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Expression, purification and crystallization of urokinase catalyst structure domain mutant

A technology of catalytic domains and mutants, applied in the fields of structural biology, biotechnology, and medicine, can solve the problems of unsuitable anti-tumor metastasis drugs, unclear interaction mechanism between urokinase and inhibitors, and achieve low cost, High stability and high expression effect

Inactive Publication Date: 2007-08-29
FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some achievements have been made in the study of urokinase inhibitors, the mechanism of the interaction between urokinase and inhibitors is still unclear
In addition, an ideal urokinase inhibitor as an anti-metastatic drug should not affect the activities of t-PA and plasmin when inhibiting urokinase, while natural urokinase inhibitors such as PAI-1 and PAI-2, etc. Kinase also strongly inhibits the activity of t-PA, so it is not suitable as an anti-metastatic drug

Method used

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  • Expression, purification and crystallization of urokinase catalyst structure domain mutant
  • Expression, purification and crystallization of urokinase catalyst structure domain mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1 Amplification of expression target gene

[0012] 1. The applicant designed and synthesized the following PCR primers by the primer synthesis company:

[0013] ①Starting primer (forward):

[0014] 5'-CCG CTCGAG AAAAGAATTATTGGGGGAGAATTCAC-3';

[0015] ②End primer (reverse): 5'-CCG CTCGAG TTATTCCTTGGTGTGACTGC-3';

[0016] ③ Mutation point (C279A) primer (forward): 5'-GACCATC GCC CTGCCCTC-3';

[0017] ④ Mutation point (N302Q) primer (reverse): 5'-GTCGGTAGA TTG CTCTTTTTCCAA-3';

[0018] Note: The underline in the ①② primer indicates the restriction endonuclease Xho I digestion site; ③④ The underline in the primer indicates the site of the mutated amino acid.

[0019] 2. Amplification and site-directed mutation of the catalytic domain of urokinase (C279A / N302Q) by overlap extension PCR (Fig.

[0020] 1): Using the full-length gene of urokinase as a template and using ③ and ④ as primers, perform the first round of PCR. The PCR conditions are: 94°C, 2 minut...

Embodiment 2

[0021] Example 2 Cloning of the target gene

[0022] Cloning of mutants in the catalytic domain of urokinase: the product was recovered by PCR, digested overnight with restriction endonuclease Xho I, and recovered by ethanol precipitation; similarly digested with restriction endonuclease Xho I to linearize Pichia pastoris expression vector pPICZαA, and remove Phosphorylated, gel recovery kit to recover linearized products. Mix the product recovered by PCR with the dephosphorylated plasmid pPICZαA, inactivate it with T4 DNA ligase, overnight at 16°C, and inactivate at 65°C for 5 minutes; take 1.5 μl of the linker and mix it with the pre-cooled competent cell TOP10F', discharge at 2.5kV, and electric shock , add 1 ml of SOC solution, incubate at 37°C for 1 hour, take 200 μl coated LB plate (25 μg / ml Zeocin), incubate at 37°C for 14 hours, select colonies, culture, small amount of recombinant plasmid extraction, enzyme digestion identification, enzyme Correctly identified clones...

Embodiment 3

[0023] Example 3 Expression and purification of mutant proteins

[0024] Expression and purification of recombinant plasmids in Pichia pastoris X-33: linearized recombinant plasmids were electrotransformed into yeast X-33 strains, coated with 100 μg / ml Zeocin YPDS plates, colonies were picked, genomes were extracted, and identified by PCR. The correct recombinant expression was induced by adding methanol at a final concentration of 0.5% every day, and the culture fluid was fermented for 4 days, and the supernatant was obtained by centrifugation, and the molecular weight was checked by 12% SDS-PAGE electrophoresis. Strains with high expression were expanded and cultured, induced by methanol for 4 days, and the supernatant was diluted to 1-fold volume and passed through a cation rapid exchange column SPFF pre-equilibrated with 20mM pH6.5 potassium phosphate buffer solution, 20mM pH6.5 potassium phosphate buffer solution (containing 0.5M chloride Sodium chloride) was eluted, and ...

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Abstract

The invneiton relates to expression, purification and crystallization of urokinase catalytic domain mutant, relating to the field of medicine, biotechnique, and structural biology. And the invention provides a techical method of expression, purification and crystallization of urokinase catalytic domain mutant in Pichia pastoris. And the cultured urokinase catalytic domain mutant has crystallographic space group R3, and unit cell parameters: a=b=120.48, c=42.45, alpha=beta=90.0deg, and gama=120.0 deg. And this protein has serine proteinase activity, appliable to high flux screening of urokinase inhibitor; and its high resolution crystal provides a research and development platform for design and development of urokinase inhibitor.

Description

Technical field: [0001] The invention relates to the fields of medicine, biotechnology and structural biology. Background technique: [0002] Urokinase is a serine protease that plays an important regulatory role in a series of physiological and pathological processes, such as: activation of plasminogen, migration of inflammatory cells, healing and remodeling of wounds, growth of trophoblast cells, etc. In recent years, research on the physiological activity of urokinase has mainly focused on cancer metastasis. Studies have shown that most cancer cells related to the epithelium can produce urokinase and transport it to the cell surface, thereby triggering a series of proteolytic reactions, resulting in the invasion and spread of cancer cells. The use of urokinase inhibitors to inhibit the activity of urokinase has been It is recognized as an effective method to inhibit the invasion and metastasis of tumor cells, and then achieve the purpose of controlling and treating tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72C12N15/09C12N15/58C12Q1/34
Inventor 黄明东赵更香袁彩
Owner FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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