Method and kit for purifying protein from fusion protein containing enzyme cleavage site

Abstract

The invention discloses a method for purifying proteins from fusion proteins containing enzyme cutting sites, comprising following steps: fixing on a first chromatographic column an enzyme that can specifically cut the enzyme cutting sites; serially connecting a second chromatographic column with the first chromatographic column which the enzyme is fixed to, this second chromatographic column being capable of adsorbing a non-target protein portion of a fusion protein that is cut, without adsorbing a target protein; enabling the fusion protein to flow sequentially through the first chromatographic column and the second chromatographic column. One-step chromatography is used in the method, omitting a purifying step and improving purification efficiency.

Description

technical field [0001] The present application relates to a method for purifying protein from a fusion protein, in particular to a method for enzymatic digestion and purification of protein that saves procedures and costs. Background technique [0002] Enzyme immobilization technology has a precedent in the field of biotechnology, such as biodiesel technology, food engineering, wastewater treatment, etc., but no relevant technical reports have been found in the field of pharmaceuticals. Fusion protein technology is a purposeful gene fusion to obtain a large number of standard proteins. Using fusion protein technology, new target proteins with multiple functions can be constructed and expressed. For example, some fusion proteins are fusion expressions of thioredoxin and target protein containing thrombin or enterokinase cleavage sites, and thrombin or enterokinase is used to cut thioredoxin and target protein in subsequent purification, and use The two proteins were separate...

AI Technical Summary

Problems solved by technology

[0003] The current enzymatic digestion technology usually adopts the static enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic cleavage, which is to mix the enzyme and the substrate protein thoroughly, and then cut the fusion protein into the target protein and the fusion head under suitable enzymatic cleavage conditions. The protein that can be touched around it will produce enzymatic cleavage, the enzymatic cleavage efficiency is low, and the amount of enzyme is large. In addition, the enzyme and protein are dissolved in a solvent at the same time, and there will be enzyme residue problems in the subsequent purification of the target protein.

Moreover, the digestion time is long, and the enzyme and protein are dissolved in a solvent at the same time, which not only affects the subsequent purification of the target protein, but also the residual enzyme will further digest the protein, making it difficult to control the end point of the digestion. increased variability

The digested protein usually needs to be purified by further chromatography. If the subsequent purification cannot effectively remove the enzyme, the residual enzyme will continue to function, thereby introducing additional impurities and cannot effectively purify the target protein.

The composition of the enzyme-cleaved protein solution is relatively complex. If the target protein is adsorbed on the chromatographic medium according to the conventional chromatography method, and then the target protein and the miscellaneous protein are separated according to their different physical properties such as surface charge and hydrophobicity, It is bound to go through multi-step chromatography. Each step of the chromatography column requires at least five solutions to complete the chromatography steps. The entire chromatography process is relatively complicated.

Each additional step of column chromatography will increase a lot of workload, and at the same time, the recovery rate of protein will also decrease. Therefore, it is the best state of purification process to achieve the same purification effect with as few chromatography steps as possible.

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