Related Experiment Video
Updated: Apr 17, 2026
GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
Published on: October 29, 2013
[The expression, purification and characterization of human His-ATG4B protein]
Rong Huang1, Xiaojie Xu2, Yingchun Liang2
1Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850; First Affiliated Hospital of China PLA General Hospital, Beijing 100048, China.
Objective:
To construct the prokaryotic expression vector of human autophagy-related protein 4 homolog B (ATG4B) labeled with His-tag, obtain the purified His-ATG4B protein, and identify its activity preliminarily.
Methods:
ATG4B coding region was amplified from human mammary gland cDNA library by PCR, and was cloned into the prokaryotic expression vector pET-28a⁺. After verification by enzyme digestion, the correct recombinant plasmid His-ATG4B was introduced into E.coli Rossate. The expressed recombinant plasmid was purified by Ni-NTA beads and identified by SDS-PAGE and Western blot analysis. The function of the purified protein His-ATG4B was detected by GST pull-down assay.
Results:
The DNA fragment of about 1100 bp was successfully amplified from human mammary gland cDNA by PCR, and inserted into pET-28a vector correctly. The results of double digestion and sequencing suggested that the His-ATG4B recombinant plasmid was successfully obtained. His-ATG4B protein of about Mr 47 000 was induced and identified by SDS-PAGE analysis. GST pull-down assay showed that His-ATG4B could interact with LC3B in vitro, suggesting that it has a good biological function.
Conclusion:
The prokaryotic expression protein of His-ATG4B has been obtained successfully, which lays a foundation for further research on the function of ATG4B in autophagy.
