Modified kappa light chain-binding polypeptides
View Patent ↗The invention discloses kappa light chain-binding polypeptide comprising a mutated binding domain of Peptostreptococcus protein L, wherein at least one asparagine residue of a parental domain defined by, or having at least 95% or 98% sequence homology with, SEQ ID NO: 2-6 or 2 has been mutated to another amino acid residue which is not asparagine, proline or cysteine.
1. A kappa light chain-binding polypeptide comprising at least one mutated binding domain of Peptostreptococcus Protein L, in which the domain has been mutated at positions:
N45A and comprising the amino acid sequence defined by SEQ ID NO: 7;
N10Q,N45A and comprising the amino acid sequence defined by SEQ ID NO: 8;
N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 9; or
N10Q,N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 11.
2. The polypeptide of claim 1 , in which the domain has been mutated at position N45A and comprising the amino acid sequence defined by SEQ ID NO: 7.
3. The polypeptide of claim 1 , in which the domain has been mutated at positions N10Q,N45A and comprising the amino acid sequence defined by SEQ ID NO: 8.
4. The polypeptide of claim 1 , in which the domain has been mutated at positions N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 9.
5. The polypeptide of claim 1 , in which the domain has been mutated at positions N10Q,N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 11.
6. The polypeptide of claim 1 , comprising a plurality of domains wherein the domains are linked by elements comprising up to 15 amino acids.
7. The polypeptide according to claim 1 , wherein the alkaline stability is improved relative to a parental polypeptide, as measured by the remaining binding capacity for kappa light chain-containing proteins after 96-100 10 min incubation cycles in 0.1M aqueous NaOH at 22+/−2° C.
8. A nucleic acid or a vector encoding a polypeptide according to claim 1 .
9. An expression system, which comprises a nucleic acid or vector according to claim 8 .
10. A separation matrix, wherein a plurality of polypeptides of claim 1 have been coupled to a solid support.
11. The separation matrix according to claim 10 , wherein the polypeptides have been coupled to the solid support by multipoint attachment.
12. The separation matrix of claim 10 , wherein the binding capacity of the matrix for kappa light chain-containing proteins after 100 10 min incubation cycles in 0.1M NaOH at 22+/−2° C. is at least 40, such as at least 50, or at least 55% of the binding capacity before the incubation.
13. A method of isolating a kappa light chain-containing protein, comprising isolating the kappa light chain-containing protein with the separation matrix of claim 10 .
14. A method of isolating a kappa light chain-containing protein comprising the steps of:
a) contacting a liquid sample comprising a kappa light chain-containing protein with a separation matrix of claim 10 ,
b) washing said separation matrix with a washing liquid,
c) eluting the kappa light chain-containing protein from the separation matrix with an elution liquid, and
d) cleaning the separation matrix with a cleaning liquid.
15. The method of claim 14 , wherein the cleaning liquid is alkaline, such as with a pH of 12-14.
16. The method of claim 14 , wherein the cleaning liquid comprises 0.01-1.0M NaOH or KOH, such as 0.05-1.0M or 0.05-0.1M NaOH or KOH.
17. The method of claim 14 , wherein steps a)-d) are repeated at least 10 times, such as at least 50 times or 50-200 times.