کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2022183 1069283 2006 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115
چکیده انگلیسی

A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7–380 mL columns and the process was performed at the pilot scale using 0.5–3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01 g of rBoNTE(Hc)/kg cells at the bench scale and 580 mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50’s of BoNT/E.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 45, Issue 2, February 2006, Pages 359–367
نویسندگان
, , , , , , , , , , , , , ,