Engineered protein A ligands, derived from a histidine-scanning library, facilitate the affinity purification of IgG under mild acidic conditions

Table 1 Mutation positions for histidine-scanning library

Number	5	6	9	10	11	13	14	15	17	24	25	27	28	31	32	35	36
Aminoacid residue of wild type	F	N	Q	Q	N	F	Y	E	L	E	E	R	N	I	Q	K	D
Distance (Å)	5.7	11	6.4	4.2	6.5	3.8	3.4	7.9	4.0	8.4	8.7	8.6	6.9	5.8	8.1	4.5	9.8
fSASA (%)	78	94	36	47	64	40	63	50	46	78	96	23	71	17	60	64	68
Mixed codon used in the library	YWT	MAY	CAW	CAW	MAY	YWT	YAT	SAW	CWK	SAW	SAW	CRY	MAY	MWT	CAW	MAW	SAT
Substituted amino acids in the library	H					H		H	H	H	H			H		H
	Y	H	H	H	H	Y	H	D	Q	D	D	H	H	N	H	N	H
	L					L		Q		Q	Q			L		Q

Distance in the table indicates the length between the PAB residue and the nearest positively charged residue in IgG-Fc. fSASA indicates the extent of solvent exposure of the side chain in wild-type amino acid residues of PAB. The seventeen mutation positions finally selected were classified into two groups: (1) the PAB residues were located within 9 Å from the positively charged IgG-Fc residues and (2) the PAB residues were located between 9 and 11 Å from the IgG-Fc residues, and with a fractional solvent-accessible surface area (fSASA) ((fSASA) = (SASA) (native)/SASA (denatured)) value of more than 68%. The following abbreviations are used for mixed bases: R = (A or G), Y = (C or T), M = (A or C), K = (G or T), S = (G or C) and W = (A or T). Substituted amino acid residues in the library indicate non-wild-type residues encoded by a mixed codon.

ISSN: 1754-1611