在生物體中，最終蛋白質的合成需要透過DNA轉錄成RNA，再經由RNA進行解碼轉譯成蛋白質來執行功能。不同的生物在表現蛋白時，會有不同的密碼子的使用偏好，這些不同的密碼子的使用，會透過影響到RNA或蛋白質的層面來進行基因的表現調控。不同生物間不同的密碼子使用偏好也會造成異源的蛋白表現困難，例如常用於蛋白表現的大腸桿菌系統，難以表現密碼使用偏好差異很大的惡性瘧原蟲蛋白質，而最常用於改善的方式是將轉譯出蛋白的基因序列，在不影響胺基酸序列的情況下，將其密碼子都改為大腸桿菌所喜好的形式，我們稱這種方式為密碼子的優化。在本次研究中，我們以惡性瘧原蟲中的DNA聚合酶為目標，嘗試了解為何在經過密碼子的優化後，就能夠順利地在大腸桿菌中合成出惡性瘧原蟲的蛋白。排除了誘導強度的影響，再藉由額外補充了大腸桿菌所缺乏的tRNA後排除了tRNA不足的因素，之後再以超高速離心的方式發現在合成蛋白的過程中核醣體似乎在轉譯的過程中”掉落”。最後我們以北方墨點法的方式發現，mRNA的合成在密碼子優化的組別有顯著的改善，似乎蛋白的表現與轉錄的過程甚至於與轉錄共轉譯的過程有所關聯。最後，我們想再進一步解，在目標聚合酶中哪一段特殊的序列，對於蛋白的調控有顯著的影響。

According to the central dogma of molecular biology, to express a protein, the corresponding gene needs to be transcribed into messenger RNA (mRNA) from DNA, and then the mRNA is translated into the polypeptides through codons on the message. Except for methionine and tryptophan, other eighteen amino acids are decoded by two to six synonymous codons. Interestingly, organisms usually prefer different synonymous codons to code a given amino acid, which is termed codon usage bias. The bias of codon usage may regulate gene expression by affecting the levels of RNA or protein. Different codon usage bias between different organisms can also cause difficulties in heterologous protein expression. For example, the Escherichia coli (E. coli) expression system that is commonly used for heterologous protein expression is difficult to express Plasmodium falciparum (P. falciparum) proteins whose codon usage is largely different from that of E. coli. This defective expression is usually fixed by the so-called codon optimization, the way to change the native codons to the preferred ones of E. coli without changing the amino acid sequence. In this study, we aimed to understand why the protein of P. falciparum can be successfully synthesized after codon optimization, using its apicoplast DNA polymerase I gene (wt-kpom1) as a model. Excluding the effect of induction strength, kpom1 translation was still hampered after E. coli rare tRNAs supplement. Ultracentrifugation of translating ribosomes revealed that the ribosomes appeared to drop off when translating kpom1. By Northern blotting analysis, we found there were much more intermediate mRNA products of kpom1 compared to its codon-optimized counterparts (op-kpom1), suggesting the kpom1 mRNA were undergone co-translational degradation. We further identified the codons between 222 to 295 in WT-Kpom1 were the most suppressive in protein translation. In conclusion, we discovered rare codons attenuated kpom1 translation through co-translational mRNA degradation, and this poor codon effect may be position dependent. The underlying mechanism is currently investigated.

Akashi, Hiroshi. 1994. 'Synonymous codon usage in Drosophila melanogaster: natural selection and translational accuracy', Genetics, 136: 927-35.
Bridgford, Jessica L, Stanley C Xie, Simon A Cobbold, Charisse Flerida A Pasaje, Susann Herrmann, Tuo Yang, David L Gillett, Lawrence R Dick, Stuart A Ralph, and Con Dogovski. 2018. 'Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome', Nature communications, 9: 3801.
Buchan, J Ross, and Ian Stansfield. 2007. 'Halting a cellular production line: responses to ribosomal pausing during translation', Biology of the Cell, 99: 475-87.
Buffet, Pierre A., Innocent Safeukui, Guillaume Deplaine, Valentine Brousse, Virginie Prendki, Marc Thellier, Gareth D. Turner, and Odile Mercereau-Puijalon. 2011. 'The pathogenesis of Plasmodium falciparum malaria in humans: insights from splenic physiology', Blood, 117: 381-92.
Burgess-Brown, N. A., S. Sharma, F. Sobott, C. Loenarz, U. Oppermann, and O. Gileadi. 2008. 'Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study', Protein Expr Purif, 59: 94-102.
Chan, Sherwin, Jun-Hong Ch’ng, Mats Wahlgren, and Jessada Thutkawkorapin. 2017. 'Frequent GU wobble pairings reduce translation efficiency in Plasmodium falciparum', Scientific Reports, 7: 723.
Dao Duc, Khanh, and Yun S. Song. 2018. 'The impact of ribosomal interference, codon usage, and exit tunnel interactions on translation elongation rate variation', PLOS Genetics, 14: e1007166.
