title: Inducible expression of calreticulin-N58 in Pichia pastoris by high density cell culture.
abstract: Calreticulin-N58 (CRT-N58), an active fragment of calreticulin with anti-angiogenesis activity, was expressed in P. pastoris by high density cell culture. Calreticulin-N58 DNA was synthesized by PCR and cloned to plasmid pPIC9 K resulting in the plasmid pPIC9 K-crt-N58 which was then transformed into P. pastoris GS115. The fermentation was carried out in a 50 l bioreactor with 20 l modified growth medium recommended by Invitrogen at 30°C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density was grown to A(600) = 135, methanol-PTM4 trace salts was added to induce the expression of calreticulin-N58. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by adding 7 M NH(4)OH. After 52 h of induction, the yield of secreted calreticulin-N58 was 70 mg/l and biomass growth was 293 as measured by absorption of 600 nm. The secreted calreticulin-N58 was purified to a purity of 100% by the use of SP-Sepharose FF ion-exchange chromatography (Pharmacia Biotech. NJ, USA) and desalted with ultrafiltration device (Millipore, Bedford, MA, USA). The recombinant calreticulin-N58 induced endothelial cell apoptosis and inhibited the angiogenesis on the CAM.
authors.affiliation: Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101 Hainan, China.
authors.forename: D X
authors.initials: DX
authors.lastname: Su
authors.forename: A L
authors.initials: AL
authors.lastname: Zhang
authors.forename: G H
authors.initials: GH
authors.lastname: Yi
authors.forename: Z W
authors.initials: ZW
authors.lastname: Liu
authors.forename: J X
authors.initials: JX
authors.lastname: Luo
authors.forename: L Y
authors.initials: LY
authors.lastname: Rao
authors.forename: T Y
authors.initials: TY
authors.lastname: Zhang
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Zhou
mesh_terms: D000818:Animals; D018533:Biomass; D037282:Calreticulin / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D037282:Calreticulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2