See also structure of the SpA (i.e., various domains)    See also Multimers of SpA

One of the problems with proteinaceous affinity ligands in large scale purification is their sensitivity to alkaline conditions. For this reasons, various protein engineering strategies have been used to improve upon affinity ligands. Uhlen (EP1123389), for example, discloses affinity separation where the asparagine (Asn) residues within the affinity ligand are deleted or replaced with a less alkaline sensitive amino acid.

Criticality of binding sites when making Variants

Using phage display technology, researchers modified the IgG binding Z domain into an IgA binding peptide designated as Affibody IgA1 or modified Z domain. This peptide binds both human IgA1 and IgA2. The original IgG binding affinity was completely lost with these modifications (WO 2007/019376).

The repetitive structure of the native SPA gene makes site specific mutagenesis technically diffiult. After mutations of one of the repeats the mismatch primer will, in the next mutagenesis step, anneal more efficiently to a mutated that to a non-mutated repeate. In addition, multiple region mutants are hard to select from a single region mutant by hydridization. These problems severely limit the value of protein engineering in the study of protein A. (Nisson, “A synthetic IgG-binding domain based on staphylococcal protin A” Protein Engineering, 1(2), 1987, 107-13)

Variants, in general

Silverman (US2006/0205016) discloses Staphylococcal protein A variants for binding Ig comprising a polypeptide which varies by one or more amino acids form the sequence of a natural variable heavy chain III (VH3) Ig-fab binding region of spA.

Ljungqvist (WO/2000/063243) describes modified polypeptides which are derivatives of the B domain or Z domain from staphylococcal protein A where between 1 and 20 amino acid residues of said B or Z domain have been substituted by other amino acid reisudes which results in interaction capacity of said polypeptide with at least one domain of human factor VIII protein.

Truncated versions/deltions from Amino or carboxyl ends:

–truncated Zvar/C domain:

Spector (US 8,895,706) discloses an alkaline stable affinity chromatography ligand which includes C domains of SpA with each domain including a G29K mutation as well as 4 amino acids deted from the N-terminus starting at position 1. 

Hansson (US 16/095,721, published as US 2020/0239514) discloses a Zvar and C-domain without the linker region amino acids 1-8 and 56-58. 

Rodrigo (US 10501557) discloses an SpA domain (E, D, A, B, C, Z and  Zvar (without linker region amino acids 1-6) where at least the asparagine or serine residue at position 11 has been mutated to an amino acid selected form the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalamine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

–Truncated version of X domain: (Peyser, WO/2008/127457) disclsoes truncated versions of protein A that include some partion but not all of the X domain of native protein A, do not include a signal sequence and bind specifically to an Fc region of IgG. The versions have the advantage of that it contains some portion of the X domain, which portion significnatly improves its ability to be immobilized for use as an affinity chromatography reagent.

Particular Amino Acids Substituted 

Asparagine (N) residues: See also particular positions below

While SPA is considered relatively stable to alkaline treatment, it has also been though beneficial to improve the stability so that it can withstand even longer exposure to harsh conditions associated with cleaning in place procedures. For this purpose, replacing the asparagine residues has been employed.

Braisted (US6013763) describe Z domain variants of staphylococcal protein A which have significantly reduced size but possess IgG binding affinity equivalent to the wild type Z domain.

Gulich (J. Biotechnology 80 (2000), 169-178) report an SpA where all four asparagine residues replaced by leucine (one residue), aspartate (two residues) and lysine (one resiude). 

Hober (WO 03/080655) teaches an immunoglobulin binding protein such as Staphylococcal protein A (SpA) derivative/analogue wherein at least one asparagine residue of the B-domain and protein Z has been mutated to amino acids other than glutamine or aspartic acid (i.e., such as lysine and leucine) resulting in a ligand with higher binding capacity under alkaline conditions. 

Johansson (WO2010/080065) also teaches ligands of a dimer of domain B of SpA or protein Z wherei the ligands are alkali-stable by mutating at least one asparagine residue to an amino acid other than glutamine. 

Li (US 2014/0031522) discloses an immunogloublin binding proteins with alkali-resistance which includes at least one asparagine residue substituted with a histidine, a serine, an aspartic acid or a threonine residue. 

Shun-Cheng (WO2012/083425 and US2014/0031522) discloses variants of the Z domain where at least one asparagine residue is substituted with a histidine, a serine, an aspartic acid or a threonine reisude. The substitution may confer to the variant an increased stability in alkaline solutions when compared to the wil type.

Uhlen (US6,831,161 and WO 00/23580) also discloses that modification of Asn residues within a protein molecule increases stability of the protein in alkaline conditions and that suitable scaffold proteins include Staphylococcal protein A (SPA) analogue Z (the “Z domain” being a derivative of the B domain of SPA) or other domains or analogues of SPA. 

Lysine Residues:

Hyogo (US 15/883,569, published as US 2018/0215836) discloses an SpA domain where the number of lysine reisudes toward the N-temrinus such as positions 1-38 is eqqual to or greater than a total number of lysine in position 39 and usbsequent positions. 

Hyogo, (US 15/887,381, published as US 2018/0170973) also disclsoes multimers that include a modified C domain where the Lysine at position 4 or 7 is modified to a different amino aicd). 

Majima (EP1992692A1 and US 12/280221, published as US 2010/0286373)  disclose a modified C-domain of SpA or Z-domain which has improved orientation for maintinating affinity for an immunoglobulin and improved chemical stability under acidic pH conditions because a ratio of the number of lysine at positions 39 onwards to the number of lysine at positions 1-38 is increased as compared to a ratio in the unmofided molecule and/or substitution of other amino acid for lysine originally present at positions 4, 7, and 35. 

Majima (US 14/916,316, published as US 2016/0215027) also teaches a multimeric immunoglobulin binding protein having 6-10 total domains from SpA such as the C domain having the general structure (R1)n-(R2m) or (R2m-(R1)n where the R1 domain is an amino acid sequence in which a non-lysine amino acid has been replaced such as the lysine being substituted with a non-lysine amino acid at 1-3 of positions 4, 7, and 35 R2 is an immunoglbulin binding domain coccuring at the N-temrinus or the C-terminus of the protein and includes an amino acid residue that covalently bonds to an insoluble support and the R2 domain is based on an amino acid sequence in which the lysine residue(s) are substituted with a non-lysine amino acid only at position 35, or at posiiton 35 and one or more positions 4, 7 and 35. In one embbodiment the R2 domain includes substitutions of 1-6 amino acid reisdues with lysine at positions 40, 43, 46, 53, 54, and 56. Because only the (R2) domains are selectively immobilized on the support via a covalent bond, it is possible to acheive a highly selective immobilization reaction trhough for example a lysine or a cysteine residue. The R1 in trun has an amino acid sequence that does not contain an amino acid that is active to the chemical reaction used for immobilization. When the immobilization reaction used to immobilzie the prtoien on the support takes place via an amino group, an R2 domain can be produce by substituting the lysine residues contained in the amino acid sequence with non-lysine aino acids only in lysine residues occurring at positiosn that interfere with bidnign to an immunoglobulin upon immobilizing the protein on the support and by substituting some of the non-lysine amino acids not involved in bidning to an immunoglobulin with lysine. In the case of immobilization via a thiol group, a new cysteine residue can be added to the R2 domain. A support immobilization reaction using a disulfide bond or a maleimide group that is highly selective to the thiol group may be used for said immobilization. 

Shima (EP1992692A1) also discloses modified protein of a C domain of Staphylococcus protein A or a Z domain which has improved affinity for an immunoglobulin and also improved chemical stability under acidic pH conditions as compared to an unmodified molecule because the number of lysines at positions 1-38 is increased as compared to the unmodified molecule.

Yoshida (US2014/0107315) discloses E, D, A, B and C domains where all lysine residues are replaced with other amino acids and adding lysine at a terminal. In an alternative embodiment, the lysines at positions 29, 33, 36 and 37 of the C domain is replaced with another amino acid. 

