Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin

Citation
L. D'Alatri et al., Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin, ANTICANC R, 18(5A), 1998, pp. 3369-3373
Citations number
19
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Onconogenesis & Cancer Research
Journal title
ANTICANCER RESEARCH
ISSN journal
0250-7005 → ACNP
Volume
18
Issue
5A
Year of publication
1998
Pages
3369 - 3373
Database
ISI
SICI code
0250-7005(199809/10)18:5A<3369:PACOAR>2.0.ZU;2-P
Abstract
We generated a recombinant immunotoxin, named scFv(MGR6)-Cla, composed of t he Fv region of an anti ErbB2 monoclonal antibody (MGR6) fused to clavin, a type I ribosome-inactivating protein (RIP) from Aspergillus clavatus. ErbB 2 is a tyrosine kinase receptor which is overexpressed in most adenocarcino mas; clavin is a 17 kDa ribonuclease which inhibits protein synthesis by in activating ribosomes. A recombinant DNA construct containing the cDNA of th e single chain Fv fragment (scFv) of the MGR6 antibody fused to the clavin cDNA, was expressed at high levels in Escherichia coli as an insoluble fusi on protein containing an N-terminal affinity tag of six consecutive histidi ne residues. Inclusion bodies were denatured and the recombinant fusion pro tein was purified under denaturing conditions by single-step purification u sing immobilised metal ion affinity chromatography (IMAC). The purified imm unotoxin was renatured at high yield and histidine tag removed by digestion with enterokinase. The purity of the immunotoxin obtained after refolding was confirmed by SDS-PAGE, PP-HPLC, GPC-HPLC and N-terminal sequence analys is. Cell-free protein synthesis inhibition and binding assays showed that b oth clavin and scFv(MGR6) maintained their properties after refolding.