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Soluble expression and purification method for recombinant murine leukemia virus reverse transcriptase (MMLV-RT) in colon bacillus

A murine leukemia virus, MMLV-RT technology, applied in the field of biochemistry, can solve the problems of easy formation of inclusion bodies, low yield, complex natural reverse transcriptase extraction technology, etc., and achieve the effect of increased solubility and simplified purification

Inactive Publication Date: 2010-03-31
孙启明 +1
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Problems solved by technology

As the most commonly used molecular biology product, reverse transcriptase (reverse transcriptase) has a huge market demand, and the extraction technology of natural reverse transcriptase is complicated and the yield is low
At present, the recombinant murine leukemia virus reverse transcriptase cloned by genetic engineering technology has a high expression level in the pET system, but it is easy to form inclusion bodies, and only a small amount of active protein can be obtained after purification

Method used

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  • Soluble expression and purification method for recombinant murine leukemia virus reverse transcriptase (MMLV-RT) in colon bacillus
  • Soluble expression and purification method for recombinant murine leukemia virus reverse transcriptase (MMLV-RT) in colon bacillus
  • Soluble expression and purification method for recombinant murine leukemia virus reverse transcriptase (MMLV-RT) in colon bacillus

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Embodiment Construction

[0037] In the present invention, through the construction of the recombinant MMLV-RT prokaryotic expression vector pET28a-MMLV-RT, the codon-optimized MMLV-RT gene is successfully cloned to the downstream of the His6-tag coding sequence and is in the same reading frame as the His6-tag and The prokaryotic expression vector of fusion protein MMLV-RT was constructed by adding enterokinase restriction sites between MMLV-RT, and the sequencing results showed that the reading frame and DNA sequence were all correct. Through comparative experiments, it was found that under low temperature conditions MMLV-RT was co-expressed with molecular chaperone TF, the protein expression was increased to 15%, and the solubility was 10 times that of the conventional method. MMLV-RT without any affinity tag was obtained through nickel affinity chromatography, EK digestion and ion exchange chromatography. Nickel-affinity chromatography removed most of the bacterial protein impurities, and the MMLV-R...

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Abstract

The invention relates to an expression and purification of murine leukemia virus reverse transcriptase (MMLV-RT), in particular to a soluble expression and a purification method for recombinant murineleukemia virus reverse transcriptase (MMLV-RT), which belongs to the field of biochemistry. MMLV-RT genes and enterokinase restriction enzyme cutting sites are cloned in a pET28a vector through vector construction; the MMLV-RT and molecular chaperones are co-expressed at the low temperature; the expression amount of protein is improved to 15 percent; and the solubility of the protein is 10 timesthat of the protein expressed by a conventional method.

Description

technical field [0001] The invention relates to the expression and purification of murine leukemia virus reverse transcriptase (MMLV-RT), in particular to a soluble expression and purification method of murine leukemia virus reverse transcriptase (MMLV-RT). Belongs to the field of biochemistry. Background technique [0002] As an important molecular biology research method, RT-PCR is a technology that combines reverse transcription (RT) of RNA and polymerase chain amplification (PCR) of cDNA. First, cDNA is synthesized from RNA by the action of reverse transcriptase, and then cDNA is used as a template to amplify and synthesize the target fragment. RT-PCR technology is sensitive and versatile, and can be used to detect the level of gene expression in cells, the content of RNA viruses in cells and directly clone the cDNA sequence of a specific gene. The process from mRNA to cDNA is called reverse transcription, which is catalyzed by reverse transcriptase. Reverse transcrip...

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Application Information

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IPC IPC(8): C12N15/70C12N9/00C12R1/19C12R1/93
Inventor 孙启明徐卫国陈宇
Owner 孙启明
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