FC binding protein and method for manufacturing same

Patent metadata

Field	Value
Publication number	US-9453066-B2
Application number	US-201113583550-A
Country	US
Kind code	B2
Filing date	Mar 10, 2011
Priority date	Mar 10, 2010
Publication date	Sep 27, 2016
Grant date	Sep 27, 2016

Abstract

Disclosed are: an Fc binding protein having increased stability with respect to heat, acid, and/or alkalinity compared with the wild type; a method for producing same; and a method for specifically isolating protein containing an Fc binding protein binding site using said Fc binding protein as a ligand for affinity chromatography.
An Fc binding protein was obtained having increased stability with respect to heat, acid, and/or alkalinity compared with the wild-type human Fc receptor by means of substituting at least one specific amino acid residue in the extracellular domain of the wild-type human Fc receptor with another amino acid residue. The Fc binding protein is useful as a ligand for affinity chromatography for example by immobilizing in a solid phase. Also, when the Fc binding protein is expressed using a host that has been transformed with an expression vector containing a polynucleotide coding for said protein, the amount of produced protein is increased compared with using a wild-type human Fc receptor.

First claim

The invention claimed is:

1. An Fc binding protein which comprises a human FcγRI α-chain region modified by substitution, wherein the unmodified human FcγRI a-chain region comprises residues 16-289 of SEQ ID NO: 1, and wherein said substitutions comprise substitution of phenylalanine at position 114 of SEQ ID NO: 1 with leucine.

2. An Fc binding protein, wherein said Fc binding protein comprises the amino acid sequence of residues 34 to 307 of any one of SEQ ID NOS: 2, 3, 4, 5, 114, 118, 130, 134, 148, 154, 164, 170, 174, and 176.

3. An Fc binding protein, wherein said Fc binding protein comprises the amino acid sequence of any one of SEQ ID NOS: 2, 3, 4, 5, 114, 118, 130, 134, 148, 154, 164, 170, 174, and 176.

4. An adsorbent for a protein containing an Fc binding protein binding site, the adsorbent obtained by immobilizing to a solid phase the Fc binding protein according to claim 1 .

5. An antibody purification method comprising:
(1) adding a solution containing an antibody to an adsorbent, the adsorbent obtained by immobilizing to a solid phase an Fc binding protein, wherein said Fc binding protein comprises the amino acid sequence of any one of SEQ ID NOS: 114, 118, 130, 134, 148, 154, 164, 170, 174, and 176; and
(2) eluting the antibody adsorbed to the adsorbent with a buffer solution of pH 3.0 to pH 4.5.

