 to 50OmM, where in the case of protease SpIA the reaction is performed in N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, TAPS, CHES, MOPS, Bis-Tris, phosphate, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine, ethanolamine or Tris buffer, whereas in the case of protease SpIB the reaction is performed in N-methyl piperazine, piperazine, propionic acid, pyridine, piperidin, acetate, citrate, lactic acid, butanedionic acid, methyl-malonic acid, formate, MES, HEPES, PIPES, ADA, ACES, BES, TES, MOPS, triethanolamine, N-methyl diethanolamine, dimethylamine, Tricine, Bicine, TAPS, ethanolamine, CHES, phosphate, Bis-Tris, CAPS or Tris buffer.
25. A method according to Claim 22, characterised in that the hydrolysis is performed in a solution containing 0 to 50OmM NaCl.
26. A protease exhibiting protease SpIA activity, characterised in that it contains an active centre encompassing a catalytic triad containing at least one of the following amino-acids: His, Asp and Ser, wherein the RMSD of Ca carbons of the main chainof the amino-acids forming the catalytic triad is no greater than 2,2 A, preferentially no greater than 1,8A, in comparison with the Ca carbons of the main chain of the amino-acids His39, Asp78 and Ser 154 contained in protease SpIA with a tertiary structure described in Table 1.
27. A protease according to Claim 26, characterised in that the RMSD of Ca carbons of the main chain in the well defined secondary structures of the molecule core is no greater than 2A, preferentially no greater than 1 ,5 A, in combination with their corresponding structural Ca carbon atoms of the main chain contained in the protease SpIA with the tertiary structure defined in Table 1.
28. A protease according to Claim 27, characterised in that the well-defined secondary structure of the molecular core contains fragments corresponding to the structure of the protein SpIA selected from among the following sequences: VaW to Glu6, Asnl6 to Ala20, Gly24 to Val29, Thr33 to Asn37, Val51 to Ala53, Asn64 to Val67, Ile70 to Glu72, Leu79 to His85, Argl l2 to Ilel l6, Metl28 to Ilel35, Phel42 to Phel45, Serl54 to Leul59, Glyl67 to Alal71, Asnl81 to Tyrl85, Glul92 to Glnl95.
29. A protease according to Claim 26, characterised in that it contains a fragment forming an α-helix corresponding to the structure of the fragment of the SpIA protein selected preferentially from among the following sequences: Lys38 to Ala41, Glul92 to Asnl96.
30. A protease according to Claim 26, characterised in that it contains a fragment forming an β- strand corresponding to the structure of the fragment of the SpIA protein selected preferentially from among the following sequences: Val4 to Lys5, VaI 18 to Ala20, Thr25 to Val28, Thr33 to Thr36, Val51 to Ala53, Asn64 to Val67, Asp69 to Glu72, Ala80 to Val84, Argl l2 to Ilel lό, Phel29 to Glyl33, Phel42 to Phel45, Vall58 to Leul59, GIy 167 to Alal71, Asnlδl to VaI 184.
31. A protease according to Claim 26, characterised in that it possesses a tertiary structure for which the RMSD of Ca carbons of the main chain is no larger than 2,5 A, preferentially no larger than 1,8 A, in comparison with the Ca carbons of the main chain contained in protease SpIA with a tertiary structure defined in Table 1.
32. A protease according to Claim 26, characterised in that it contains structural elements preferentially selected from among:
- at the position corresponding to GIu 1 of the sequence of SpIA it contains an amino-acid selected from among: GIu, Asp, GIn, Asn;
- at the position corresponding to Val28 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val29 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile34 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val35 it contains an amino-acid selected from among: VaI, Leu, He, Ala; '
- at the position corresponding to Thr36 it contains an amino-acid selected from among: Ser, Thr;
- at the position corresponding to His39 it contains His;
- at the position corresponding to Val67 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile70 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Asp78 it contains Asp;
- at the position corresponding to Leu79 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile81 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Val82 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Val98 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Ser, Thr;
- at the position corresponding to GIy 117 it contains GIy;
- at the position corresponding to Tyrl 18 it contains an amino-acid selected from among: Tyr, Phe, Trp; - at the position corresponding to Met 128 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Alal49 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Glnl50 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Prol 51 it contains Pro;
- at the position corresponding to GIy 152 it contains GIy;
- at the position corresponding to Asn 153 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Serl54 it contains Ser;
- at the position corresponding to GIy 155 it contains GIy;
- at the position corresponding to Serl56 it contains an amino-acid selected from among: VaI, Ala, Ser, Thr, GIy;
- at the position corresponding to Prol 57 it contains Pro;
- at the position corresponding to GIy 167 it contains GIy;
- at the position corresponding to Ilel68 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Leul69 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Tyrl70 it contains an amino-acid selected from among: Tyr, Phe, Tip;
- at the position corresponding to Alal71 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to GIy 172 it contains GIy;
- at the position corresponding to Glul77 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Serl78 it contains an amino-acid selected from among: Ser, Thr, VaI, Leu, He, Ala;
- at the position corresponding to Asnl81 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Phe 193 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to He 194 it contains an amino-acid selected from among: VaI, Leu, He, Ala.
