Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin.
We generated a recombinant immunotoxin, named scFv(MGR6)-Cla, composed of the Fv region of an anti ErbB2 monoclonal antibody (MGR6) fused to clavin, a type 1 ribosome-inactivating protein (RIP) from Aspergillus clavatus. ErbB2 is a tyrosine kinase receptor which is overexpressed in most adenocarcinomas; clavin is a 17 kDa ribonuclease which inhibits protein synthesis by inactivating ribosomes. A recombinant DNA construct containing the cDNA of the 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 fusion protein containing an N-terminal affinity tag of six consecutive histidine residues. Inclusion bodies were denatured and the recombinant fusion protein was purified under denaturing conditions by single-step purification using immobilised metal ion affinity chromatography (IMAC). The purified immunotoxin 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, RP-HPLC, GPC-HPLC and N-terminal sequence analysis. Cell-free protein synthesis inhibition and binding assays showed that both clavin and scFv(MGR6) maintained their properties after refolding.
D'Alatri
L
L
Menarini Ricerche S.p.A., Department of Biotechnology, Pomezia, Rome, Italy.
Di Massimo
A M
AM
Anastasi
A M
AM
Pacilli
A
A
Novelli
S
S
Saccinto
M P
MP
De Santis
R
R
Mele
A
A
Parente
D
D
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D005822:Genetic Vectors / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D000922:Immunotoxins / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D011500:Protein Synthesis Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012260:Ribonucleases
D000911:Antibodies, Monoclonal; D005656:Fungal Proteins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011500:Protein Synthesis Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012260:Ribonucleases; C100767:CLA protein, Aspergillus clavatus