ArticleScientific reports2017
Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.
Article in Scientific reports, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed, 20 citations in OpenAlex.
- Genotyped functional screening of soluble Fab clones enables in-depth analysis of mutation effects.Scientific reports · 2023Article
- Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins.ACS synthetic biology · 2022Article
- Fragment antigen binding domains (FSynthetic and systems biotechnology · 2022Article
- Genetic and metabolic engineering challenges of C1-gas fermenting acetogenic chassis organisms.FEMS microbiology reviews · 2021Review
- Application of antibody phage display to identify potential antigenic neural precursor cell proteins.Journal of biological research (Thessalonike, Greece) · 2020Article
- Evolution ofInternational journal of molecular sciences · 2020Review
- Phage Display Derived Monoclonal Antibodies: From Bench to Bedside.Frontiers in immunology · 2020Review
- Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries.Microbial cell factories · 2019Article
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.
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