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Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof

A polynucleotide kinase and phage technology, applied in the field of biochemistry, can solve the problems of a small amount of active protein, easy formation of inclusion bodies, large market demand for T4 bacteriophage polynucleotide kinase, etc., and achieve the effect of simplified purification and increased solubility

Inactive Publication Date: 2014-12-03
孙启明
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Problems solved by technology

As a commonly used molecular biology product, T4 phage polynucleotide kinase is in great demand in the market. At present, the recombinant T4 phage polynucleotide kinase cloned by genetic engineering technology has a high expression level in the pET system, but it is easy to form inclusion bodies, and it can only be purified after purification. active protein

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  • Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof
  • Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof
  • Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof

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Embodiment Construction

[0025] Through the construction of recombinant T4 polynucleotide kinase prokaryotic expression vector pGEX-T4 PNK, the present invention successfully clones the codon-optimized T4 polynucleotide kinase gene to the downstream of the GST tag coding sequence and is in the same reading frame as it, and constructs a fusion The prokaryotic expression vector of protein GST-T4 PNK, the sequencing results show that the reading frame and DNA sequence are all correct. Through comparative experiments, it was found that the expression of pGEX-T4 polynucleotide kinase protein reached 15% and the solubility reached 95% under low temperature induction conditions. GST affinity chromatography removed most of the foreign proteins of the thalline, ion exchange further removed foreign proteins and trace DNA, and about 7 mg of T4 polynucleotide kinase (purity about 95%) was obtained in one liter of fermentation broth. 12% SDS-PAGE analysis showed that the molecular weight of the purified T4 polynuc...

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Abstract

The invention relates to expression and purification of T4 bacteriophage polynucleotide kinase (T4 PNK) and particularly relates to soluble expression and a purification method of the T4 PNK, belonging to the field of biochemistry. According to the invention, a T4 PNK gene is cloned into a pGEX vector through vector construction, the T4 polynucleotide kinase and GST undergo fused expression under the condition of low temperature, then, the protein expression level reaches 15%, and the solubility reaches 95%.

Description

technical field [0001] The present invention relates to the expression and purification of T4 bacteriophage polynucleotide kinase (T4 PNK), in particular to a soluble expression and purification method of T4 bacteriophage polynucleotide kinase (T4 PNK). Belongs to the field of biochemistry. Background technique [0002] T4 bacteriophage polynucleotide kinase (T4 bacteriophage polynucleotide kinase, T4 PNK), is a polynucleotide 5'hydroxyl kinase that can catalyze ATP? Transfer of a phosphate group to the 5' hydroxyl of single- or double-stranded DNA, RNA, oligonucleotides, or mononucleotides with a 3' phosphate group. Other NTPs can also produce the same reaction: 5'-OH+NTP? 5'-P+NDP. T4Polynucleotide Kinase also has 3'phosphatase activity, which can catalyze the dephosphorylation of 3'phosphorylated polynucleotides: 3'-P? 3'-OH+Pi. T4 phage polynucleotide kinase is widely used, such as oligonucleotide, DNA or RNA 5' end labeling, used as probes for Southern, Northern, E...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70
CPCC12N9/1252C12Y207/07007
Inventor 孙启明张亚徐卫国陈远
Owner 孙启明
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