title: [The expression, purification and characterization of human His-ATG4B protein].
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850; First Affiliated Hospital of China PLA General Hospital, Beijing 100048, China.
authors: Rong
authors: R
authors: Huang
authors: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
authors: Xiaojie
authors: X
authors: Xu
authors: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
authors: Yingchun
authors: Y
authors: Liang
authors: Health Division of Guard Bureau, Headquarters of General Staff of PLA, Beijing 100017, China.
authors: Li
authors: L
authors: Zhang
authors: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
authors: Tao
authors: T
authors: Wang
authors: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
authors: Liying
authors: L
authors: Zhou
authors: First Affiliated Hospital of China PLA General Hospital, Beijing 100048, China.
authors: Nan
authors: N
authors: DU
authors: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
authors: Qinong
authors: Q
authors: Ye
mesh_terms: D000071183:Autophagy-Related Proteins; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000071183:Autophagy-Related Proteins; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases