McDonald J R, Ong M, Shen C, Parandoosh Z, Sosnowski B, Bussell S, Houston L L
PRIZM Pharmaceuticals, 11035 Roselle Street, San Diego, California, 92121, USA.
Protein Expr Purif. 1996 Aug;8(1):97-108. doi: 10.1006/prep.1996.0079.
In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rFGF-2-SAP was cloned into the inducible pET 11a expression vector and transformed into Escherichia coli strain BL21 (DE3). The transformants were grown using a fed-batch fermentation until the A600 reached 85. At this stage, induction of the expression of the fusion protein led to the production of approximately 2.2 mg/liter per A600 unit. The soluble mitotoxin was purified to homogeneity from cell lysates via expanded bed adsorption chromatography followed by cation-exchange, heparin-affinity, and size-exclusion chromatography. Purified rFGF-2-SAP contained less than 0.5 EU/mg of endotoxin, as determined by gel clot analyses. The highly purified rFGF-2-SAP retained the toxin's ability to inhibit protein synthesis as measured in a cell-free system and was cytotoxic to a number of normal and neoplastic cell lines bearing FGF receptors. Binding studies establish that the fusion protein exerts its effects via the FGF high-affinity receptor.
为了生产足够数量的成纤维细胞生长因子-皂草毒素（rFGF-2-SAP）促细胞分裂毒素，用于在癌症和再狭窄等疾病模型中进行临床前评估，我们对该重组分子进行了大规模表达、纯化和特性鉴定。将编码rFGF-2-SAP的融合基因克隆到可诱导的pET 11a表达载体中，并转化到大肠杆菌BL21（DE3）菌株中。使用补料分批发酵培养转化体，直到A600达到85。在此阶段，诱导融合蛋白表达导致每A600单位产生约2.2毫克/升的产量。通过扩张床吸附色谱，随后进行阳离子交换、肝素亲和和尺寸排阻色谱，从细胞裂解物中纯化可溶性促细胞分裂毒素至同质。通过凝胶凝块分析测定，纯化的rFGF-2-SAP的内毒素含量低于0.5 EU/毫克。在无细胞系统中测量，高度纯化的rFGF-2-SAP保留了毒素抑制蛋白质合成的能力，并且对许多带有FGF受体的正常和肿瘤细胞系具有细胞毒性。结合研究表明，融合蛋白通过FGF高亲和力受体发挥其作用。
