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A kind of separation and purification method of Trichoderma reesei recombinant t-pa

A technology for separation and purification of Trichoderma reesei, which is applied in the field of separation and purification of t-PA, and achieves the effects of low operation cost, simple operation steps, and improved specific activity.

Active Publication Date: 2020-01-03
HUNAN UNIV OF SCI & ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the separation and purification of t-PA that have been reported are all isolated from biological tissues and genetically recombined animal cells, and there is no report on the isolation of t-PA from genetically recombined microorganism Trichoderma reesei cells

Method used

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  • A kind of separation and purification method of Trichoderma reesei recombinant t-pa
  • A kind of separation and purification method of Trichoderma reesei recombinant t-pa
  • A kind of separation and purification method of Trichoderma reesei recombinant t-pa

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Embodiment 1

[0043] 1. Materials:

[0044] The genetically engineered bacteria Trichoderma reesei (Trichoderma reesei) 306 was purchased from the Laboratory of Applied Microbiology, Tianjin University of Science and Technology. Agarose was purchased from Sino-American Biotec; fibrinogen, sodium dodecyl sulfate (SDS), acrylamide, and methylene bisacrylamide were all purchased from Sigma; thrombin was purchased from the Institute of Blood, Chinese Academy of Medical Sciences; Low relative molecular mass standard protein (Marker, 14400-97400) and isoelectric point protein standard (pI3.5-9.3) were purchased from Shanghai Sebas Biotechnology Development Co., Ltd.; Q-Sepharose High Performance and Superdex 75PrepGrade were purchased from From Pharmacia Biotech; the rest of the reagents are imported or domestic chromatographically pure or analytically pure reagents. AKTA prime protein purification system was purchased from GE, USA.

[0045] 2. Method

[0046] 2.1 Preparation of enzyme solutio...

Embodiment 2

[0082] A method for separating and purifying Trichoderma reesei recombinant t-PA, comprising the following steps:

[0083] 1, prepare crude enzyme liquid, preparation method is with embodiment 1;

[0084] 2. Decolorization of crude enzyme solution: pass the clarified enzyme solution centrifuged to remove bacteria and solids through D296 strong anion exchange resin column (Φ1.5±0.2×30±1cm), control the linear velocity to less than 0.5cm / min, when the resin is The pigment adsorption is close to saturation, stop adding the enzyme solution, and elute with 0.018mol / L phosphate buffer, and the resin of the eluted column is regenerated for reuse;

[0085] 3. Ammonium sulfate salting-out: Adjust the saturation of the decolorized fermentation broth to 40% with ammonium sulfate, then refrigerate it in a refrigerator (2-6°C) for 9 hours, centrifuge at 2°C and 7000r / min for 30min, and separate the supernatant and the precipitate Separate, collect the precipitate and dissolve it with pH 7...

Embodiment 3

[0090] A method for separating and purifying Trichoderma reesei recombinant t-PA, comprising the following steps:

[0091] 1, prepare crude enzyme liquid, preparation method is with embodiment 1;

[0092] 2. Decolorization of the crude enzyme solution: Pass the clarified enzyme solution centrifuged to remove bacteria and solids through a D296 strong anion exchange resin column (Φ1.5±0.2×30±1cm), control the linear velocity to less than 0.5cm / min, when the resin is The pigment adsorption is close to saturation, stop adding the enzyme solution, and elute with 0.022mol / L phosphate buffer, and the resin of the eluted column is regenerated for reuse;

[0093] 3. Ammonium sulfate salting-out: adjust the saturation of the decolorized fermentation broth to 50% with ammonium sulfate, then refrigerate it in a refrigerator (2-6°C) for 7 hours, centrifuge at 6°C and 8000r / min for 20min, and separate the supernatant and the precipitate Separate, collect the precipitate and dissolve it wit...

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Abstract

The invention relates to the field of separation of purification of t-PA, particularly to a separation and purification method of Trichoderma reeseire combination t-PA. The separation and purification method comprises the following steps: pretreating t-PA-containing crude enzyme liquid prepared by fermenting Trichoderma reesei; decoloring the crude enzyme liquid with D296 strong anion exchange resin column, eluting the crude enzyme liquid with a phosphatic buffer solution to obtain eluent, and salting out the eluent with ammonium sulfate to obtain precipitates; dissolving the precipitates with the phosphatic buffer solution, and performing dialysis desalting; separating and purifying desalted enzyme liquid with Q-Sepharose-High-Performance ion exchange chromatography, performing gradient elution, and collecting to obtain active component liquid; freeze-drying to obtain a t-PA finished product. The whole method is simple and feasible; the molecular mass A of the obtained t-PA is about 65,000; the isoelectric point is about 4.24; the specific activity is greater than 3,200 U / mg; the purification factor is greater than 51.5, and the recycling rate is over 75 percent.

Description

technical field [0001] The invention relates to the field of separation and purification of t-PA, in particular to a method for separation and purification of Trichoderma reesei recombinant t-PA. Background technique [0002] Thrombotic disease is common disease, frequently-occurring disease, has become one of the most serious diseases that endanger people's health now. Thrombolytic therapy in modern medicine is a safe and effective treatment for thrombotic diseases. Although the thrombolytic agents that have been used in clinical use at home and abroad have good drug effects, they all have side effects to varying degrees and are expensive. [0003] Tissue-type plasminogen activator (tissue-type plasminogen activator, t-PA) is an enzyme contained in various tissues in the body, which can selectively convert plasminogen in thrombus into plasmin to make thrombus The thrombolysis caused by t-PA is generally not accompanied by the tendency of systemic bleeding, so it has becom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/58C12R1/885
CPCC12N9/58
Inventor 邵金华
Owner HUNAN UNIV OF SCI & ENG
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