Modified biotin-binding protein, fusion proteins thereof and applications
The disclosure provides modified biotin-binding proteins which can be expressed in soluble form in high yield in bacteria. Also provided are fusion proteins comprising the modified biotin-binding protein and an antigen. The disclosure further provides non-hemolytic variants of alpha-hemolysin from S. aureus and fusion protein comprising non-hemolytic variant of alpha-hemolysin and a biotin-binding domains. Immunogenic compositions comprising the proteins are also disclosed and use of such immunogenic compositions for inducing an immune response or for vaccinating a subject are also disclosed.
1. A polypeptide comprising an alpha-hemolysin (Hla) polypeptide, comprising a mutation at amino acid residue 205, 213 or 209-211 of wild-type S. aureus alpha-hemolysin polypeptide, wherein the alpha-hemolysin polypeptide has lower hemolytic activity than an equivalent titer of wild-type Hla polypeptide.
2. The polypeptide_of claim 1 , wherein the hemolytic activity of the alpha-hemolysin polypeptide is at least 25% lower than an equivalent titer of wild-type Hla polypeptide.
3. The polypeptide of claim 1 , wherein the alpha-hemolysin polypeptide comprises one of the following mutations in the wild-type S. aureus alpha-hemolysin polypeptide: (i) residue 205 W to A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD to AAA.
4. The polypeptide of claim 1 , wherein the alpha-hemolysin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.
5. A composition comprising the polypeptide of claim 1 .
6. A method inducing an immune response in a subject, comprising administering to the subject a composition of claim 5 .
7. The method of claim 6 , wherein the subject is a human.
8. The method of claim 6 , wherein the immune response is an antibody/B-cell response.
9. The method of claim 6 , wherein the immune response is a CD4+ T-cell response, including Th1, Th2, or Th17 response, and/or a CD8+ T-cell response.
10. The polypeptide of claim 1 , wherein the wild-type Hla polypeptide comprises the amino acid sequence as set forth below:
(SEQ ID NO: 59)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKK
LLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYY
PRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDF
KTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSM
KAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDD
YQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN
wherein the amino acid sequence of SEQ ID NO: 59 corresponds to amino acids 27-319 of the wild-type Hla mature polypeptide.
11. The polypeptide of claim 1 , further comprising a biotin-binding domain.
12. The polypeptide of claim 11 , wherein the biotin-binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 1.
13. The polypeptide of claim 11 , comprising a peptide linker between the biotin-binding domain and the alpha-hemolysin polypeptide.
14. The polypeptide of claim 13 , wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 22.
15. The polypeptide of claim 11 , comprising a bacterial signal sequence at the N-terminus.
16. The polypeptide of claim 11 , comprising a purification tag at the C-terminus.
17. A composition comprising the polypeptide of claim 11 .
18. The polypeptide of claim 11 , wherein the biotin-binding domain comprises the amino acid sequence of SEQ ID NO: 1.