 |
 |
 |
 |
 |
 |
 |
Gutman G. and Hatfield G.W. "Nonrandom Utilization of Codon Pairs in Escherichia coli." The Proceedings of the National Academy of Sciences USA, 86:3699-3703. (1989). We analyzed protein-coding sequences of Escherichia coli and found that codon-pair utilization is highly biased, reflecting over-representation or under-representation of many pairs compared with their random expectations. This is the seminal paper that is the basis for CODA Genomics’ IP portfolio and technology for optimized protein expression, structure and function. (view/download PDF)
Hatfield G. W. and Gutman G. A. “Codon Pair Utilization Bias in Bacteria, Yeast and Mammals.” In Transfer RNA in Protein Synthesis. (Edited by D. L. Hatfield, B. J. Lee, and R. M. Pirtle). Published by CRC Press, Boca Raton, FL, 1993. A comprehensive review of codon pair bias and its relationship to codon context effects on translation. (view/download PDF)
Irwin B., Heck J., Hatfield G.W. "Codon Pair Utilization Biases Influence Translational Elongation Step Times." The Journal of Biological Chemistry, 270:22801-22806 (1995). Two independent assays capable of measuring the relative in vivo translational step times across a selected codon pair in a growing polypeptide in Escherichia coli were developed and then employed to demonstrate that codon pairs observed in protein coding sequences occur more frequently than predicted (over-represented codon pairs) and are translated more slowly (translational pausing) than pairs observed less frequently than expected (under-represented codon pairs). (view/download PDF)
Hatfield G. W. “Codon Context, Translational Step-time, and Attenuation.” In Gene Expression in Escherichia coli. (Edited by E. C. C. Lin). Published by R. G. Landes Company, Austin, Texas, 1995. A review of the role of codon pair bias in the bacterial transcription attenuation mechanism.
Kittle, J.D. "Radical Changes in the Engineering of Synthetic Genes for Protein Expression.” BioPharm International. 2006:12-18 (2006). New concepts in gene design emphasize the control of protein translation kinetics as a means to improve yield and to alter activity. (view/download PDF)
David A. Roth, Liza S. Z. Larsen, and G. Wesley Hatfield. “Translation Engineering™ and Synthetic Biology” In Cell-Free Expression. (Edited by W. Antoni Kudlicki). Published by Landes Bioscience, Georgetown, Texas, 2007. Translation Engineering TM combined with synthetic biology (gene synthesis) techniques makes it possible to deliberately alter the presumed translation kinetics of genes without altering the amino acid sequence. CODA Genomics, Inc. has developed proprietary technologies that design and assemble synthetic genes for high expression and enhanced protein production, and offers new insights and methodologies for affecting protein structure and function in a variety of heterologous host and cell-free expression systems. (view/download PDF)
G. Wesley Hatfield and David A. Roth. “Optimizing Scaleup Yield for Protein Production: Computationally Optimized DNA Assembly (CODA) and Translation Engineering™" in Biotechnology Annual Review. Edited by: M. Raafat El-Gewely, Ph.D. Published by Elsevier, B.V. 2007. We describe novel technologies that will enable pharmaceutical and biotechnology researchers to routinely assemble synthetic genes to produce large amounts of functional proteins in traditional and developing expression systems that can employ large scale bioreactor and fermentor technologies. This technology allows scalable, rational creation of functional proteins for developing protein and antibody-based drugs that may safely treat or prevent formidable diseases with few side effects at reasonable costs for manufacturers, health organizations and patients. (In Press)
Hans Bügl, John P. Danner, Robert J. Molinari, John Mulligan, David A. Roth, Ralf Wagner, Bruce Budowle, Robert M. Scripp, Jenifer A. L. Smith, Scott J. Steele, George Church, & Drew Endy “A Practical Perspective on DNA Synthesis and Biological Security.” December 4, 2006 [version 3D, for distribution] (view/download PDF)
Other Scientists
Trinh R., Gurbaxani B., Morrison S., Seyfzadeh M. "Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression." Molecular Immunology 40: 717-722 (2004). A single silent mutation predicted by the work of Hatfield and Gutman in a recombinant antibody gene that replaced an over-represented (slowly translated) codon pair with an under-represented (rapidly translated) codon pair resulted in a 30-fold increase in active protein expression. (view/download PDF)
Cortazzo, P., Cervenansky, C., Marin, M., Reiss, C., Ehrlich, R. and Deana, A. “Silent mutations affect in vivo protein folding in Escherichia coli.” Biochemical and Biophysical Research Communications 293:537–541 (2002). These authors report the effects of codon substitutions in the EgFABP1 (Echinococcus granulosus fatty acid binding protein1) gene that replaced five codons with their synonymous ones. The altered region corresponded to a turn between two short alpha helices. One of the silent mutations markedly decreased the solubility of the protein when expressed in Escherichia coli. Expression of this protein also caused strong activation of a reporter gene designed to detect misfolded proteins, suggesting that the turn region has special translation kinetic requirements that ensure proper folding of the protein. They interpret their results in terms of the importance of codon context (codon pairs) for in vivo protein folding. (view/download PDF)
J. Ross Buchan, Lorna S. Aucott1 and Ian Stansfield "tRNA properties help shape codon pair preferences in open reading frames" Nucleic Acids Research, 34:1015–1027. (2006). This paper confirms the early observations of Gutman and Hatfield and interpretations about codon pair bias and tRNA incompatibilities in the A and P sites of a translating ribosome. However, because like Gutman and Hatfield they also find little correlation between codon pair bias and codon usage, they prefer to suggest that that the dinucleotide bias observed between adjacent codons is responsible for "translational efficiency". (view/download PDF)
Karlin, S., Mrazek, J., and Campbell, A.M. “Codon Usage in Different Gene Classes of Genes in the Escherichia coli Genome” Mol. Micro. 29:1341-1355 (1998). This study carefully considers the earlier work of Irwin, Heck, and Hatfield [Journal of Biological Chemistry, 270:22801-22806 (1995)]. It is a comprehensive study and independent discussion by eminent Stanford scientists of the relative importance of codon usage and dicodon (codon pair) bias for the control of protein expression levels and for protein function. (view/download PDF)
Miller, J.H. and Albertini, A. M. “Effects of surrounding sequence on the suppression of nonsense codons” Journal of Molecular Biology 164:59-71 (1983). This a classic early paper that led to the suggestion that the structural compatibility of adjacent tRNA isoacceptors on the surface of translating ribosomes affect translational step-times. Miller and Abertini used a lacI-Z fusion system, to determine the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying β-galactosidase activity. They examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons. The largest effects were found with the amber tRNA suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency. They found that nonsense suppression efficiency was correlated with the codon on the 3′ side of the codon being suppressed. (view/download PDF).