–Gln or Lys to Ala, Ser or Thr:

Hyogo (US 15/876615, published as US 2018/0215785) discloses substitution of a Glutamine (Gln)  or lysine (Lys) in the Fc binding site of an ABP to Ala, Ser or Thr such that the ligand has a lower antibody binding capacity in an acidic pH range. In one e;mbodiment, the substiutions in the C domain are Q9A/G29A, Q9S/G29A, Q9T/G29A and G29A/Q32A.

Methionine and Asparagine-glycine substitutions: 

The methionine residues present in the domains E, D and A make protein A sensitive to cyanogen bromide treatment. In addition, the asparagine-glycine sequences present in all domains makes the protein sensitive to hydroxylamine treatment. Nilsson designed a synthetic IgG binding fragment lacking both the methionine residue and the asparagine-glycine sequence. The synthetic fragment was based on the sequence of domain B, which lacks methionine residues and is thus cyanogen bromide resistant. Finally, a non-palindromic AccI was ntroduced in the 5 end of the fragment to enable polymerization of the fragment. This site introduces a valine residue instead of an alanine residue in position 1. The AccI site is also present in the 3 end of the synthesized Z fragment, thus encoding two additional amino acid residues.  Nilsson “A synthetic IgG-binding domain based on staphylococcal protein A” Protein Engineering, 107-13, 1987).  See also Abrahmsen (US 5,143,844 and EP0230869) for the patent covering this invention (discloses a recombinant DNA fragment Z coding for an immunoglobuilin G binding domain related to SpA where the methionine codon has been replaced with a different amino acid such as valine codon to enable expression of a Met free protein; also discloses G-A substitution).

Proline residues:

Bjorkman (WO 2012/074463) discloses one or mroe of a protein A domain (E, D, A, B, C) or protein Z or a functional variant thereof with at least one of the monomers having a substitution of the C-temrinal most proteline residue after teh third alpha-ehlix. 

Hydrophobic Amino Acids for other Hydrophobic or polar uncharged amino acids

Hyogo (US 15/876597, published as 2018/0215795) discloses engineered protein A ligands dervied from one of the E, D, A, B and C domains in which a hydrophobic amino acid residue in an Fc binding site (e.g., Phe at position 5 or 13, Leu at position 17 or Ile at position 31) is substituted by a different hydrophobic amino acid (Gly, Ala, Val, Leu, Ile, Met, Phe, or Trp) or polar uncharged amino acid residue (Ser, Thr, Gln, Asn, Tyr, and Cys) where the protein has a reduced antibody-binding capacity in an acidic pH range as compared to teh wild type protein. 

Affinity tags and Coupling elements (cysteine, Histidine , arginine, lysine) 

Protein A/G with Basic amino acid (e.g., histidine) tags:  See also modified Z domains with Histidine linkers below

Katayose (US2010/0105879A1) discloses immobilization of a protein having affinity for an antibody molecule such as Protein A/G having a tag sequencefrom preferably 5-30 amino acids  that includes a basic amino acid such as a histidine to a support which has at least one functional group such as carboxyl, epoxy or tosyl group.

Ljungquist (Eur. J. Biochem. 186, 557-561 (1989) discloses introduction of a cysteine into the C temrinal part of SpA, containing one, two or 5 IgG binding domains and subsequently immobilizing the domains on thiopropyl-Sepharose. The gel was then used for affinity purificaiton of human IgG. 

Rodrigo (Wo2106/079033) eaches protein A mutants which include coupling elements such as cysteine residues, a pluarlity of lysine resiudes and a plurality of histidine residues. The coupling elements can be located within 1-5 amino acid residues from teh C or N terminal end. 

Wagner (US6,406,921B1) disclose immobilization of protein via an affinity tag which may comprise a poly-cysteine, poly-lysine, poly-arginine or poly-histidine. The position of the amino acid tag can be at the amino or carboxy-terminus of the protein. An adaptor molecule such as protein A or G that links the affinity tag to the immobilized protein can also be used.

