Abstract
Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its "codon-preferred" DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ~2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10(-8) m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.
Highlights
Synonymous codon usage affects protein properties in a given organism
We have shown that a scFv encoded by different synonymous codons could be synthesized in E. coli with considerably changed solubility and antigen-binding activity while retaining the identical, original amino acid sequence
Our CD analysis from affinity-improved variants detected the structural/conformational changes (Fig. 4), directly demonstrating that synthesis and folding of proteins in E. coli is controlled by genetic codes
Summary
Synonymous codon usage affects protein properties in a given organism. Results: A total of 342 antibody codon variants were identified, differing significantly in solubility and functionality while retaining the identical original amino acid sequence. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence.
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