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BMC Proceedings

Open Access

Gene expression level: is it an important factor in codon optimization for overexpression of recombinant proteins?

  • Juliana da Costa Ramos1,
  • Jennifer Malaguez Oliveira1,
  • Carlos Frederico Ceccon Lanes2,
  • Marcio de Azevedo Figueiredo2 and
  • Luis Fernando Marins1
BMC Proceedings20148(Suppl 4):P224

Published: 1 October 2014


Green Fluorescent ProteinRecombinant ProteinCodon UsageHeterologous ProteinGene Table


Microalgae are becoming a viable model for the expression of recombinant proteins. However, suitable levels of expression for commercial production have not yet been obtained. One way to increase the heterologous protein production is the codon optimization of the target gene so that it presents the preferential codons used by the host organism according to its own pool of transfer RNA (tRNA). For codon optimization the Codon Usage Database (CUD; has been used. The database provides codon frequency tables based on gene transcripts, regardless of their level of expression. Therefore, the aim of this study was to determine if there are changes in the frequency of codons used when taking into account the level of expression of genes, considering the 50 most expressed genes and 50 least expressed genes from the microalgae Chlamydomonas reinhardtii.


The transcriptome data, published by Lv et al. (2013), was used to select the genes according to their expression level. The 50 most expressed transcripts (RPKM>2900) were selected from the four stages of growth of C. reinhardtii: Log (LP), stationary (SP), lipid accumulation (LAP), and cellular decline (CDP). As a negative control, we selected the 50 least expressed transcripts (RPKM<15) from the LP growth phase. The sequences of the selected genes were obtained from the website The codon frequency tables were created using the tool Gene to Codon Usage ( Tables, including the one provided by CUD, were compared via the UPGMA method ( and by the statistical test of chi-square using the software R.

Results and conclusion

The tables created with the 50 most expressed genes in the four stages of growth of the microalgae were not different by analysis of UPGMA. Thus, the LP stage table was used for all other comparisons. The UPGMA method distinguished the three tables. The main difference among the tables was observed in the AAG codon, with a frequency of 98.6 per thousand in the 50 most expressed genes table, compared with 43.3 per thousand from the CUD, and 28.0 from the negative control. Moreover, codons with zero frequency were only identified in table from 50 most expressed genes: AUC, AUA, GGA, and AGA. The chi-square test shows that there is enough evidence to support the hypothesis that the frequencies of the codons are not homogeneous among the three tables. Therefore, we conclude that there is a change in the codon frequency dependent on the level of gene expression. The next step is the production of three genetic constructs with the green fluorescent protein (GFP) gene, optimized by each of the codon frequency tables in order to observe, in vivo, whether there is an improvement in the production of recombinant proteins.



CNPq - Brazil Science without Borders Program (COCBI).

Authors’ Affiliations

Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, FURG, Rio Grande, Brazil
Insituto de Oceanografia, Universidade Federal do Rio Grande, FURG, Rio Grande, Brazil


  1. Lv H, Qu G, Qi X, Lu L, Tian C, Ma Y: Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation. Genomics. 2013, 10: 229-37.Google Scholar
  2. Specht E, Miyake-Stoner S, Mayfield S: Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett. 2010, 32: 1373-1383.PubMed CentralView ArticlePubMedGoogle Scholar


© Costa Ramos et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.