title: [Expression of recombinant human ZP3 protein using the baculovirus expression system].
abstract: OBJECTIVE
  To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.
  
  
  METHODS
  The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.
  
  
  RESULTS
  The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.
  
  
  CONCLUSIONS
  It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.
authors.affiliation: Department of Urology, Shanghai Jiatong University, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Hua
authors.initials: H
authors.lastname: Diao
authors.forename: Yu-fang
authors.initials: YF
authors.lastname: Xiao
authors.forename: He-guo
authors.initials: HG
authors.lastname: Yu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
authors.forename: Hui-juan
authors.initials: HJ
authors.lastname: Shi
mesh_terms: D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D000072216:Zona Pellucida Glycoproteins
chemical_list: D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins