Construction and optimization of a CC49-Based scFv-β-lactamase fusion protein for ADEPT
| Autor: | Melodie Estabrook, Volker Schellenberger, Pete Gualfetti, Regina Chin, Martin Roberge, Joshua Basler, Amy Liu, Stephanie Wong, M. Harunur Rashid, Lilia Maria Babe, Tom Graycar |
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| Rok vydání: | 2006 |
| Předmět: |
Antibodies
Neoplasm Recombinant Fusion Proteins Molecular Sequence Data Bioengineering Protein Engineering medicine.disease_cause Biochemistry beta-Lactamases Epitope Antigens Neoplasm Peptide Library Consensus Sequence Escherichia coli medicine Combinatorial Chemistry Techniques Prodrugs Amino Acid Sequence Peptide library Molecular Biology Peptide sequence Glycoproteins Thermostability Chemistry Antibodies Monoclonal Adept Protein engineering Fusion protein Mutagenesis Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection. 19:141-145 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzj012 |
| Popis: | CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT). |
| Databáze: | OpenAIRE |
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