System and method for in vitro detection of activity of Ub (ubiquitin)-conjugating enzyme

Abstract

The invention discloses a system and a method for in vitro detection of activity of a Ub (ubiquitin)-conjugating enzyme. The system comprises a prokaryotic expression strain C1 competent cell capable of expressing E1 protein, a prokaryotic expression bacterium C2 competent cell capable of expressing the E1 protein and Ub protein simultaneously and a plasmid P3 having different replication origins from those of plasmids expressing the E1 protein and Ub protein and being capable of expressing to-be-detected E2 protein in vitro of protokaryon. P3 is transferred into the C1 competent cell and the C2 competent cell, named as C3CK (negative control) and C3 respectively, amplified and cultured respectively, the bacteria are collected after induction of protein expression, and protein western blot is performed; a migration stripe of the to-be-detected E2 protein is detected in a C3 sample, and the migration size conforms to the Ub molecular weight, therefore, the condition that the to-be-detected E2 protein has activity of the Ub-conjugating enzyme is proved. According to the method, neither lysis of cells for protein extraction and purification nor in vitro response is required, the method is quite convenient, and results are more stable.

Description

technical field [0001] The invention relates to a stable and rapid in vitro detection system and method for ubiquitin-conjugating enzyme activity without lysing cells to purify proteins, and belongs to the field of biotechnology. Background technique [0002] The ubiquitin (Ub)-mediated 26S proteasome system provides sufficient amino acid supply for organisms by participating in the degradation of abnormal proteins in growth and development, and precisely regulates the physiological processes of organisms by degrading some rate-limiting steps of proteins, providing It is very important for organisms to maintain normal physiological functions to facilitate the growth and development of organisms and to respond to changes in the external environment. The main molecules involved in the ubiquitin-mediated 26S proteasome degradation pathway include ubiquitin, ubiquitin activating enzyme (Ub-activating enzyme) E1, ubiquitin-conjugating enzyme (Ub-conjugating enzyme) E2, ubiquitin ...

AI Technical Summary

Problems solved by technology

The current ubiquitin-conjugating enzyme in vitro ubiquitination reaction system (such as patent 201310007041.9, invention name: in vitro plant ubiquitin protein degradation system and its application) has the following disadvantages: the main molecules required for ubiquitin-conjugating enzyme in vitro ubiquitination detection include Ub, E1, and E2 proteins all need to be expressed in Escherichia coli in vitro, and then the cells are lysed to extract the protein, and some of them need to be purified. The purified protein needs to be stored in a -80°C refrigerator to maintain its activity

The reaction buffer must contain ATP to provide energy. The chemical properties of ATP are unstable, and it is difficult for the reaction buffer to maintain activity for a long time; the amount of various proteins added to the system and the reaction time and conditions are also different, and each needs to explore the conditions and workload. huge

If the activity of ubiquitin-conjugating enzymes cannot be detected later, it is impossible to distinguish whether it is due to problems in protein expression, purification, storage, freeze-thawing, and reaction steps, or the protein itself does not have the function of ubiquitin-conjugating enzymes, which may easily cause false negatives

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