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.
Highlights
Antibody fragments are increasingly used for a variety of applications in diagnostics, basic life science research, and as therapeutics[1]
In order to restore cell viability and Fab production, several DNA segments of the Fab gene were redesigned with synonymous codon substitutions (Supplementary Fig. 2a)
In the Angov method the heterologous gene is engineered with synonymous codons to obtain a similar positional codon usage profile as is present in the original host
Summary
Antibody fragments are increasingly used for a variety of applications in diagnostics, basic life science research, and as therapeutics[1]. The small size of antibody fragments makes them more amenable to genetic modifications (e.g. for fusion proteins and site-specific chemical tagging)[5]. Another useful property of antibody fragments is their easy expression in E. coli, which enables easier production with lower costs, and compatibility with phage display[4, 6]. The low expression levels of Fab fragments have been attributed to toxicity effects, intracellular degradation, aggregation and inefficient translocation[11, 12]. To alleviate these problems different vector elements, bacterial strains, and cultivation conditions can be tested[7, 13]. In multi-domain proteins, such as Fab, the translation speed of the succeeding domains may be a crucial parameter for successful expression[29]
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