Histidine Linker-Z domain: 

Anderson (US2011/0136169A1) teaches a vector with N- or C terminal His tags fused to the upstream synthetic protein A IgG binding (“Z”) domains.  Separating the ZZ domains from the cloning site is an intervening linker region containing coding sequences for a tobacco etch virus (TEV) protease cleavage recogniction site which allows cleavage and release of the cloned target. Targets fused to His-Tags (at the C terminus) can thus be purified by metal affinity chromatography.

Chen, (“Immobilized protein ZZ, an affinity tool for immunoglobulin isolation and immunological experimentation” Biottechnol. Appl. Biochem. (2006) 45, 87-92) discloses recombinant ZZ protein with a hexahistidine tag at the N-terminus which exhibited good performance in affinity purification of IgG.

Tamori (US13/36410) discloses the formula R-R2 where R represents an amino acid sequence consisting of 4-300 amino acid residues containing a region of 4-20 contiguous histidine residues (also referred to as a “histidine linker”) and R2 represents an amino acid sequence consisting of 40-500 amino acid residues containing Z domain of Protein A. The ligand is immobized on a carrier and showed excellent slakli resistance. It is thought that when a regio of continguous histidine residues is added to the Z domain, the position of binding between the carrier and the Z domain leads to a structural change leading to an increase of alkli resistance. 

Particular Amino Acids Inserted or Deleted

Acidic amino acids (e.g., aspartic acid or glutamic acid):

Yoshida (US 14/916263, published as WO/2015/034056) discloses increasing the number of acidic amino acids in the C or Z domains of Protein A. Acidic amino acids include an amino acid having a carboxyl group in a side chain such as aspartic acid or glutamic acid. In one embodiment, the amino acid substituted by the acidic amino acid is at least one at positions 7, 18, 40, 46 or 55. 

Histidine amino acids:

Masaya (JP2010081866) discloses an improved protein A that is obtained by replacing an amino acid resiude having an exposed surface area ration of greater or equal 35% with a histidine residue. 

Shinya (US 9382297 and EP 2728000) discloses a protein A mutant protein having reduced affinity in acidic region where any one of Phe5, Asn6 Gln9, Gln10, Asn11, Phe13, Tyr14, Glu15, Leu17, Glu24, Glu25, Arg27, Asn28, Ile31, Gln 32, Lys35 and Asp36 are substiuted by a histidine residue. 

amino acids added to either end: Martin (EP2066419) discloses a modified Spa Domain C where the Asn-Lys_Phe-Asn in positions 3-6 are deleted or where one or more amino acids are added to either end of the WT Domain C. In some embodiments, the addition comprises the mutation G29A.

Spector (US8,754,196 and US 8, 895, 706) teaches a modified SpA ligand from the B, D and Z domains where at least 3 consecutive amino aicds form the N-terminus are deleted. 

Particular Positions (by type of amino acid (1 letter code)):

D36:

Knick (US 2019/0300568) discloses Fc binding prtoeins which remainstable even after akaline treatment. In one emdobiment, the Fc binding protein includes 2-6 Fc binding domains linked to each other. In one obdiment, the ligand include D36H. 

Tsukamoto “Engineered protein A ligands, derived form a histidne-scanning library, facilitate the affinity purificaiton of IgG under mild acidc conditions” J Biological Engineering, 2014, 8: 15) disclsoes a D36H mutation whose binding of IgG was highly senstive to pH change. The antibodies could be leuted at a higher pH when used as a ligand. 

E24: Bian (US 12/653888) discloses generation of an SpA B domain variants with the glutamic acid at position 24 mutated to all other naturally occurring amino acids except cysteine (C), serine  (S), alanine (A), glycine (g), asparagine (N), and glutamine (Q).

F5 or F13: Hyogo (US 15/876597, published as 2018/0215795) discloses substitution of the Phe at a position 5 or 13 of the C domain C, B, Z, E, D or A domains with a different hydrophobic amino aicd such as Gly, Ala, Val, Leu, Ile, Met, Phe, or Trup or by a polar uncharged amino acid such as Ser, Thr, Gln, Asn, Tyr or Cys. 

