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Protein cross linking agent and method therefor

A cross-linking agent and protein technology, which is applied in the field of pharmaceutical protein modification research and protein modification research, to achieve the effect of improving controllability

Inactive Publication Date: 2004-09-01
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no cross-linking agent that can meet the above requirements at the same time

Method used

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  • Protein cross linking agent and method therefor
  • Protein cross linking agent and method therefor
  • Protein cross linking agent and method therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Preparation of intramolecular hemoglobin by ethylene glycol diglycidyl ether (EGDE)

[0032] The hemoglobin concentration is 20 mg / ml, the solution system is 50 mM phosphate buffer solution with pH 7.5, the total solution system is 5 ml, the molar ratio of ethylene glycol diglycidyl ether to hemoglobin is 25:1, and the reaction is performed on a water bath shaker at 37° C. for 1 hour. The cross-linked product was detected by SDS-PAGE electrophoresis, and the result showed a darker band of about 32KD, indicating that there was cross-linking of two subunits ( figure 1 ). At the same time, the sample was taken through the Superdex 200 gel filtration column, and its peak time was the same as that of natural hemoglobin, indicating that its molecular weight was 64KD. Indicates the generation of intramolecular hemoglobin ( figure 2 ). The cross-linked hemoglobin kept the biological activity well, the P50 was 23.2, and the Hill coefficient was 2.42 ( image 3...

Embodiment 2

[0033] Embodiment 2: Preparation of polymerized hemoglobin by ethylene glycol diglycidyl ether (EGDE)

[0034] The hemoglobin concentration is 100 mg / ml, the solution system is 50 mM borax-sodium hydroxide buffer solution with pH 9.5, the total solution system is 5 ml, the molar ratio of ethylene glycol diglycidyl ether to hemoglobin is 500:1, and the reaction is performed on a water bath shaker at 37°C 12 hours. The cross-linked product was detected by SDS-PAGE electrophoresis, and the results showed that the protein subunit bands were mainly concentrated between 32kD and 96kD, indicating that a cross-linked product of two to six subunits was formed ( Figure 4 ). At the same time, the sample is taken through the Superdex 200 gel filtration column, the peak eluting time is earlier than that of the hemoglobin standard sample, and the cross-linking rate is as high as 98%. The molecular weight of polymerized hemoglobin is about 128KD, and its peak area accounts for about 80% o...

Embodiment 3

[0035] Example 3: One-step cross-linking of hemoglobin and serum albumin with ethylene glycol diglycidyl ether

[0036] The concentration of hemoglobin is 2mg / ml, the concentration of albumin is 2.092mg / ml, the solution system is 50mM boric acid buffer solution with pH8.7, the total solution system is 5ml, the molar ratio of ethylene glycol diglycidyl ether to hemoglobin is 100:1, 25°C React in a water bath shaker for 24 hours. The cross-linked product was detected by SDS-PAGE electrophoresis, and the results showed that 83kD and 97kD bands were generated. The molecular weight of hemoglobin single subunit is about 16kD, the molecular weight of albumin is 67kD, 83kD is a cross-linked product of a single hemoglobin subunit and serum albumin, 97kD is a conjugate of two cross-linked hemoglobin subunits and a serum albumin, It shows that the conjugate of hemoglobin and serum albumin is generated.

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Abstract

The invention provides a method for using homotype double functional cross linking agent with epoxy radicals for protein and polypeptide cross bonding, the cross linking agent used by the present invention can realize an ideal reaction velocity for the cross bonding between proteins and polypeptides with good controllability. When applied to the hemoglobin cross bonding under controlled conditions, the method can raise the yield for inter-molecule cross bonding to above 90%.

Description

technical field [0001] The invention belongs to the field of protein modification research, in particular to the field of medicinal protein modification research. Background technique [0002] As drugs, polypeptides and proteins have been widely used in the treatment of diseases. Proteins are natural antigens, and heterologous proteins entering the human body will cause antibodies to be produced. Through the antigen-antibody reaction, the heterologous protein cannot effectively perform its therapeutic function because it is cleared, and sometimes the heterologous protein even produces an allergic reaction. Some peptides and proteins with therapeutic prospects cannot achieve therapeutic effects due to their short circulation half-life in the body. Chemical modification of proteins (polypeptides) can effectively solve the above problems. The chemical modification of proteins refers to the modification of protein molecules at the molecular level by chemical methods, that is,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107C07K14/76C07K14/805C07K19/00C08H1/00
Inventor 苏志国徐宇红路秀玲郑春杨
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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