Abstract
High levels of translational errors, both truncation and misincorporation in an Fc-fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified. For an Fc-fusion protein expressed in Escherichia coli, the targeted strategy, in combination with appropriate fermentation conditions, virtually eliminated misincorporation (proteins produced with a wrong amino acid sequence), and reduced the level of truncation. The use of full optimization using commercially available strategies reduced the initial errors, but introduced different misincorporations. However, truncation was higher using the targeted strategy than for most of the full optimization strategies. This targeted approach, along with monitoring of translation fidelity and careful attention to fermentation conditions is key to minimizing translational error and ensuring high-quality expression. These findings should be useful for other biopharmaceutical products, as well as any other transgenic constructs where protein quality is important.
References
Jan 1, 1977·Cell·P Edelmann, J Gallant
May 11, 1992·Nucleic Acids Research·K WadaT Ikemura
Jul 23, 1991·Biochimica Et Biophysica Acta·A K UlrichJ Parker
Nov 20, 1991·Journal of Molecular Biology·M A Sørensen, S Pedersen
Dec 1, 1989·Medical Hypotheses·J C ToháJ Diaz-Valdes
May 20, 1988·Journal of Molecular Biology·M J TothP Schimmel
Dec 29, 1971·Nature: New Biology·L Gorini
Dec 15, 1984·Journal of Molecular Biology·S VarenneC Lazdunski
Feb 15, 1981·Journal of Molecular Biology·T Ikemura
Jan 1, 1982·Molecular & General Genetics : MGG·N Ellis, J Gallant
Jan 11, 1980·Nucleic Acids Research·R GranthamA Pavé
Aug 1, 1996·Protein Science : a Publication of the Protein Society·T A Thanaraj, P Argos
May 9, 1998·Applied and Environmental Microbiology·M J Weickert, I Apostol
Feb 25, 2003·Protein Expression and Purification·Dean E McNultyJames F Kane
Feb 5, 2004·Molecular Cell·Kirill B Gromadski, Marina V Rodnina
Jul 16, 2004·Analytical Chemistry·Zhongqi Zhang
Jul 27, 2005·Journal of Molecular Evolution·Hans K Stenøien, Wolfgang Stephan
Oct 1, 2005·Analytical Chemistry·Zhongqi Zhang
Apr 20, 2006·The Journal of Biological Chemistry·Naomi HirabayashiTsutomu Suzuki
Oct 21, 2006·RNA·Narayanaswamy KirthiGloria M Culver
Nov 11, 2006·RNA·Emily B Kramer, Philip J Farabaugh
Apr 4, 2007·Genes & Development·Asa FredrikssonThomas Nyström
Feb 26, 2009·Cell·Hani S Zaher, Rachel Green
Apr 8, 2009·Molecular Biology and Evolution·Tong ZhouClaus O Wilke
Jun 26, 2009·The Journal of Biological Chemistry·John B JordanBarbra J Sasu
Sep 22, 2009·Analytical Chemistry·Zhongqi Zhang
Citations
Sep 6, 2015·Interdisciplinary Sciences, Computational Life Sciences·Rui-Fang LiZhi-Fang Lu
May 21, 2013·Bioengineered·Long LiuJian Chen
Feb 16, 2015·Interdisciplinary Sciences, Computational Life Sciences·Rui-Fang LiZhi-Fang Lu
Feb 21, 2013·Electrophoresis·M Eleanor LeKatariina M Hutterer
Aug 4, 2020·MAbs·Aming ZhangNing Li
Oct 24, 2018·ACS Synthetic Biology·Zoltan LipinszkiZsuzsanna Gyorfy
Nov 13, 2018·Biochemistry·H Edward WongZhongqi Zhang
Oct 17, 2013·Biochemistry·Zhongqi ZhangPavel V Bondarenko