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Construction and optimization of a CC49-Based scFv-β-lactamase fusion protein for ADEPT
- Source :
- Protein Engineering, Design and Selection. 19:141-145
- Publication Year :
- 2006
- Publisher :
- Oxford University Press (OUP), 2006.
-
Abstract
- 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).
- Subjects :
- 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
Subjects
Details
- ISSN :
- 17410134 and 17410126
- Volume :
- 19
- Database :
- OpenAIRE
- Journal :
- Protein Engineering, Design and Selection
- Accession number :
- edsair.doi.dedup.....b970ff0a566f09e4802f3a07c473dc43