Allophycocyanin (APC) and Annexin V protein coupling method

A technology for coupling allophycocyanin and protein, which is applied in the field of coupling allophycocyanin and AnnexinV protein, can solve the problems of incapable mass production and achieve the effect of reducing purification cost, less purification steps and good biological activity

Pending Publication Date: 2020-01-10
杭州联科生物技术股份有限公司
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Problems solved by technology

[0006] Molecular sieve high-pressure liquid chromatography column purification has high purification purity and high resolution effi

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  • Allophycocyanin (APC) and Annexin V protein coupling method

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Embodiment 1

[0036] A method for coupling allophycocyanin (APC) to Annexin V protein, comprising the following steps:

[0037] Step 1: AnnexinV protein expression and purification:

[0038] 1.1: Gene sequence optimization: Find the gene sequence of human AnnexinV protein, optimize the human AnnexinV gene sequence, remove the stop codon sequence, connect it to the prokaryotic expression vector pET28a, keep the front and rear His tag proteins, and prepare for protein purification and subsequent coupling Preparation for fluorescein purification;

[0039] 1.2 Induced expression of AnnexinV protein: Pick a single colony of pET28a-AnnexinV plasmid, inoculate it into 10ml LB liquid medium containing 50μg / ml kanamycin, and cultivate overnight at 37°C with shaking at 200rpm; inoculate the cultured bacterial solution at a ratio of 1:100 Into the LB liquid medium containing 50μg / ml kanamycin, shake culture at 200rpm at 37°C to the bacterial concentration OD 600 04-0.6; take out 1ml of bacterial liq...

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Abstract

The invention discloses an allophycocyanin (APC) and Annexin V protein coupling method. The APC and Annexin V protein coupling method comprises the following steps: 1, an Annexin V protein is expressed and purified: specifically, 1.1, a gene sequence is optimized, specifically the gene sequence of the human Annexin V protein is looked up, the human Annexin V gene sequence is optimized, a termination codon sequence is removed, the human Annexin V gene sequence is connected into a prokaryotic expression vector, front and back His tag proteins are retained, and preparation is made for protein purification and subsequent coupled fluorescein purification; and 1.2, the Annexin V protein is subjected to inducible expression, specifically, a recombinant plasmid single colony is picked and inoculated into an LB liquid culture medium containing kanamycin, and culturing is conducted until the concentration OD600 of bacterial liquid is 0.4-0.6, and an inducer is added into the bacterial liquid forinduction culture. When the Annexin V gene sequence is designed, His tags at the two ends of the protein are reserved, the Annexin V protein and an Annexin V-APC conjugate are convenient to purify, only conventional nickel column purification is needed, special purification equipment is not needed, the purification steps are few, the purification time is short, the purification cost is lowered, the good biological activity of the protein is maintained, and the Annexin V protein can be used for cell apoptosis detection.

Description

technical field [0001] The invention relates to a coupling method of allophycocyanin (APC) and Annexin V protein. Background technique [0002] Annexin V is a human endogenous protein, which belongs to the calcium ion-dependent phospholipid-binding protein family, has anticoagulant effect, is widely distributed, and is expressed in various tissue cells (such as cardiomyocytes, liver cells, endothelial cells, etc.). AnnexinV is encoded by the AnnexinV gene located on the fourth human chromosome 4q26-q28 on the chromosome, with a total of 320 amino acids, including repeating units of 70-80 amino acids, and a molecular weight of about 36kD. In the process of cell apoptosis, the apoptotic cell membrane changes, and the migration of PS from the inner layer of the lipid bilayer to the outer layer of the cell membrane is the initial event of the apoptosis cascade reaction. AnnexinV protein has a high affinity with PS on the cell membrane. Therefore, the use of AnnexinV high-affini...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/107C07K1/22C07K1/34C07K14/47C07K14/795
CPCC07K14/4747C07K14/795C07K2319/00
Inventor 郭爱龙
Owner 杭州联科生物技术股份有限公司
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