Large-scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin.
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.
McDonald
J R
JR
PRIZM Pharmaceuticals, 11035 Roselle Street, San Diego, California, 92121, USA.
Ong
M
M
Shen
C
C
Parandoosh
Z
Z
Sosnowski
B
B
Bussell
S
S
Houston
L L
LL
D001667:Binding, Competitive; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents / Q000378:metabolism; D003603:Cytotoxins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics; D000922:Immunotoxins; D013058:Mass Spectrometry; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects; D000076985:Saporins
D004396:Coloring Agents; D003603:Cytotoxins; D000922:Immunotoxins; D010940:Plant Proteins; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C078442:basic fibroblast growth factor-saporin mitotoxin, recombinant; D016222:Fibroblast Growth Factor 2; D009699:N-Glycosyl Hydrolases; D000076985:Saporins