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Preparation method of recombinant staphylokinase dimer and site-specific modificaton of polyethylene glycol

A staphylokinase dimer, staphylokinase technology, applied in the field of protein chemistry, can solve the problems of staphylokinase biological activity reduction, staphylokinase activity reduction, interference with staphylokinase plasminogen interaction, etc.

Inactive Publication Date: 2012-08-22
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, PEG modification results in decreased biological activity of staphylokinase
Since PEG is a chain-like macromolecule, while shielding the antigenic determinant, it also interferes with the interaction between staphylokinase and its receptor plasminogen, resulting in a decrease in the activity of staphylokinase

Method used

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  • Preparation method of recombinant staphylokinase dimer and site-specific modificaton of polyethylene glycol
  • Preparation method of recombinant staphylokinase dimer and site-specific modificaton of polyethylene glycol
  • Preparation method of recombinant staphylokinase dimer and site-specific modificaton of polyethylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction, expression and purification of recombinant staphylokinase (SAK) containing cysteine ​​at the C-terminus

[0034] 1 Experimental method

[0035] 1.1 Construction of recombinant staphylokinase (SAK) gene

[0036] Design and synthesize the following 2 primers:

[0037] Primer (pF) sequence: 5'-atgcccatgggcaaaggcgatgacgcg-3'

[0038] Antisense primer (pR) sequence: 5'-gctaggatccttagcagccacctttcttttctataacaacctttgta-3'

[0039] Using SakSTAR as a template, -Gly-Gly-Cys was introduced into the C-terminus by PCR method to construct recombinant staphylokinase. The denaturation, annealing, and extension temperatures of a single cycle of PCR were 94°C, 55°C, and 72°C, respectively, and the reaction time for each temperature was 30 seconds. Perform 30 cycles to obtain PCR amplification products.

[0040] 1.2 Construction of recombinant staphylokinase expression vector

[0041] NcoI / BamHI double enzyme digestion reaction system was used to digest the gen...

Embodiment 2

[0049] Example 2 PEG site-directed modification and product identification of recombinant staphylokinase dimer

[0050] 1 Experimental method

[0051] 1.1 Dimerization reaction of staphylokinase with different ratios of 1,4-bismaleimidobutane

[0052] 1,4-Bismaleimidobutane (BMB) was dissolved in N,N-dimethylformamide to prepare a solution with a concentration of 4mM. The concentration of the purified staphylokinase is 0.2-0.3mM, and the buffer system used is 20mM phosphate buffer (pH 7.4). Staphylokinase and BMB were reacted overnight at 4°C at 1:0.5, 1:1, 1:2 and 1:5 (molar ratio), and the obtained products were identified by Superdex 200 gel filtration column.

[0053] 1.2 PEG-aldehyde (20K) site-directed modification of staphylokinase and its dimer (dSAK)

[0054] The reaction buffer system was 20 mM acetic acid-sodium acetate buffer (pH 5.0). SAK:PEG:NaCNBH 3 = 1:6:60, dSAK:PEG:NaCNBH 3 =1:8:80. The final reaction concentration of staphylokinase was 0.1 mM, and the...

Embodiment 3

[0064] In vitro activity assay of embodiment three PEG-modified staphylokinase dimer (PEG-dSAK)

[0065] 1 Experimental method

[0066] The in vitro activity of staphylokinase was accomplished by the lysosomal circle assay. The buffer used in the experiment was 50 mM Tris-hydrochloric acid buffer (pH 7.5) containing 0.15 M NaCl. Weigh 0.1 g of human fibrinogen, dissolve it in 10 ml of the above buffer solution, and place it in a thermostat at 37° C. for 1 hour to fully dissolve it. Weigh 0.15 g of agarose, dissolve it in 10 ml of the above buffer solution, dissolve it with a microwave, and store it at 55° C. for 20 minutes. Add 10 U of thrombin into the completely dissolved fibrinogen solution, mix well and pour into the agarose solution, mix well and pour the plate. Leave the plate for 10 minutes to fully solidify, and punch holes on the plate. Add an equal amount of staphylokinase sample (0.2 mg / ml×20 μl) to each well, invert at 37° C. for 20 hours, observe the fibrinoly...

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Abstract

The invention discloses a staphylokinase mutant, wherein cysteine is introduced to the C-end of the staphylokinase mutant. Compared with the wild type staphylokinase, the C-end of the mutant has three more amino acids, namely glycine-glycine-cysteine (Gly-Gly-Cys). The invention also discloses a preparation method of the dimer of the staphylokinase mutant. The dimerization method of the staphylokinase is that the two maleimide groups of the homotype bifunctional binder 1,4-dimaleimide butane and the cysteine at the C-end of the staphylokinase are utilized to form a stable covalent bond. The invention also discloses a preparation method for realizing the N-end site-specific modificaton of polyethylene glycol by using the dimer of the staphylokinase mutant. Under special conditions, methoxypolyethylene glycol-propionaldehyde is connected with one N-end of the staphylokinase dimer molecule through the covalent bond. The biological activity of the polyethylene glycol modified product of the staphylokinase dimer is higher than that of the modified product of the staphylokinase monomer.

Description

technical field [0001] The invention belongs to the field of protein chemistry, and relates to a preparation method of recombinant staphylokinase dimer and its N-terminal polyethylene glycol site-specific modification, which can prolong the circulation half-life of staphylokinase in vivo and improve the biological activity of staphylokinase. Background technique [0002] Staphylokinase (SAK) is a single-chain protein containing 136 amino acids with a relative molecular weight of 15.5kDa. As a new generation of thrombolytic drugs, its thrombolytic efficacy is comparable to that of the second generation of thrombolytic drugs tissue plasminogen activator (tissue plasminogen activator, t-PA), but the price is much lower than t-PA and its derivatives. At present, the research on recombinant staphylokinase at home and abroad has entered the phase III clinical trial stage. Staphylokinase, as an activator of plasminogen (Plg), can form a 1:1 complex with Plg. Under the action of tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/48
Inventor 胡涛苏志国马光辉刘如艳
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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