G29A:

Hall (US 9290549) disclsoes one or more Domain C units form SpA with the mutation G29A which after 5 hours incubation in 0.5 M NaOH has retained at least 95% of its orignal binding capacity. 

Q9 Q32 or Q40 or Q55: Rogrigo (US 2016/0159857 and WO 2015/005859) disclsoes an Fc binding polypeptide with improved alkaline stability such as the B or C domain of SpA where the glutamine residue at position 9 has been substituted with an amino acid other than asparagine, proline or cysteine, such  as to a tryptophan, leucine, glutamic acid, valine or lysine. This has the advantage that deamidation sensitive asparagines are not introduced, that the chain cofrmation is not disturbed by introduction of prolines and sites for disulfide bridges are not introduced. In another embodiment the glutamine 32 has been mutated to an amino acid other than asparagine, glutamine, proline or cysteins, such as an alanine.  In some embodiments, the glutamine residue at position 40 is the one mutated.

Hyogo (US 15/876,615, published as US 2018/0215785) disclsoes methods for purifying antibodies using a ligand from the D, A, B and C domains of Protein A where the Gln at positions 9, 10 and 32 of for example the C domain has been mutated to Ala, Ser, or Thr. 

L17: Hyogo (US 15/876597, published as 2018/0215795) discloses substitution of the Leu at a position 17 of the C domain C, B, Z, E, D or A domains with a different hydrophobic amino aicd such as Gly, Ala, Val, Leu, Ile, Met, Phe, or Trup or by a polar uncharged amino acid such as Ser, Thr, Gln, Asn, Tyr or Cys.

H18:  

Ander (US14/385,336) discloses an immunoglobulin or Fc binidng protein that has one or more domains of Spa A (i.e., the E, D, A B and/or C domains) or protein Z domain where the Asparagine or Histidine at the position corresponding to H18 of the B or Z domain has been substituted with an amino acid which is not proline or Asparagine. Further, if the amino acid at position corresponding to P57 of the B or Z domain is Proline and the amino acid residue at position N28 of the B or Z domain is Asparagine, then the aino acid at position H18 of the B or Z domain is not Serine, Threonine or Lysine. An advantage of the mtuated protein is that it binds Fc containing proteins and rleases them at pH levels higher than Fc binding proteins not have the H18 mutation. 

I31: Hyogo (US 15/876597, published as 2018/0215795) discloses substitution of the Ile31 at a position 17 of the C domain C, B, Z, E, D or A domains with a different hydrophobic amino acid such as Gly, Ala, Val, Leu, Ile, Met, Phe, or Trup or by a polar uncharged amino acid such as Ser, Thr, Gln, Asn, Tyr or Cys.

N11 (or N3 in truncated Z or C domain):

Rodrigo (US 10501557) discloses an SpA domain (E, D, A, B, C, Z and  Zvar (without linker region amino acids 1-6) a where at least the asparagine or serine residue at position 11 has been mutated to an amino acid selected form the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalamine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Rogdrigo (US 15/282367, published as US 20170327534) discloses SpA analogues where at least the asparagine or serine in the case of Domain D of SpA has residue at position 11 in the B, C or Z domain has been mutated to an amino acid such as glutamic acid, lysine, tyrosine, threonine, phenylalamine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine. The mutation of N11 in these domains confers an improved alkali stability. 

Rodrigo/Hansson (US 16/095721, published as US 2020/0239517) see also US 10703774) discloses a Zvar and C-domain without the linker region amino acids 1-8 and 56-58 where the amino acid residues at positions 13 and 44 are asparagine and wherein at least hte asparagine residue at position 3 (corresponds to position 11  for the B, C or Z domains) has been mutated to an amino acid selected form the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine alanine, histidine and argineine. In one embodiment the matrix is provided in a stroage liquid that includes at least 50% by volume of an aqueous alkali metal hydroxide solution and storing the matrix for at least 5 days. 

