Purifying method for inhibiting degradation of mechanical sensing protein PDLIM3

Abstract

The invention provides a purification method for inhibiting the degradation of the mechanical sensing protein PDLIM3, first expressing the mechanical sensing protein PDLIM3; then breaking the bacteria, centrifuging to collect the supernatant; then gravity flow Ni gel affinity chromatography; The ultrafiltration membrane with a cut-off of 30kDa achieves the purpose of inhibiting the degradation of the mechanosensory protein PDLIM3, and finally obtains a high-concentration target protein solution. The invention reduces the environmental external force applied to the mechanical sensing protein PDLIM3 during the concentration process, that is, reduces the shearing force, thereby inhibiting the degradation of the mechanical sensing protein PDLIM3, increasing the concentration, and maintaining its activity. The invention greatly shortens the concentration time of the mechanosensory protein PDLIM3, remarkably improves its concentration efficiency, and reduces time and economic cost.

Description

technical field [0001] The invention belongs to the field of protein purification and crystallization, and relates to a method for inhibiting the degradation of mechanosensing protein PDLIM3 in the purification process. Background technique [0002] PDLIM3 protein is mainly expressed in cardiac and skeletal muscles, can bind to α-actinin, and enhance the cross-linking of α-actinin and actin fibers. PDLIM3 protein plays a key role in muscle mechanical perception and signal transduction. Mutations in the PDLIM3 protein can cause muscle-related diseases such as muscular dystrophy, cardiomyopathy, and myotonic dystrophy. [0003] Usually, protein molecules are unstable and easily affected by external environmental factors, which can easily lead to protein degradation or deformation during the purification and concentration process, and it is extremely difficult to obtain high-concentration proteins for subsequent crystallization. Document 1: "Purification, characterization, an...

AI Technical Summary

Problems solved by technology

Since the mechanical sensing protein PDLIM3 has a mechanical sensing function, it is more sensitive to force. The sample obtained with a 10kDa ultrafiltration membrane is commonly used in the purification and concentration process. Under the action of force, the degradation is serious, the activity is very low, and the purity is very low. Obviously, this conventional selection method cannot be applied to the purification method of the mechanosensing protein PDLIM3

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