Method for constructing a novel affinity peptide library for binding immunoglobulin G based on a protein affinity model of protein A

Patent metadata

Field	Value
Publication number	US-10001489-B2
Application number	US-201314654731-A
Country	US
Kind code	B2
Filing date	Aug 19, 2013
Priority date	Dec 20, 2012
Publication date	Jun 19, 2018
Grant date	Jun 19, 2018

Abstract

An affinity ligand peptide library of IgG constructed on the basis of Protein A affinity model and the application of a design method thereof. According to the Molecular Mechanics—Poisson-Boltzmann surface area (MM/PBSA) method and on the basis of the known human IgG-Protein A complex structure, the hot spots of Protein A that have high affinity for human IgG are obtained analytically, and a Protein A simplified affinity model is built thereof. An affinity peptide library of IgG is constructed including heptapeptide and octapeptide structural modes. On the basis of the peptide structural modes, the types of inserted amino acids that ‘X’ residues represent are further identified using amino acid location method. Then, molecular docking and molecular dynamics simulation methods are used to screen the candidate peptides successively. Finally, the affinity peptide ligands that can effectively purify IgG are identified using affinity chromatography.

First claim

The invention claimed is:

1. A method for constructing a novel affinity ligand peptide library for binding immunoglobulin G on the basis of an affinity model of Protein A, comprising:
adding a cysteine to a middle region of a peptide to obtain the following peptides:

FYCHXXXE, FYXHCXXE, FYCHXXR, FYXHCXR, FYXCRXE,

YFXCRXE, HXYFCXR,

and

HXYFCXK;

wherein ‘X’ represents the common 19 amino acids except cysteine; and synthesizing one or more of the peptides.

2. The method of claim 1 , wherein, the constructing is on the basis of six Staphylococal Protein A hot spots: F132, Y133, H137, E143, R146, and K154.

3. The method of claim 1 , further comprising determining the kinds of amino acids ‘X’ in the peptides that are within the scope of the peptide library using an amino acid location method.

4. The method of claim 3 , wherein, a candidate peptide library is obtained and shown below:

Peptide &

Number of

peptides
Amino acids ‘X’ represents

FYCHX 1 X 2 X 3 E
X 1 : A, R, N, D, Q, E, H, I, L, K, M,

number: 990
T, W, Y, V

X 2 : A, Q, E, I, L, K

X 3 : A, R, D, Q, E, L, K, M, P, S, T

FYX 1 HCX 2 X 3 E
X 1 : A, R, N, D, I, P, T, W, Y

number: 594
X 2 : A, Q, E, I, L, K

X 3 : A, R, D, Q, E, L, K, M, P, S, T

FYCHX 1 X 2 R
X 1 : N, D, Q, E, H, I, K, M, F, P, T,

number: 112
W, Y, V

X 2 : Q, E, H, I, K, M, F, W

FYX 1 HCX 2 R
X 1 : A, R, N, D, I, P, T, W, Y

number: 72
X 2 : Q, E, H, I, K, M, F, W

FYX 1 CRX 2 E
X 1 : A, R, N, D, Q, E, G, H, I, L, K,

number: 153
M, F, P, W, Y, V

X 2 : N, D, Q, E, L, M, S, T, W

YFX 1 CRX 2 E
X 1 : N, D, E, H, I, L, K, M,P, S, T,

number: 117
Y, V

X 2 : N, D, Q, E, L, M, S, T, W

HX 1 YFCX 2 R
X 1 : A, R, N, D, I, P, T, W, Y

number: 54
X 2 : Q, H, K, S, W, Y

HX 1 YFCX 2 K
X 1 : A, R, N, D, I, P, T, W, Y

number: 81
X 2 : A, R, Q, H, K, F, W, Y, V.

5. The method of claim 4 , wherein the peptides in the candidate peptide library are screened by molecular docking, RMSD comparison, and rescreened by molecular dynamics simulation, obtaining peptide ligands possessing high affinity for human immunoglobulin G, which are
SEQ ID NO: 1499, FYWHCLDE;
SEQ ID NO: 1883, FYFCRWE;
SEQ ID NO: 1309, FYIHCLPE;
SEQ ID NO: 1576, FYYHCKKE;
SEQ ID NO: 859, FYCHWALE;
SEQ ID NO: 798, FYCHWQDE;
SEQ ID NO: 757, FYCHTIDE;
SEQ ID NO: 1073, FYRIICQRE;
SEQ ID NO: 397, FYCHHKTE;
SEQ ID NO: 606, FYCHLQKE;
SEQ ID NO: 122, FYCHRKAE;
SEQ ID NO: 147, FYCHNQDE;
SEQ ID NO: 82, FYCHRQEE; and
SEQ ID NO: 1133, FYNHCASE.

6. The method of claim 2 , further comprising determining the kinds of amino acids ‘X’ in the peptides that are within the scope of the peptide library using an amino acid location method.

7. The method of claim 6 , wherein, a candidate peptide library is obtained and shown below:

Peptide &

Number of

peptides
Amino acids ‘X’ represents

FYCHX 1 X 2 X 3 E
X 1 : A, R, N, D, Q, E, H, I, L, K, M,

number: 990
T, W, Y, V

X 2 : A, Q, E, I, L, K

X 3 : A, R, D, Q, E, L, K, M, P, S, T

FYX 1 HCX 2 X 3 E
X 1 : A, R, N, D, I, P, T, W, Y

number: 594
X 2 : A, Q, E, I, L, K

X 3 : A, R, D, Q, E, L, K, M, P, S, T

FYCHX 1 X 2 R
X 1 : N, D, Q, E, H, I, K, M, F, P, T,

number: 112
W, Y, V

X 2 : Q, E, H, I, K, M, F, W

FYX 1 HCX 2 R
X 1 : A, R, N, D, I, P, T, W, Y

number: 72
X 2 : Q, E, H, I, K, M, F, W

FYX

Assignees

• Univ Tianjin

Inventors

• Sun Yan
• Zhao Weiwei
• Liu Fufeng
• Shi Qinghong

Classifications

• G06F19/18

  Physics · mapped topic

• C40B30/02

  Chemistry & Metallurgy · mapped topic

• C40B50/02

  Chemistry & Metallurgy · mapped topic

• G01N33/6854 Primary

  Immunoglobulins · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title