N6 or 11:

Hober (US 20160152668) discloses a plurality of lgiands coupled to a solid support wherein at least hte asparagine residue at position 6 or 11 or a parental immunoglublin binding protein has been mutated to an amino acid other than glutamine which confers increased chemical stability at alkaline pH. 

N23T:

Johansson (US 20140329995) disclsoes multimers of Protein Z or C domain of Protein A such as the Z prteoin with an N23 mutation. 

Hober (US 7834158, US 8198404, US 8354510, US 9296791, US 9534023, US 10918971 and US 9156892) disclsoes a modified B or Z domain where one asparagine residue has been mtuated with an amino acid other than glutamine. In advantageous embodiments, the mutations are N23T N23T and N43E, N23A, N6A, N11S, N11S and N23% and N6A and N23%. The mutations(s) confer an increased chemical stability at alkaline pH value.

Linhult (“Improving the tolerance of a Protein A analogue to repeated alkaline exposures using a bypass mutagenesis approach, Proteins 55: 2004, 407-416) discloses an engineered SPA Z form where the asparagine 23 is exchanged for a threonine, thereby increaseing the stability to alkaline treatment compared to the native Z molecule.

N28X:

Bjorkman (US13/996023 & WO2013147691) teaches modified SpaA B and Z domains where the Asparagine at position 28 has been substituted with another amino acid (i.e., N28X).

Jendeberg (J. Molecular Recognition, 8, 270-278 (1995) dicloses mutant Z proteins with an N28A substitution.

G29A:

–Z domain: (A1V/removal of Methionine & G29A): 

Protein Z is a synthetic construct derived from the B domain of SpA, wherein the glycine in position 29 has been exchanged for alanine (Ander, US 14,385, 336, citing Stahl et al., 1999). 

The Z domain of SpA is actually an engineered analogue of the B domain of SpA and includes an alanine instead of glycine residue at position 29 (G29A). The 1st amino acid ala is also replaced by Val (A1V).  Thus the Z-domain is a synthetic IgG binding protein, consisting of 58 amino acids, which are derived from the B domain of staphylococcal Protein A. It is designed to lack the Asp-Gly dipeptide sequence as well as methionine residues, so it is resistant to treatment with hydroxylamine and CNBr, in contrast with native Protein A (Chen, “Immobilized protein ZZ, an affinity tool for immunoglobulin isolation and immunological experimentation” (Biottechnol. Appl. Biochem. (2006) 45, 87-92). The Z domain binds to the Fc region of immunoglobulins as do the 5 homologous SPA domains, but unlike the parental domain it does not bind to the Fab region. The term “affibody” is used to define a class of engineered proteins selected fro their specific binding activity towards a desired target and based on the Z domain.

In the Z domain, another mutation is simultaneously introduced to substitute Ala at position 1 of the B domain by Val. This mutation is intedended to facilitate the genetic engineering preparation of a gene encocoding multiple domains linked together and does not affect the domain functions. (WO 2012/165544)

Nilsson showed that the asparagine side chain of SPA forms a hydrogen bond to the side chain of a glutamine in the Fc fragment which suggested that substitution of this asparagine residue in domain B would be likely to affect the IgG interaction. In contrast, the neighboring glycine residue (Gly 29), unusually placed in an alpha helix, did not seemed to be involved in the IgG interaction. Thus a methyl group was added to the glycine converting it into an alanine. The substitution was found to be sterically aceptable to the structure. This “Z domain” thus lacked methionine residues being based on fragment B and the glycine residue at position 29 had been substituted by an alanine to change the asparagine-glycine sequence. Finally, a non-palindromic AccI site had been introduced in the 5 end of the fragment to enable polymerization of the fragment. This site introduces a valine residue instead of an alanine residue in position 1. The AccI site is also present in the 3 end of the synthesized Z fragment, thus encoding two additional amino acid residues.  Nilsson “A synthetic IgG-binding domain based on staphylococcal protein A” Protein Engineering, 107-13, 1987).  See also Abrahmsen (US 5,143,844 and EP0230869) for the patent covering this invention (discloses a recombinant DNA fragment Z coding for an immunoglobuilin G binding domain related to SpA where the methionine codon has been replaced with a different amino acid such as valine codon to enable expression of a Met free protein; also discloses G-A substitution).

