title: [Expression and affinity purification of recombinant human epidermal growth factor receptor-2 affibody with C-terminal cystein].
abstract: OBJECTIVE
  To prepare the modified ZHER2V2 affibody with amino-terminal HEHEHE sequence and carboxyl-terminal GGGC sequence by gene recombinant expression,which is the basis for invasive HER2 imaging with affibody.
  
  
  METHODS
  The encoded affibody gene was optimized by codon preference of E. coli with gene designer software. The N-terminal of affibody was fused with HEHEHE sequence,while the C-terminal was fused with GGGC sequence. The synthetic gene was confirmed by Hind 3 endonuclease restriction and gene sequencing. The human epidermal growth factor receptor-2(HER2)affibody gene was sub-cloned into pET22b(+)plasmid and transformed into competent BL21(DE3)bacteria. The expression of modified affibody was induced with isopropyl Β-D-1-thiogalactopyranoside(IPTG)and identified by SDS-PAGE. The affibody was purified by nickel affinity binding and imidazole elution. The purified affibody was labeled with (68)Ga and its affinity was determined by saturation analysis with HER2-positive cells MDA-MB-361.
  
  
  RESULTS
  The affibody gene containing N-terminal HEHEHE and C-terminal GGGC sequences were confirmed by Hind 3 endonuclease restriction and gene sequencing. A newly expressed 8×10(3) protein was expressed from the induced recombinant bacteria identified by SDS-PAGE after sub-cloning HER2 affibody gene into pET22b(+)plasmid,transforming recombinant plasmid into competent BL21(DE3)bacteria and inducing the recombinant bacteria with IPTG. The expressed protein was purified from nickel agarose by 60 mmol/L imidazole eluting. The affinity Kd value of (68)Ga labeled affibody to HER2 positive MDA-MB-361 cells was 1.5 nmol/L.
  
  
  CONCLUSIONS
  The affiibody ZHER2V2 containing N-terminal HEHEHE and C-terminal GGGC was successfully prepared by gene optimization,recombinant expression and affinity purification.
authors.affiliation: Department of Nuclear Medicine, PUMC Hospital, CAMS and PUMC, Beijing, China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Cai
authors.forename: Kun
authors.initials: K
authors.lastname: Zheng
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Dang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
mesh_terms: D000345:Affinity Labels / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
chemical_list: D000345:Affinity Labels; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C508053:ERBB2 protein, human