title: Production and characterization of scFvA33T1, an immunoRNase targeting colon cancer cells.
abstract: Within the last 10 years, the use of different RNases as therapeutic agents for various diseases has been pursued. Furthermore, the advancements of recombinant technology have allowed the assembly of proteins with different functions. In this regard, immunoribonucleases (immunoRNases) stand out as some of the most promising therapeutic candidates given their enzymatic and non-mutagenic character. Accordingly, the work reported here describes fusing RNase T1, one of the most studied members of the microbial RNase family, to the single-chain variable fragment (scFv) of a monoclonal antibody that targets the glycoprotein A33 antigen (GPA33) from human colon cancer cells. A heterologous production system, which employs the yeast Pichia pastoris, has been optimized to produce this immunoRNase (scFvA33T1) with yields of ∼ 5-10 mg · L(-1). The purified protein appears to be correctly folded as it retains its antigen specificity and ribonucleolytic activity. Finally, it also shows specific binding to, internalization into and toxicity against GPA33-positive cell lines compared with the control, GPA33-negative cells. Overall, it can be concluded that scFvA33T1 is a promising therapeutic fusion protein with the additional advantage that presumably it can be produced and purified in large amounts using an easily scalable yeast-based system.
authors: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.
authors: Jaime
authors: J
authors: Tomé-Amat
authors: Aida
authors: A
authors: Menéndez-Méndez
authors: Lucía
authors: L
authors: García-Ortega
authors: Carl A
authors: CA
authors: Batt
authors: Mercedes
authors: M
authors: Oñaderra
authors: Alvaro
authors: A
authors: Martínez-del-Pozo
authors: José G
authors: JG
authors: Gavilanes
authors: Javier
authors: J
authors: Lacadena
mesh_terms: D002942:Circular Dichroism; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D006801:Humans; D006163:Ribonuclease T1 / Q000378:metabolism*; D057127:Single-Chain Antibodies; D013056:Spectrophotometry, Ultraviolet
chemical_list: D057127:Single-Chain Antibodies; D006163:Ribonuclease T1