Hober (US2006/0194950) discloses alkali stable domain that can be further modified such that the ligands lack affinity for Fab but retains Fc affinity, for example by a G29A mutation. 

Jansson (FEMS Immunology and Medical Microbiology 20 (1998) 69-78 disclose an engineered domain Z, similar to fragment B but with a single glycine to alanine amino acid substitution.

Bjorkman (US 2013/0274451) discloses a ligand form SpA that is made of multimer copies of domain C, with Arginine residue at N28 mutated and optionally, the ligand also contains a G29A mutation.

–Replacement of G29 with amino acid other than alanine or tryptophan:

Spector (EP2157099A1; US8,592,555, US2010/0063256) discloses modified immunoglobulin binding proteins such as SpA based on one or more domains of SpA (i.e., E, D, A, B, C and Z) which are modified to replace at least the amino acid at position 29. In the case of the domains E, D, A, B and C, the amino acid at position 29 is a glycine, which is replaced with an amino acid other than alanine or tryptophan. In the case of domain Z, the amino acid at position 29 is an alanine, which is replaced with an amino acid other than glcine or tryptophan. Spector (US8,592,555) discloses a modified C domain of SpA where the glycine residue at position 29 is replaced with a lysine amino acid residue such that the SpA binds an Fc portion of an immunoglbulin but exhibits reduced binding to a Fab portion of an immunoglobulin. Spector (US 14/061,080, published as US 2014/0046037 teaches B and C domains where the glycine (G) at position 29 is substituted with lysin (K), Leucine (L) or arginine (R) and where the alanine (A) at position 29 of the Z domain is substituted with lysine (K), Leucine (L) or arginine (R). 

Q9:

Rodrigo (US 20160159855, 20160159857) disclsoes a mtutant B or C domain of SpA wherein at least the glutamine at position 9 has been mutated to an amino acid other than asparagine. 

V40 (C domain): Hyogo discloses a C domain ligand for affinity chromatography where the valine is exchange for a polar uncharged amino acid such as a thr, Ser, Gln, Asn or Cys or a basic amino acid such as a his, lys or Arg. 

Methods of Generating

Align sequences of domains (in silico) – statistically fragment (in silico) –reassemble so order remains is maintained

Ulrich (US 15/744,147, published as US 2018/0305463)discloses a method of generating a non-natural Ig binding protein from known sequences of protein A domains (E, D, A, B and C) by alignment of the known sequences, statistical fragmentation in silico (performed on a computer) , and then in silico assembly of new sequences from the various fragments such that the relative order of the statistically fragmented sequences in the amino acid sequence of the non-natural immunoglobulin binding protein is maintained as said statistically fragmented sequences present in the amino acid sequences of Ig binding domains of the Protein A polypeptides. 

Mutate domain (B2 domain of protein G)  –Express gene sequences –Purify by affinity chromatography –Mass Spectromety

Honda (US 2015/0183820) discloses a method of generating a variant Protein G which includes the steps of selection of the target part for teh mutation based on an alysis of teh surface bound to the Fc region (3D coodinate dats of the complex of the B2 domain of the protein G and the Fc region of Ig1 downloaded from Protein Data Bank (PDB) and amino acdi residues of the B2 domain within a set angstrom distance from teh Fc region with an exposed surface area ratio was selected as the target parts for the mutation), disgning the base sequences of the genes encoding the variant proting G (using Gene Designer), synthezing plasmid vectors which contain the genes encoding the variant protin G, transformation of E coli with the vectors, purifying the variants with affinity chromatography (IgG Sepharose  Fast flow column), measuring MW of the improved protein G with MALDI-TOF type MS and comparing MW of a peak detected form the MS and theoretial MS calculated form teh amino acid sequence of the produced mutant prtoin to oconfirm target protein.