Large-scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin.

Abstract:

: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.

journal_name

Protein Expr Purif

authors

McDonald JR,Ong M,Shen C,Parandoosh Z,Sosnowski B,Bussell S,Houston LL

doi

10.1006/prep.1996.0079

subject

Has Abstract

pub_date

1996-08-01 00:00:00

pages

97-108

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(96)90079-0

journal_volume

8

pub_type

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