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Protein A domain Z derivative with specific binding effect on antibody and application thereof

A technology of binding action and domain, applied in the field of affinity chromatography in biotechnology, can solve the problems of reduction, destruction of the three-dimensional structure of the chromatographic ligand of protein A, loss of antibody binding ability, etc., and achieves good chemical stability and high chemical stability. sexual effect

Active Publication Date: 2017-03-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This is still very harsh for the current commercial protein A chromatography medium, which will lead to the destruction or even loss of the three-dimensional structure of the protein A chromatography ligand and the reduction of antibody binding ability [Protein Science, 2013,22(9):1230 -1238】

Method used

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  • Protein A domain Z derivative with specific binding effect on antibody and application thereof
  • Protein A domain Z derivative with specific binding effect on antibody and application thereof
  • Protein A domain Z derivative with specific binding effect on antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1 sequence number is the preparation and stability of the domain Z derivative of SEQ ID No.:2

[0029] The structural domain Z with the sequence number of SEQ ID No.: 1 and the protein with the sequence number of SEQ ID No.: 2 were respectively synthesized by using a polypeptide solid-phase chemical synthesis method. The above proteins were dissolved in 20mmol / L PBS buffer (pH 6.0) containing 100mmol / L NaCl to prepare a protein solution with a protein concentration of 1mg / mL. The denaturation temperature of the protein in the above two solutions was carried out in a VP-DSC differential scanning calorimeter. First, after the sample cell and reference cell of VP-DSC were washed with 100mL ultrapure water, the buffer solution (20mmol / L PBS buffer solution containing 100mmol / L NaCl, pH 6.0) and about 0.75 mL of the protein solution were added to the reference pool and the sample pool (the volume of the reference pool and the sample pool was 0.5282 mL). After th...

Embodiment 2

[0030] Example 2 The sequence number is the stability of the domain Z derivative of SEQ ID No.: 2 in alkaline solution

[0031] The preparation method described in Example 1 was used to synthesize the domain Z with the sequence number of SEQ ID No.: 1 and the protein with the sequence number of SEQ ID No.: 2. The above-mentioned protein was dissolved in 0.1 mol / L sodium hydroxide solution to prepare a protein solution with a protein concentration of 1 mg / mL. The denaturation temperatures of the proteins in the above two solutions were analyzed using the differential scanning calorimetry method as in Example 1. The solution system is 0.1mol / L sodium hydroxide solution. Inject the 0.1mol / L sodium hydroxide solution that has been vacuum degassed for more than 15 minutes into the reference cell. The temperature scanning range of differential scanning calorimetry is 25-120°C, and the heating rate is 1°C / min. Before heating starts, the test temperature equilibration time is 8min....

Embodiment 3

[0032] Example 3 The sequence number is the preparation and stability of the domain Z derivative of SEQ ID No.:3

[0033] The protein whose sequence number is SEQ ID No.:3 was synthesized by the preparation method described in Example 1. The denaturation temperature of the above proteins under neutral conditions was analyzed by differential scanning calorimetry as in Example 1. The result is as figure 1 shown. The results show that the denaturation temperature of the domain Z derivative whose sequence number is SEQ ID No.: 3 is 76.9°C. This shows that the domain Z derivative with the sequence number of SEQID No.: 3 is more stable than domain Z under neutral conditions.

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Abstract

The invention relates to an protein A domain Z derivative with a specific binding effect on an antibody and an application thereof. According to the invention, mutation of N23T and mutation of at least one amino acid residue are generated in a domain Z amino acid sequence. The derivative is obtained through a strategy of mutation of at least one amino acid residue in Helix1 and 2 in an protein A mutation domain Z. In addition, the invention also provides a preparation method for an affinity chromatography matrix by utilizing the domain Z derivative as a ligand. Compared with the affinity chromatography matrix by utilizing the domain Z derivative as the ligand, the domain Z derivative provided by the invention has higher chemical stability, and has a protein denaturation temperature in alkaline solution increased by 4 DEG C; and the synthesized affinity chromatography matrix maintains considerable antibody adsorption capacity.

Description

technical field [0001] The invention relates to a derivative of protein A mutant domain Z with improved chemical stability under alkaline conditions and a method for its application, in particular to an affinity chromatography medium using mutant domain Z derivatives as ligands The preparation method belongs to the field of affinity chromatography in biotechnology. Background technique [0002] Monoclonal antibody drug preparation technology has developed rapidly in the past 30 years. This is mainly due to the construction of high-expression cell lines, the improvement of medium and the continuous expansion of reactor scale. At present, reactors of more than 20,000 liters have been promoted, and the antibody titer has increased by more than 30 times, and the antibody titer of 25g / L has also been reported [New Biotechnol,2011,28(5):458-63; Adv Drug Deliv Rev, 2006, 58(5-6):671-85]. Protein A chromatography is currently the most widely used and mature technology in the proc...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K16/00C07K1/22B01J20/291B01J20/285
CPCB01J20/285B01J20/291B01J2220/44C07K1/22C07K14/31C07K16/00
Inventor 史清洪管志龙白姝孙彦
Owner TIANJIN UNIV
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