6. An Fc binding protein which comprises a human FcγRI α-chain region modified by substitution, wherein the unmodified human FcγRI α-chain region comprises residues 16-289 of SEQ ID NO: 1, and wherein the substitution(s) are selected from the group consisting of (1) to (167), with the proviso that at least phenylalanine at position 114 of SEQ ID NO: 1 is substituted by leucine:
(1) threonine at position 20 of SEQ ID NO: 1 is substituted by proline;
(2) threonine at position 25 of SEQ ID NO: 1 is substituted by lysine;
(3) threonine at position 38 of SEQ ID NO: 1 is substituted by alanine or serine;
(4) leucine at position 46 of SEQ ID NO: 1 is substituted by arginine or proline;
(5) alanine at position 62 of SEQ ID NO: 1 is substituted by valine;
(6) threonine at position 63 of SEQ ID NO: 1 is substituted by isoleucine;
(7) serine at position 69 of SEQ ID NO: 1 is substituted by phenylalanine or threonine;
(8) arginine at position 71 of SEQ ID NO: 1 is substituted by histidine;
(9) valine at position 77 of SEQ ID NO: 1 is substituted by alanine or glutamic acid;
(10) asparagine at position 78 of SEQ ID NO: 1 is substituted by aspartic acid;
(11) aspartic acid at position 94 of SEQ ID NO: 1 is substituted by glutamic acid;
(12) isoleucine at position 100 of SEQ ID NO: 1 is substituted by valine;
(13) serine at position 110 of SEQ ID NO: 1 is substituted by asparagine;
(14) phenylalanine at position 114 of SEQ ID NO: 1 is substituted by leucine;
(15) histidine at position 125 of SEQ ID NO: 1 is substituted by arginine;
(16) leucine at position 131 of SEQ ID NO: 1 is substituted by arginine or proline;
(17) tryptophan at position 149 of SEQ ID NO: 1 is substituted by leucine;
(18) leucine at position 156 of SEQ ID NO: 1 is substituted by proline;
(19) isoleucine at position 160 of SEQ ID NO: 1 is substituted by methionine;
(20) asparagine at position 163 of SEQ ID NO: 1 is substituted by serine;
(21) asparagine at position 195 of SEQ ID NO: 1 is substituted by threonine;
(22) threonine at position 199 of SEQ ID NO: 1 is substituted by serine;
(23) asparagine at position 206 of SEQ ID NO: 1 is substituted by lysine, serine, or threonine;
(24) leucine at position 207 of SEQ ID NO: 1 is substituted by proline;
(25) leucine at position 218 of SEQ ID NO: 1 is substituted by valine;
(26) asparagine at position 240 of SEQ ID NO: 1 is substituted by aspartic acid;
(27) leucine at position 248 of SEQ ID NO: 1 is substituted by serine;
(28) leucine at position 283 of SEQ ID NO: 1 is substituted by histidine;
(29) leucine at position 285 of SEQ ID NO: 1 is substituted by glutamine;
(30) valine at position 17 of SEQ ID NO: 1 is substituted by glycine or glutamic acid;
(31) threonine at position 19 of SEQ ID NO: 1 is substituted by isoleucine;
(32) threonine at position 20 of SEQ ID NO: 1 is substituted by isoleucine;
(33) threonine at position 25 of SEQ ID NO: 1 is substituted by methionine or arginine;
(34) glutamine at position 27 of SEQ ID NO: 1 is substituted by proline or lysine;
(35) glutamine at position 35 of SEQ ID NO: 1 is substituted by leucine, methionine, or arginine;
(36) glutamic acid at position 36 of SEQ ID NO: 1 is substituted by glycine;
(37) leucine at position 41 of SEQ ID NO: 1 is substituted by methionine;
(38) histidine at position 42 of SEQ ID NO: 1 is substituted by leucine;
(39) glutamic acid at position 44 of SEQ ID NO: 1 is substituted by aspartic acid;
(40) valine at position 45 of SEQ ID NO: 1 is substituted by alanine;
(41) leucine at position 46 of SEQ ID NO: 1 is substituted by alanine, asparagine, aspartic acid, glutamine, glycine, histidine, lysine, serine, or tryptophan;
(42) histidine at position 47 of SEQ ID NO: 1 is substituted by glutamine, leucine, or asparagine;
(43) proline at position 49 of SEQ ID NO: 1 is substituted by serine or alanine;
(44) glycine at position 50 of SEQ ID NO: 1 is substituted by arginine or glutamic acid;
(45) serine at position 51 of SEQ ID NO: 1 is substituted by alanine, threonine, leucine, proline, or valine;
(46) serine at position 52 of SEQ ID NO: 1 is substituted by glycine;
(47) serine at position 53 of SEQ ID NO: 1 is substituted by leucine, threonine, or proline;
(48) glutamine at position 55 of SEQ ID NO: 1 is substituted by arginine;
(49) phenylalanine at position 57 of SEQ ID NO: 1 is substituted by tyrosine;
(50) leucine at position 58 of SEQ ID NO: 1 is substituted by arginine;
(51) glycine at position 60 of SEQ ID NO: 1 is substituted by aspartic acid;
(52) threonine at position 61 of SEQ ID NO: 1 is substituted by alanine or serine;
(53) alanine at position 62 of SEQ ID NO: 1 is substituted by glutamic acid;
(54) threonine at position 63 of SEQ ID NO: 1 is substituted by leucine, or phenylalanine;
(55) glutamine at position 64 of SEQ ID NO: 1 is substituted by proline, histidine, leucine, or lysine;
(56) threonine at position 65 of SEQ ID NO: 1 is substituted by alanine or valine;
(57) serine at position 66 of SEQ ID NO: 1 is substituted by threonine;
(58) threonine at position 67 of SEQ ID NO: 1 is substituted by alanine or serine;
(59) serine at position 69 of SEQ ID NO: 1 is substituted by alanine;
(60) tyrosine at position 70 of SEQ ID NO: 1 is substituted by histidine or phenylalanine;
(61) arginine at position 71 of SEQ ID NO: 1 is substituted by tyrosine;
(62) threonine at position 73 of SEQ ID NO: 1 is substituted by alanine or serine;
(63) serine at position 74 of SEQ ID NO: 1 is substituted by phenylalanine;
(64) serine at position 76 of SEQ ID NO: 1 is substituted by asparagine;
(65) valine at position 77 of SEQ ID NO: 1 is substituted by aspartic acid or lysine;
(66) asparagine at position 78 of SEQ ID NO: 1 is substituted by serine or glycine;
(67) serine at position 80 of SEQ ID NO: 1 is substituted by alanine;
(68) arginine at position 84 of SEQ ID NO: 1 is substituted by serine;
(69) glycine at position 88 of SEQ ID NO: 1 is substituted by serine;
(70) leucine at position 89 of SEQ ID NO: 1 is substituted by glutamine or proline;
(71) serine at position 90 of SEQ ID NO: 1 is substituted by glycine;
(72) arginine at position 92 of SEQ ID NO: 1 is substituted by cysteine or leucine;
(73) isoleucine at position 96 of SEQ ID NO: 1 is substituted by valine or lysine;
(74) glutamine at position 97 of SEQ ID NO: 1 is substituted by leucine or lysine;
(75) histidine at position 101 of SEQ ID NO: 1 is s

Assignees

• Hatayama Kouta
• Asaoka Yoshiharu
• Tanaka Toru
• Ide Teruhiko
• Sagami Chemical Res Inst
• Tosoh Corp

Inventors

• Hatayama Kouta
• Asaoka Yoshiharu
• Tanaka Toru
• Ide Teruhiko

Classifications

• C07K17/08

  the carrier being a synthetic polymer · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• C07K14/70535 Primary

  Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F) · CPC title