Johnson Scott K, Zhang Wenhui, Smith Leonard A, Hywood-Potter Karen J, Todd Swanson S, Schlegel Vicki L, Meagher Michael M
Biological Process and Development Facility, University of Nebraska-Lincoln, Lincoln, NE 68583-0919, USA.
Protein Expr Purif. 2003 Nov;32(1):1-9. doi: 10.1016/j.pep.2003.07.003.
A recombinant heavy chain fragment C of botulinum neurotoxin serotype F (BoNTF(Hc)) has been expressed in Pichia pastoris for use as an antigen in a proposed human vaccine. P. pastoris cells were grown using glycerol batch, glycerol fed-batch, and methanol fed-batch methods to achieve high cell densities. The total cellular protein recovered after homogenization was 72 mg/g of cell paste. BoNTF(Hc) was purified from soluble Pichia cell lysate employing ion-exchange chromatographic (IEC) and hydrophobic interaction chromatographic (HIC) methods developed at the bench scale using 10-100 mL columns. The process was performed at the pilot scale using 1-4L columns for evaluation of scale up. The purification process resulted in greater than 98% pure product consisting of at least three forms of BoNTF(Hc) based on mass spectrometry and yielded up to 205 mg/kg cells at the bench scale and 170 mg/kg cells at the pilot scale. Full-length BoNTF(Hc) is present based on mass spectrometry and SDS-PAGE, however is postulated to be N-terminally blocked by acetylation. N-terminal sequencing showed that two of the three forms are missing the first 11 (80%) and 14 (20%) amino acids of the N-terminus from the full-length form. The ratios of the two clipped forms were consistent from the bench to pilot scales. Purified BoNTF(Hc) at the pilot scale was found to sufficiently protect mice against a high dose of BoNTF neurotoxin.
肉毒杆菌神经毒素F型重组重链片段C（BoNTF(Hc)）已在毕赤酵母中表达，用作一种拟用人类疫苗的抗原。采用甘油分批培养、甘油补料分批培养和甲醇补料分批培养方法培养毕赤酵母细胞，以实现高细胞密度。匀浆后回收的总细胞蛋白为72 mg/g细胞糊。采用在实验室规模使用10 - 100 mL柱开发的离子交换色谱（IEC）和疏水相互作用色谱（HIC）方法，从可溶性毕赤酵母细胞裂解物中纯化BoNTF(Hc)。该工艺在中试规模下使用1 - 4L柱进行，以评估放大效果。纯化过程得到了纯度大于98%的产物，基于质谱分析，该产物至少由三种形式的BoNTF(Hc)组成，在实验室规模下产量高达205 mg/kg细胞，在中试规模下产量为170 mg/kg细胞。基于质谱分析和SDS - PAGE表明存在全长BoNTF(Hc)，但推测其N端被乙酰化封闭。N端测序表明，三种形式中的两种缺失了全长形式N端的前11个（80%）和14个（20%）氨基酸。两种截短形式的比例从实验室规模到中试规模是一致的。发现在中试规模下纯化的BoNTF(Hc)能充分保护小鼠免受高剂量的BoNTF神经毒素侵害。