33. A protease exhibiting protease SpIB activity, characterised in that it contains an active centre formed by, among others, the catalytic triad His, Asp and Ser, wherein the RMSD of all atoms forming the amino-acids of the catalytic triad is no greater than 2.0A, preferentially no greater than 1.5 A, in comparison with His 39, Asp 77 and Ser 157 contained in protease SpIB with a tertiary structure defined in Table 2.
34. A protease according to Claim 33, characterised in that the RMSD of Ca carbons of the main chain within well defined secondary structures of the molecular core is no greater than 2.5A, preferentially no greater than 2.0A, in combination with their corresponding structural Ca carbon atoms of the main chain contained in the protease SpIB with the tertiary structure defined in Table 2.
35. A protease according to Claim 34, characterised in that in that the well-defined secondary structure of the molecular core contains fragments corresponding to the structure of the protein SpIB preferentially selected from among the following sequences: Val4 to Lys6, Thrlό to Ala20, Ala24 to Val29, Thr33 to Val40, Ile50 to Ala52, Ile63 to Asn71, Val78 to Glu84, Argl l5 to Ilel l9, Leul31 to Vall38, Serl45 to Tyrl48, Thrl52 to Leul62, GIy 170 to Serl75, Alal85 to Tyrl89, Lysl96 to Alal99.
36. A protease according to Claim 33, characterised in that it contains a fragment forming an α-helix corresponding to the structure of the fragment of the SpIB protein selected preferentially from among the following sequences: Lys38 to Ser41, Lysl96 to Glu200.
37. A protease according to Claim 33, characterised in that it contains a fragment forming a β- strand corresponding to the structure of the fragment of the SpIB protein selected preferentially from among the following sequences: Val4 to Thr5, VaI 18 to Ala20, Thr25 to Val28, Thr33 to Thr36, Arg49 to Ala52, Ile63 to Asn71, Ser79 to Val83, Argl l5 to Ilel l9, Tyrl32 to Glyl36, Serl45 to Tyrl48, Vallόl to Leul62, Glyl70 to Serl75, Alal85 to Vall88.
38. A protease according to Claim 33, characterised in that it possesses a tertiary structure for which the RMSD of Ca carbons of the main chain is no larger than 3,0 A, preferentially no larger than 2,θA, in comparison with the Ca carbons of the main chain contained in protease SpIB with a tertiary structure defined in Table 1.
39. A protease according to Claim 33, characterised in that it contains structural elements preferentially selected from among:
- at the position corresponding to GIu 1 of the sequence of SpIB it contains an amino-acid selected from among: GIu, Asp, GIn, Asn
- at the position corresponding to Val28 it contains an amino-acid selected from among: VaI, Leu, He, Ala; - at the position corresponding to Val29 it contains an amino-acid selected from among: VaI, Leu, lie, Ala;
- at the position corresponding to Ile34 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Leu35 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Thr36 it contains an amino-acid selected from among: Ser, Thr;
- at the position corresponding to His39 it contains His;
- at the position corresponding to Ile66 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Ile69 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Asp77 it contains Asp;
- at the position corresponding to Val78 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Val80 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to Ileδl it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to VaIl 18 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Ser, Thr;
- at the position corresponding to GIy 120 it contains GIy;
- at the position corresponding to Tyrl21 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to Leul31 it contains an amino-acid selected from among: VaI, Leu, He, Ala, Met;
- at the position corresponding to GIy 155 it contains GIy;
- at the position corresponding to Asnl56 it contains an amino-acid selected from among: Asn, GIn, Asp, GIu;
- at the position corresponding to Serl57 it contains Ser;
- at the position corresponding to GIy 158 it contains GIy;
- at the position corresponding to Ser 159 it contains an amino-acid selected from among: VaI, Ala, Ser, Thr, GIy;
- at the position corresponding to Pro 160 it contains Pro; - at the position corresponding to GIy 170 it contains GIy;
- at the position corresponding to Ilel71 it contains an amino-acid selected from among: VaI, Leu, He, Ala;
- at the position corresponding to Phel97 it contains an amino-acid selected from among: Tyr, Phe, Trp;
- at the position corresponding to He 198 it contains an amino-acid selected from among: VaI, Leu, He, Ala.
40. A method of storage of protease exhibiting protease SpIA activity according to Claims
26-32 or of protease exhibiting protease SpIB activity according to claims 33-39 wherein the said enzymes are preferentially stored for more than 12 hours: a.) in solution preferentially at temperatures from +250C to O0C b.) in solution containing antifreezing agent at temperatures from +25°C to -85°C c.) frozen at the temperatures form O0C to -2000C preferentially in a -2O0C freezer, -7O0C freezer, dry ice or liquid nitrogen d.) liophylized (freeze-dried) preferentially in moisture free containers or moisture free, vacuum sealed containers or moisture free, inert gas filled containers.
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PL382638A PL214451B1 (en) | 2007-06-11 | 2007-06-11 | Proteinase Sp1B and peptides identified by it and their applications |
PLPL382638 | 2007-06-11 | ||
PL382770A PL221052B1 (en) | 2007-06-28 | 2007-06-28 | Proteinase Sp1A and peptides recognized by it and their application |
PLPL382770 | 2007-06-28 |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9138455B2 (en) | 2013-03-15 | 2015-09-22 | Mead Johnson Nutrition Company | Activating adiponectin by casein hydrolysate |
US9289461B2 (en) | 2013-03-15 | 2016-03-22 | Mead Johnson Nutrition Company | Reducing the risk of autoimmune disease |
US9352020B2 (en) | 2013-03-15 | 2016-05-31 | Mead Johnson Nutrition Company | Reducing proinflammatory response |
US9457058B2 (en) | 2013-03-15 | 2016-10-04 | Mead Johnson Nutrition Company | Nutritional composition containing a peptide component with anti-inflammatory properties and uses thereof |
CN108794570A (en) * | 2018-06-15 | 2018-11-13 | 华南理工大学 | A kind of xanthine oxidase inhibitor and application thereof containing phenylalanine |
WO2022125928A1 (en) * | 2020-12-11 | 2022-06-16 | Bayer Animal Health Gmbh | Methods and compositions for improved production of an antigen for use in an s. aureus vaccine |
EP4389865A1 (en) * | 2022-12-21 | 2024-06-26 | Novozymes A/S | Recombinant protease for cell detachment |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070065878A1 (en) * | 2005-08-31 | 2007-03-22 | Daugherty Patrick S | Cellular libraries of peptide sequences (CLiPS) and methods of using the same |
-
2008
- 2008-06-11 WO PCT/PL2008/000042 patent/WO2008153429A2/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070065878A1 (en) * | 2005-08-31 | 2007-03-22 | Daugherty Patrick S | Cellular libraries of peptide sequences (CLiPS) and methods of using the same |
Non-Patent Citations (3)
Title |
---|
BOULWARE K.T. ET AL.: "Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)" P.N.A.S, vol. 103, 16 August 2006 (2006-08-16), pages 7583-7588, XP002503593 * |
DUBIN G ET AL: "Enzymatic Activity of the Staphylococcus aureus SplB Serine Protease is Induced by Substrates Containing the Sequence Trp-Glu-Leu-Gln" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 379, no. 2, 29 May 2008 (2008-05-29), pages 343-356, XP022648469 ISSN: 0022-2836 [retrieved on 2008-04-03] * |
POPOWICZ ET AL: "Functional and Structural Characterization of Spl Proteases from Staphylococcus aureus" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 358, no. 1, 21 April 2006 (2006-04-21), pages 270-279, XP005345774 ISSN: 0022-2836 * |
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US9138455B2 (en) | 2013-03-15 | 2015-09-22 | Mead Johnson Nutrition Company | Activating adiponectin by casein hydrolysate |
US9289461B2 (en) | 2013-03-15 | 2016-03-22 | Mead Johnson Nutrition Company | Reducing the risk of autoimmune disease |
US9352020B2 (en) | 2013-03-15 | 2016-05-31 | Mead Johnson Nutrition Company | Reducing proinflammatory response |
US9457058B2 (en) | 2013-03-15 | 2016-10-04 | Mead Johnson Nutrition Company | Nutritional composition containing a peptide component with anti-inflammatory properties and uses thereof |
CN108794570A (en) * | 2018-06-15 | 2018-11-13 | 华南理工大学 | A kind of xanthine oxidase inhibitor and application thereof containing phenylalanine |
CN108794570B (en) * | 2018-06-15 | 2021-08-06 | 华南理工大学 | Xanthine oxidase inhibitor containing phenylalanine and application thereof |
WO2022125928A1 (en) * | 2020-12-11 | 2022-06-16 | Bayer Animal Health Gmbh | Methods and compositions for improved production of an antigen for use in an s. aureus vaccine |
EP4389865A1 (en) * | 2022-12-21 | 2024-06-26 | Novozymes A/S | Recombinant protease for cell detachment |
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