Alkali-tolerant affinity chromatography ligands
View Patent ↗This disclosure relates to chromatography ligands comprising one or more domains of Staphylococcal Protein A (SPA), or any functional variants thereof, as well as methods of making and using the same.
1 . A polypeptide comprising a first immunoglobulin (Ig)-binding domain, wherein the first Ig-binding domain comprises:
(1) the amino acid sequence that is identical to SEQ ID NO: 1, except that the amino acid that corresponds to N55 of SEQ ID NO: 1 is Val or Leu and the amino acid that corresponds to S44 of SEQ ID NO: 1 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(2) the amino acid sequence that is identical to SEQ ID NO: 2, except that the amino acid that corresponds to N54 of SEQ ID NO: 2 is Val or Leu and the amino acid that corresponds to S43 of SEQ ID NO: 2 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(3) the amino acid sequence that is identical to SEQ ID NO: 3, except that the amino acid that corresponds to N52 of SEQ ID NO: 3 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 3 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(4) the amino acid sequence that is identical to SEQ ID NO: 4, except that the amino acid that corresponds to N52 of SEQ ID NO: 4 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 4 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(5) the amino acid sequence that is identical to SEQ ID NO: 5, except that the amino acid that corresponds to N52 of SEQ ID NO: 5 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 5 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp; or
(6) the amino acid sequence that is identical to SEQ ID NO: 6, except that the amino acid that corresponds to N52 of SEQ ID NO: 6 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 6 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp.
2 . The polypeptide of claim 1 , wherein the amino acid that corresponds to S44 of SEQ ID NO: 1, S43 of SEQ ID NO: 2, S41 of SEQ ID NO: 3, S41 of SEQ ID NO: 4, S41 of SEQ ID NO: 5, or S41 of SEQ ID NO: 6 is Ala or Val.
3 . The polypeptide of claim 1 , wherein the first Ig-binding domain comprises one of the following amino acid sequences:
(1) the amino acid sequence that is identical to SEQ ID NO: 1, except that the amino acid at position 44 is Ala, Val or Leu and the amino acid at position 55 of SEQ ID NO: 1 is Val or Leu;
(2) the amino acid sequence that is identical to SEQ ID NO: 2, except that the amino acid at position 43 is Ala, Val or Leu and the amino acid at position 54 of SEQ ID NO: 2 is Val or Leu;
(3) the amino acid sequence that is identical to SEQ ID NO: 3, except that the amino acid at position 41 is Ala, Val or Leu and the amino acid at position 52 of SEQ ID NO: 3 is Val or Leu;
(4) the amino acid sequence that is identical to SEQ ID NO: 4, except that the amino acid at position 41 is Ala, Val or Leu and the amino acid at position 52 of SEQ ID NO: 4 is Val or Leu;
(5) the amino acid sequence that is identical to SEQ ID NO: 5, except that the amino acid at position 41 is Ala, Val or Leu and the amino acid at position 52 of SEQ ID NO: 5 is Val or Leu; and
(6) the amino acid sequence that is identical to SEQ ID NO: 6, except that the amino acid at position 41 is Ala, Val or Leu and the amino acid at position 52 of SEQ ID NO:
6 is Val or Leu.
4 . The polypeptide of claim 1 , wherein the polypeptide further comprises a second Ig-binding domain, wherein the second Ig-binding domain comprises
(1) the amino acid sequence that is identical to SEQ ID NO: 1, except that the amino acid that corresponds to N55 of SEQ ID NO: 1 is Val or Leu and the amino acid that corresponds to S44 of SEQ ID NO: 1 is Ser, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(2) the amino acid sequence that is identical to SEQ ID NO: 2, except that the amino acid that corresponds to N54 of SEQ ID NO: 2 is Val or Leu and the amino acid that corresponds to S43 of SEQ ID NO: 2 is Ser, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(3) the amino acid sequence that is identical to SEQ ID NO: 3, except that the amino acid that corresponds to N52 of SEQ ID NO: 3 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 3 is Ser, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(4) the amino acid sequence that is identical to SEQ ID NO: 4, except that the amino acid that corresponds to N52 of SEQ ID NO: 4 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 4 is Ser, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp;
(5) the amino acid sequence that is identical to SEQ ID NO: 5, except that the amino acid that corresponds to N52 of SEQ ID NO: 5 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 5 is Ser, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp; or
(6) the amino acid sequence that is identical to SEQ ID NO: 6, except that the amino acid that corresponds to N52 of SEQ ID NO: 6 is Val or Leu and the amino acid that corresponds to S41 of SEQ ID NO: 6 is Ser, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp.
5 . The polypeptide of claim 4 , wherein the sequence of the first Ig-binding domain and the second Ig-binding domain are identical.
6 . The polypeptide of claim 4 , wherein the sequence of the first Ig-binding domain and the second Ig-binding domain are different.
7 . The polypeptide of claim 4 , wherein the first Ig-binding domain and the second Ig-binding domain are separated by a spacer domain.
8 . The polypeptide of claim 1 , wherein the polypeptide further comprises an additional 1 to 10 lg-binding domains, wherein each of the 1 to 10 Ig-binding domains comprises an amino acid sequence that is identical to SEQ ID NO: 6, except that the amino acid that corresponds to S41 of SEQ ID NO: 6 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp in each of the 1 to 10 lg-binding domains and the amino acid that corresponds to N52 of SEQ ID NO: 6 is Val or Leu in each of the 1 to 10 lg-binding domains.
9 . The polypeptide of claim 1 , wherein the polypeptide further comprises a second Ig-binding domain, a third Ig-binding domain, a fourth Ig-binding domain, a fifth Ig-binding domain, and a sixth Ig-binding domain, wherein each of the second, third, fourth, fifth and sixth Ig-binding domain comprises an amino acid sequence that is identical to SEQ ID NO: 6, except that the amino acid that corresponds to S41 of SEQ ID NO: 6 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp in each of the second, third, fourth, fifth and sixth Ig-binding domain and the amino acid that corresponds to N52 of SEQ ID NO: 6 is Val or Leu in each of the second, third, fourth, fifth and sixth Ig-binding domain.
10 . The polypeptide of claim 1 , wherein the polypeptide can retain 60% to 100% of its Ig-binding capability after contacting a 0.1-0.5 M alkali solution for a time of 48 hours or less.
11 . A vector comprising a polynucleotide encoding the polypeptide of claim 1 .
12 . A chromatography ligand comprising the polypeptide of claim 1 .
13 . An affinity chromatography matrix comprising a plurality of the polypeptide of claim 1 .
14 . The affinity chromatography matrix of claim 13 , wherein the plurality of polypeptides are covalently coupled to a solid support, wherein the solid support is selected from the group consisting of polysaccharides, co-polymers, natural or synthetic polymers, silica, glass, plastic, metal and ceramics.
15 . The affinity chromatography matrix of claim 13 , wherein the plurality of polypeptides are covalently coupled to agarose, cellulose, dextran, alginate, or starch.
16 . A polypeptide comprising an immunoglobulin (Ig)-binding domain, wherein the Ig-binding domain comprises SEQ ID NO: 6 with a mutation at N52, wherein N52 is substituted with a Val or Leu amino acid residue and wherein the Ig-binding domain further comprises a mutation at S41, wherein S41 is substituted with a hydrophobic amino acid residue selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp.