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Expression and purification method of recombinant human tissue-type plasminogen activator

A technology for expression and purification of plasminogen, which is applied in the field of biomedicine, can solve the problems of low protein yield and complicated purification steps, and achieve the effects of simplified purification steps, simple operation, and increased yield

Active Publication Date: 2020-11-10
武汉人福药业有限责任公司
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Problems solved by technology

However, its purification steps are relatively complicated, and it is easy to cause the problem of low yield of protein preparation, which needs further improvement

Method used

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  • Expression and purification method of recombinant human tissue-type plasminogen activator

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Embodiment 1

[0030] Example 1 A method for expression and purification of recombinant human tissue-type plasminogen activator

[0031] 1. The t-PA gene is amplified by PCR using the cDNA library, and the general method is to design primers to connect the t-PA gene to the genetic engineering vector pET30a to construct the pET30a-tPA plasmid.

[0032] 2. Transform the constructed pET30a-tPA plasmid into E.coli DH5α competent cells, and spread it on the LB medium plate containing kanamycin. The composition of the LB medium is as follows:

[0033] Yeast extract 5g / L Tryptone 10g / L NaCl 10g / L

[0034] Add 200 μL of 10M NaOH to each liter of medium to adjust the pH to neutral, and then sterilize under high temperature and high pressure. After overnight culture, select a single colony and culture it in ZYM-505 liquid medium containing kanamycin. The composition of ZYM-505 liquid medium is as follows:

[0035] Nitrogen source N-Z-amine(%) 1 Yeast extr...

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Abstract

The present invention provides an expression and purification method of a recombinant human tissue-type plasminogen activator. The expression and purification method comprises the following steps: inducing a prokaryotic system to express human tissue-type plasminogen activator protein molecules through a self-induction culture system; purifying the recombinant human tissue-type plasminogen activator expressed by the prokaryotic system by using a serine protease affinity chromatography column; and renaturing the human tissue-type plasminogen activator protein molecules by using a dilution dialysis technology. The provided expression and purification method of the recombinant human tissue-type plasminogen activator is simple to operate, low in cost, large in protein expression quantity and high in protein yield.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for expressing and purifying recombinant human tissue-type plasminogen activator. Background technique [0002] Tissue plasminogen activator, also known as tissue-type plasminogen activator (tissue-type plasminogen activator, t-PA) or plasminogen activator (tissue plasminogen activator), is a physiological agonist of the fibrinolytic system in vivo , plays a key role in the balance regulation of human fibrinolysis and blood coagulation. t-PA belongs to the serine proteases in glycoproteins. It is a protein unique to the human body. It consists of 527 amino acids, including 35 cysteine ​​residues. They participate in the formation of 17 disulfide bonds and can specifically hydrolyze fibrinolysis. The zymogen forms plasmin, activates the fibrinolytic system, and plays an important role in clearing insoluble fibrin in blood vessels and ensuring smooth blood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/70A61K38/48A61P7/02
CPCC12N9/6459C12N15/70A61K38/482A61P7/02C12Y304/21068
Inventor 黄俊杰王静陈诗熊心磊邓霞飞
Owner 武汉人福药业有限责任公司
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