Drott, Donald. 2001. Overcoming the codon bias of E. coli for enhanced protein expression.
Fichera, M. E., and D. S. Roos. 1997. 'A plastid organelle as a drug target in apicomplexan parasites', Nature, 390: 407-9.
Gouy, Manolo, and Christian Gautier. 1982. 'Codon usage in bacteria: correlation with gene expressivity', Nucleic acids research, 10: 7055-74.
Grosjean, Henri, and Walter Fiers. 1982. 'Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes', Gene, 18: 199-209.
Gustafsson, C., S. Govindarajan, and J. Minshull. 2004. 'Codon bias and heterologous protein expression', Trends Biotechnol, 22: 346-53.
Hanson, G., and J. Coller. 2018. 'Codon optimality, bias and usage in translation and mRNA decay', Nat Rev Mol Cell Biol, 19: 20-30.
Hershberg, Ruth, and Dmitri A Petrov. 2008. 'Selection on codon bias', Annual review of genetics, 42: 287-99.
Ikemura, T. 1985. 'Codon usage and tRNA content in unicellular and multicellular organisms', Mol Biol Evol, 2: 13-34.
Ikemura, Toshimichi. 1981. 'Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system', J Mol Biol, 151: 389-409.
Kalanon, M., and G. I. McFadden. 2010. 'Malaria, Plasmodium falciparum and its apicoplast', Biochem Soc Trans, 38: 775-82.
Kane, J. F. 1995. 'Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli', Curr Opin Biotechnol, 6: 494-500.
Kennedy, S. R., C. Y. Chen, M. W. Schmitt, C. N. Bower, and L. A. Loeb. 2011a. 'The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum', J Mol Biol, 410: 27-38.
Kennedy, Scott R, Cheng-Yao Chen, Michael W Schmitt, Cole N Bower, and Lawrence A Loeb. 2011b. 'The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum', Journal of molecular biology, 410: 27-38.
Lim, Liting, and Geoffrey Ian McFadden. 2010. 'The evolution, metabolism and functions of the apicoplast', Philosophical Transactions of the Royal Society B: Biological Sciences, 365: 749-63.
Menzella, H. G., E. A. Ceccarelli, and H. C. Gramajo. 2003. 'Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer', Biotechnol Bioeng, 82: 809-17.
Pop, Cristina, Silvi Rouskin, Nicholas T Ingolia, Lu Han, Eric M Phizicky, Jonathan S Weissman, and Daphne Koller. 2014. 'Causal signals between codon bias, mRNA structure, and the efficiency of translation and elongation', Molecular systems biology, 10: 770.
Presnyak, V., N. Alhusaini, Y. H. Chen, S. Martin, N. Morris, N. Kline, S. Olson, D. Weinberg, K. E. Baker, B. R. Graveley, and J. Coller. 2015. 'Codon optimality is a major determinant of mRNA stability', Cell, 160: 1111-24.
Ralph, S. A., M. C. D'Ombrain, and G. I. McFadden. 2001. 'The apicoplast as an antimalarial drug target', Drug Resist Updat, 4: 145-51.
Rosano, G. L., and E. A. Ceccarelli. 2014. 'Recombinant protein expression in Escherichia coli: advances and challenges', Front Microbiol, 5: 172.
Sorensen, H. P., and K. K. Mortensen. 2005. 'Advanced genetic strategies for recombinant protein expression in Escherichia coli', J Biotechnol, 115: 113-28.
van Hemert, Formijn, Antoinette C van der Kuyl, and Ben Berkhout. 2016. 'Impact of the biased nucleotide composition of viral RNA genomes on RNA structure and codon usage', Journal of General Virology, 97: 2608-19.
Welch, Mark, Sridhar Govindarajan, Jon E. Ness, Alan Villalobos, Austin Gurney, Jeremy Minshull, and Claes Gustafsson. 2009. 'Design Parameters to Control Synthetic Gene Expression in Escherichia coli', PloS one, 4: e7002.
Wu, Xiaoqiu, Hans Jörnvall, Kurt D Berndt, and Udo Oppermann. 2004. 'Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance', Biochemical and biophysical research communications, 313: 89-96.
Yu, Chien-Hung, Yunkun Dang, Zhipeng Zhou, Cheng Wu, Fangzhou Zhao, Matthew S Sachs, and Yi Liu. 2015. 'Codon usage influences the local rate of translation elongation to regulate co-translational protein folding', Molecular cell, 59: 744-54.
Zalucki, Yaramah M, Ifor R Beacham, and Michael P Jennings. 2011. 'Coupling between codon usage, translation and protein export in Escherichia coli', Biotechnology journal, 6: 660-67.
Zhou, Z., Y. Dang, M. Zhou, L. Li, C. H. Yu, J. Fu, S. Chen, and Y. Liu. 2016. 'Codon usage is an important determinant of gene expression levels largely through its effects on transcription', Proc Natl Acad Sci U S A, 113: E6117-e25.