Effects of codon optimization on expression in Escherichia coli of protein-coding DNA sequences from the protozoan Eimeria

Authors: Jenkins, Mark; Parker, Carolyn; Obrien, Celia; CAMPOS, PHILIP - Orise Fellow; Tucker, Matthew; Miska, Kate

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2023
Publication Date: 6/2/2023
Citation: Jenkins, M.C., Parker, C.C., Obrien, C.N., Campos, P., Tucker, M.S., Miska, K.B. 2023. Effects of codon optimization on expression in Escherichia coli of protein-coding DNA sequences from the protozoan Eimeria. Journal of Microbiological Methods. 211. Article 106750. https://doi.org/10.1016/j.mimet.2023.106750.
DOI: https://doi.org/10.1016/j.mimet.2023.106750

Interpretive Summary: Characterizing proteins originating from eukaryotic or prokaryotic organisms often requires inserting the genes coding for these proteins in the bacterium Escherichia coli. The advantage of this approach is that large amounts of the protein can be made by simply growing up E. coli harboring the gene in a culture flask. The E. coli-derived protein is termed a recombinant protein because it was produced using recombinant DNA technology. A problem often encountered with this method is that the organism from which the gene originated may use a slightly different sequence than E. coli. Proteins are composed of a string of amino acids and each amino acid may is encoded by 1-6 different 3 letter codons. Through natural selection, organisms prefer using certain codons for each amino acid, while infrequently using other codons (termed “rare”) for the same amino acid. When one tries to produce a protein from one organism in E. coli, the bacterium often has trouble producing the protein due to differences in codon usage. One way to overcome this is to synthesize the gene and replace the original codons with those used most frequently by E. coli. However, in our experience, this approach often doesn’t work. In this paper, we demonstrate the 3 possible outcomes in taking this approach – increased or decreased or no change in protein production. This information should be helpful to researchers attempt to use E. coli to produce recombinant proteins for diagnostic or vaccine purposes.

Technical Abstract: The objective of this study was to compare the levels of recombinant protein from three Eimeria genes before and after optimization of codons for expression in Escherichia coli. Protein coding sequences from Eimeria maxima (EmaxSO7, EmaxIMP1) and E. acervulina (EAH00033530) were cloned with or without prior codon optimization and expressed as polyHis fusion proteins. All three outcomes: higher, lower, or no change in the yield of amount of recombinant protein were observed suggesting that codon optimization alone for expression in Es. coli does not inevitably lead to higher expression levels. Analysis of codon usage for each gene sequence revealed that, similar to other organisms, Eimeria intersperses rare and frequently used codons in protein-coding sequences. However, no relationship was observed between the predicted protein structure and the location of major and minor codons, suggesting that codon selection in this apicomplexan parasite is influenced by factors other than regional secondary protein structure.