title: Expression and identification of type 1 diabetes associated autoantigen IA-2.
abstract: OBJECTIVE
  To obtain prokaryotic expressed IA-2 recombinant protein and to identify its immunological activity.
  
  
  METHODS
  The complimentary DNA (cDNA) coding for the intracytoplasmic part of IA-2 (IA-2ic) was amplified from human fetal brain RNA, and was subcloned into the PinPoint Xa-1 T vector to construct recombinant expression plasmid, and was then expressed in E. coli JM109 cells as a fusion protein with a biotinylated peptide sequence at the aminoterminus. The biotinylated fusion protein was then purified by affinity chromatography and was subsequently dialyzed. Finally, its immunogenicity was evaluated by enzyme linked immunosorbent assay (ELISA).
  
  
  RESULTS
  The purified IA-2ic fusion protein resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single Coomassie brilliant blue stained band with a molecular weight of 59 kDa and its immunogenicity was confirmed by ELISA.
  
  
  CONCLUSIONS
  E. coli expressed IA-2ic fusion protein has immunological activity. It can be used for detection of IA-2 autoantibodies (IA-2A) and for further studies on type 1 diabetes in future.
authors.affiliation: Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China. xiujuanjia@yahoo.com.cn
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Jia
authors.forename: Guo
authors.initials: G
authors.lastname: Li
authors.forename: Zhan
authors.initials: Z
authors.lastname: Chen
authors.forename: Guangwu
authors.initials: G
authors.lastname: Xu
authors.forename: Chao
authors.initials: C
authors.lastname: Xie
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.forename: Wenzhong
authors.initials: W
authors.lastname: Zhou
authors.forename: Sheng
authors.initials: S
authors.lastname: Zheng
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Xie
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.forename: Jiping
authors.initials: J
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Luo
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000032:analysis; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D001324:Autoantigens; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8