MUTANT FRAGMENTS OF OspA AND METHODS AND USES RELATING THERETO
The present invention relates to a polypeptide comprising a mutant fragment of an outer surface protein A (OspA), a nucleic acid coding the same, a pharmaceutical composition (particularly for use as a medicament of in a method of treating or preventing a Borrelia infection) comprising the polypeptide and/or the nucleic acid, a method of treating or preventing a Borrelia infection and a method of immunizing a subject.
1 - 17 . (canceled)
18 . A method for producing a polypeptide comprising a mutant fragment of an outer surface protein A (OspA), characterized by the following steps:
a) introducing a vector encoding the polypeptide into a host cell,
b) growing the host cell under conditions allowing for expression of said polypeptide,
c) homogenizing said host cell, and
d) subjecting the host cell homogenate to purification steps.
19 - 48 . (canceled)
49 . The method according to claim 18 , wherein the polypeptide comprises a polypeptide selected from the group of amino acid sequences of SEQ ID NO: 185 to 208; or any functional variant of said amino acid sequences, wherein said functional variant has a sequence identity of at least 80% to any of the sequences of SEQ ID NO: 185 to 208.
50 . The method according to claim 18 , wherein the polypeptide comprises a heterodimer selected from the group consisting of Lip-S1D4-S2D4 (SEQ ID NO: 185), Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S3D4-S4D4 (SEQ ID NO: 187), Lip-S3D1-S4D1 (SEQ ID NO: 188), Lip-S5D4-S6D4 (SEQ ID NO: 189), Lip-S5D1-S6D1 (SEQ ID NO: 190), Lip-S2D4-S1D4 (SEQ ID NO: 191), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S4D4-S3D4 (SEQ ID NO: 193), Lip-S4D1-S3D1 (SEQ ID NO: 194), Lip-S6D4-S5D4 (SEQ ID NO: 195), Lip-S6D1-S5D1 (SEQ ID NO: 196), Lip-S1D4-S2D1 (SEQ ID NO: 197), Lip-S1D1-S2D4 (SEQ ID NO: 198), S3D4-S4D1 (SEQ ID NO: 199), S3D1-S4D4 (SEQ ID NO: 200), S5D4-S6D1 (SEQ ID NO: 201), S5D1-S6D4 (SEQ ID NO: 202), S2D4-S1D1 (SEQ ID NO: 203), S2D1-S1D4 (SEQ ID NO: 204), S4D4-S3D1 (SEQ ID NO: 205), S4D1-S3D4 (SEQ ID NO: 206), S6D4-S5D1 (SEQ ID NO: 207) and Lip-S6D1-S5D4 (SEQ ID NO: 208).
51 . The method according to claim 18 , wherein the polypeptide consists of a heterodimer selected from the group consisting of Lip-S1D4-S2D4 (SEQ ID NO: 185), Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S3D4-S4D4 (SEQ ID NO: 187), Lip-S3D1-S4D1 (SEQ ID NO: 188), Lip-S5D4-S6D4 (SEQ ID NO: 189), Lip-S5D1-S6D1 (SEQ ID NO: 190), Lip-S2D4-S1D4 (SEQ ID NO: 191), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S4D4-S3D4 (SEQ ID NO: 193), Lip-S4D1-S3D1 (SEQ ID NO: 194), Lip-S6D4-S5D4 (SEQ ID NO: 195), Lip-S6D1-S5D1 (SEQ ID NO: 196), Lip-S1D4-S2D1 (SEQ ID NO: 197), Lip-S1D1-S2D4 (SEQ ID NO: 198), S3D4-S4D1 (SEQ ID NO: 199), S3D1-S4D4 (SEQ ID NO: 200), S5D4-S6D1 (SEQ ID NO: 201), S5D1-S6D4 (SEQ ID NO: 202), S2D4-S1D1 (SEQ ID NO: 203), S2D1-S1D4 (SEQ ID NO: 204), S4D4-S3D1 (SEQ ID NO: 205), S4D1-S3D4 (SEQ ID NO: 206), S6D4-S5D1 (SEQ ID NO: 207) and Lip-S6D1-S5D4 (SEQ ID NO: 208).
52 . The method according to claim 18 , wherein the polypeptide consists of a heterodimer selected from the group consisting of Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S4D1-S3D1 (SEQ ID NO: 194) and Lip-S5D1-S6D1 (SEQ ID NO: 190).
53 . The method according to claim 18 , wherein the vector comprises a nucleic acid molecule encoding said polypeptide.
54 . The method according to claim 18 , wherein the vector is pET28b(+).
55 . The method according to claim 18 , wherein the host cell is E. coli.
56 . The method according to claim 55 , wherein the E. coli is an E. coli BL21 or an E. coli BL21™ Star cell.
57 . The method according to claim 18 , wherein the purification steps comprise enriching the polypeptide in the lipid phase by phase separation and purifying over a gel filtration column.
58 . The method according to claim 57 , wherein the purification steps further comprise processing over a buffer exchange column.