CYP101 enzyme recombinant vector, construction method thereof and CYP101 enzyme high-efficiency expression and purification method

Abstract

The invention relates to a CYP101 enzyme recombinant vector, a construction method thereof and a CYP101 enzyme high-efficiency expression and purification method, and belongs to the field of biotechnology. A PET28a-CYP101 high-efficiency expression vector is constructed and transformed into BL21 plysS strain, and high-purity enzyme with purity higher than 95% is obtained by a simple two-step purification method including affinity chromatography and ion exchange chromatography after inducible expression. Compared with the prior art, CYP101 is simply and efficiently produced, the cost is low, purification is facilitated, and 23mg of target protein with the purity higher than 95% can be obtained by per liter of culture media. The CYP101 enzyme high-efficiency expression and purification method can be used for expressing and purifying CYP101 enzyme, the production efficiency of a laboratory is greatly improved, and experimental cost is greatly reduced. The CYP101 enzyme high-efficiency expression and purification method has an excellent application prospect for large-scale CYP101 enzyme production and purification.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a CYP101 enzyme recombination vector and a construction method, and a CYP101 enzyme high-efficiency expression and purification method. Background technique [0002] Cytochrome P450 (cytochrome P450, referred to as CYP450) is a superfamily of heme-thiolate proteins, which widely exist in various organisms in nature. Metabolism of xenobiotics. Its participation process can be roughly divided into three aspects: the synthesis of hormone substances in the body, the metabolism of exogenous substances and the decomposition of fatty acids. They were first discovered by Garfinkel and Klingenberg from mammalian liver microsomes in 1958. They were named because they can bind CO in a reduced state and detect an obvious absorption peak at a wavelength of 450nm after binding. Cytochrome P450 enzymes. Molecular biology techniques have been applied to the study of cytochrome P450, and the use o...

AI Technical Summary

Problems solved by technology

[0004] In traditional purification methods, in order to obtain high-purity enzymes, more than three purification methods are often used. The purification time is long, resulting in greatly reduced enzyme activity, and multi-step purification also leads to low recovery efficiency.

Fewer purification methods will lead to relatively low purity of the purified target protein, which cannot meet the ideal requirements of technicians

According to previous literature reports, in the construction of vectors for CYP101, in order to facilitate cloning and purification, longer purification tags, such as GST, MBP, Halo and other fusion expression tags, containing tens to hundreds of amino acid residues were used; or using Short purification tags, such as 6*His (6 amino acid residues), flag (8 amino acid residues), avidin (15 amino acid residues), and a longer linker peptide is added between the short tag and the target protein Due to the introduction of these short tags, the final target protein has at least 10 more amino acids than the natural protein, which is different from the natural structure and may affect the properties or functions of the target protein

[0005] In the purification process of CYP101 enzyme, a tag is introduced for the convenience of purification. Although protease can be used to remove the tag after purification to ensure the similarity with the natural protein, the enzyme digestion operation is cumbersome and costly, and it is often difficult to use in a large number of purified samples.

At the same time, the enzyme digestion step increases the purification time, making it difficult to guarantee the activity of the enzyme

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