title: [The expression and purification of TRPM2 protein in E.coli and preparation of its polyclonal antibody].
abstract: OBJECTIVE
  To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific polyclonal antibody against human transient receptor potential melastatin 2(TRPM2) protein.
  
  
  METHODS
  The DNA fragment encoding evolutionarily conserved N-terminus (1-334 amino acids) of TRPM2 was amplified by PCR. The amplicon was then subcloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL21 (DE3) cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with 1 mmol/L IPTG and purified by GST affinity chromatography. The purified protein was mixed with complete Freund's adjuvant and used to immunize the New Zealand white rabbits with classical 4-injection protocol to generate specific anti-TRPM2 polyclonal antibody. The specificity and titer of the anti-TRPM2 antibody was analyzed by Western blotting.
  
  
  RESULTS
  The cDNA fragment encoding N-terminus of human TRPM2 was amplified by PCR and directionally subcloned into pGEX-4T3 plasmid. Under the induction of IPTG, we observed the expression of GST-TRPM2N fusion protein. Polyclonal antibody against human TRPM2 protein was successfully prepared in the rabbits immunized with the purified GST-TRPM2N fusion protein. And the preliminary analysis showed that the anti-TRPM2 antibody could specifically identify transiently expressed TRPM2-EE in HEK293 cells.
  
  
  CONCLUSIONS
  The specific polyclonal antibody against human TRPM2 protein has been successfully prepared, which facilitates our future research on the function of TRPM2 channel.
authors: Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, China. shw1552128@163.com
authors: Hongwei
authors: H
authors: Sun
authors: Haihua
authors: H
authors: Luo
authors: Ni
authors: N
authors: Wang
authors: Xiaoli
authors: X
authors: Ou
authors: Xiaoli
authors: X
authors: Wen
authors: Yong
authors: Y
authors: Jiang
authors: Zhuzhong
authors: Z
authors: Mei
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D051152:Clusterin / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins
chemical_list: D000906:Antibodies; D051152:Clusterin; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins