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US 2010/0143415 A1Published application
Streptococcus Pneumoniae Antigens
AssigneeProvalis UK Limited
InventorsAllan William Cripps, Jennelle Maree Kyd, Maha Jomaa, Jeremy Mark Wells, Phillip Michael Hansbro
Life of this patent
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Abstract
There are provided various novel antigens from Streptococcus pneumoniae, as well as homologues, derivatives and fragments thereof. The use of these in medicine is described, particularly in the treatment or prophylaxis of S. pneumoniae infections. The use of the antigens in diagnosis is also described.
Claims24
A protein or polypeptide obtainable from S. pneumoniae selected from:
- (i) one having a molecular weight of 55 kDa, as determined by SDS/PAGE, and having the N-terminal sequence VEPKAKPADPSVV;
- (ii) one having a molecular weight of 50 kDa, as determined by SDS/PAGE, and having the N-terminal sequence NDRLVATQSADGRNESVLMSIET;
- (iii) one having a molecular weight of 85 kDa, as determined by SDS/PAGE, and having the N-terminal sequence EDTTNSRFGSQFDKYRQPNAEPDHSHDAVSADNSTAHNRFGYGFAIGSKYIRY D;
- (iv) one having a molecular weight of 38 kDa, as determined by SDS/PAGE, and having the N-terminal sequence DKYRQPNAEPDDHHYAV;
- (v) one having a molecular weight of 30 kDa, as determined by SDS/PAGE, and having the N-terminal sequence DAVSAD or SETNVY;
- (vi) one having a molecular weight of 32 kDa, as determined by SDS/PAGE, and having the N-terminal sequence DKVDGLSAKPDILKP;
- (vii) one having a molecular weight of 43 kDa, as determined by SDS/PAGE, and having the N-terminal sequence ELKEEG(W)VVK;
- (viii) one having a molecular weight of 100 kDa, as determined by SDS/PAGE, and having the N-terminal sequence EVHA;
- (ix) one having a molecular weight of <14 kDa, as determined by SDS/PAGE, and having the N-terminal sequence MKLNEVKEFVKELRAET;
- (x) one having a molecular weight of <14 kDa, as determined by SDS/PAGE, and having the N-terminal sequence AKYEILYIERPNIEEFAK;
- (xi) one having a molecular weight of <14 kDa, as determined by SDS/PAGE, and having the N-terminal sequence I(R)LTRM(E)GGKKKP(K)FYY;
- (xii) one having a molecular weight of 16 kDa, as determined by SDS/PAGE, and having the N-terminal sequence VMTDPIADXLXRI;
- (xiii) one having a molecular weight of 27.5 kDa, as determined by SDS/PAGE, and having the N-terminal sequence (VA)(KE)LVFARHGE(LT)E(NK);
- (xiv) one having a molecular weight of 44 kDa, as determined by SDS/PAGE, and having the N-terminal sequence IITDVYAREVLDSRGNPTL.
- (xv) one having a molecular weight of 12-14 kDa as determined by SDS PAGE under reducing conditions and has the following amino terminal sequence:
- A L N I E N I I A E I K
- Ala Leu Asn Ile Glu Asn Ile Ile Ala Glu Ile Lys
- I A S
- Ile Ala Ser
- (xvi) is a reduced toxicity variant or fragment of the protein defined in (xv) above;
- (xvii) one having a molecular weight of about 16 kDa as determined by SDS PAGE under reducing conditions;
- or
- (xviii) has a molecular weight of about 57 kDa as determined by SDS PAGE under reducing conditions and has the following amino terminal sequence:
- R I I K F V Y A K
- Arg Ile Ile Lys Phe Val Tyr Ala Lys.
22 more elements
A process for the preparation of an isolated and purified protein the process comprising the following steps:
- (a) preparing cultures of S. pneumoniae, growing the cultures under appropriate conditions and harvesting them, followed by washing with centrifugation to yield a washed cell pellet;
- (b) resuspending the washed cells in an appropriate buffer followed by disruption of the cells;
- (c) centrifuging to remove cell debris and obtaining the supernatant containing soluble cell proteins;
- (d) subjecting the solution obtained to anion exchange chromatography with a sodium chloride gradient elution, and pooling the fractions corresponding to each separate peak;
- (e) suspending the protein fractions in a buffer comprising 0.5M Tris HCl pH 6.8;
- 10% (v/v) glycerol;
- 10% (w/v) SDS;
- 0.05% (w/v) bromophenol blue;
- and 0.05% (v/v) mercaptoethanol;
- boiling the mixture and then purifying by SDS-PAGE using a 12% (w/v) acrylamide/BIS separating gel with a 4% (w/v) acrylamide/BIS stacking gel, nm at 16 mA in the stacking gel and 24 mA in the resolving gel;
- (f) selecting a fraction containing a protein having a molecular weight of 12-14 kDa, 16 kDa, 34 kDa or 57 kDa and isolating the protein from the selected fraction.
8 more elements
Dependencies are inferred from the claim text.