Boycheva, S., Chkodrov,G. and Ivanov, I. “Codon pairs in the genome of Escherichia coli.” Bioinformatics 19:987–998 (2003). As reported 14 years earlier by Gutman and Hatfield, these authors report a significant difference between the real and statistically predicted frequency of occurrence of codon pairs between highly expressed and poorly expressed E.coli genes. Using an empirically defined criterion (deltaREG), they classify these codon pairs are classified as ‘hypothetically attenuating’ or ‘hypothetically non-attenuating’ (and discuss their possible effects on translation. (view/download PDF). In the following letter Hatfield and Gutman present alternative analyses and interpretations of the data reported by Boycheva et al, (view/download PDF).
Beliakova-Bethell N., Beckham C., Giddings T, Winey M., Parker R., and Sandmeyer S. "Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components." RNA 12:94-101. (2006). A CODA Genomics, Inc. translationally engineered red fluorescent protein was used to demonstrate the assembly of yeast Ty3 virus-like particles in association with intracellular p-bodies (view/download PDF)
Kim, Chang H., Oh, Younghoon, and Lee, Tai H. " Codon optimization for high-level expression of human erythropoietin (EOP) in mammalian cells" GENE 199:293-301. (1997). (view/download PDF)
Wu, Xiaoqiu, Jornvall, Hans, Berndt, Kurt D., and Oppermann, Udo " Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but no tRNA abundance. " BBRC 313:89-96. (2004). (view/download PDF)
Wu, Gang, Zheng, Yuanpu, Qureshi, Htar T. Z., Beck, Tyler, Bulka, Blazej and Freeland, Stephen J. " SDGB: a database of synthetic genes re-designed for optimizing protein over-expression " Nucleic Acids Research 00:database issue. (2006). (view/download PDF)
Chava Kimchi-Sarfaty, Jung Mi Oh, In-Wha Kim, Zuben E. Sauna, Anna Maria Calcagno, Suresh V. Ambudkar, Michael M. Gottesman "A “Silent” Polymorphism in the MDR1 Gene Changes Substrate Specificity" Sciencexpress 10.1126/science.1135308. (view/download PDF)
CODA in the Lab
A Red Fluorescent Protein for Hydra
The freshwater polyp, Hydra, has been an important biological model system for nearly 250 years. Recently it has become important for such studies as pattern formation, limb regeneration, and stem cells. To take advantage of recently developed molecular genetics methods, a fluorescent protein was needed to expand several recent studies. However, because the Hydra genome is 71% A+T rich, commercially available fluorescent proteins did not express well in this organism. This prompted Professor Robert Steele at the University of California, Irvine, and his collaborators, Professor Diane Bridge, at Elizabethtown College in Elizabethtown Pennsylvania, and Professor Thomas Bosch at Kiel University in Germany to turn to CODA Genomics, Inc.
[full text PDF]
When Gene Fusions Don’t Express
Researchers in the laboratory of Professor Hung Fan of the UCI Cancer Research Institute at the University of California, Irvine, attempted to express the cytoplasmic tail of a retroviral envelope protein as a GST fusion protein in E. coli without success. The results of a SpeedPlot™ of this fusion gene performed by CODA scientists suggested that the presence a highly over-represented (slowly translated) codon pair generated by the fusion was the culprit.
[full text PDF]
A Red Fluorescent Protein for Yeast
Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated, is poorly understood.
[full text PDF]
CODA Genes Produce Active Enzymes in Eukaryotes: Trypanosomes
Trypanosomatids are protozoan hemoflagellates responsible for a variety of human diseases, including Chagas’ disease, leishmaniasis, and African sleeping sickness, which account for nearly one million deaths per year. Because of trypanosomes’ diploidy and asexual life cycle, forward genetics studies in these eukaryotes has been a challenge, prompting a development of selection, gene knockouts, functional complementation, and RNA interference experimental approaches.
[full text PDF]
Misplaced Codon Pairs Inhibit Translation in Mammalian Cells
Early in 2004, before the founding of CODA Genomics, Inc., researchers in the Molecular Biology Institute at UCLA attempted to express a human protein in which a single Fc chain specific for the HER2/neu gene containing VH and VL regions joined by a flexible (GGGGS)3 linker fused to a human anti-rat transferrin receptor IgG3 heavy chain with the same flexible (GGGGS)3 linker. In initial experiments they were unable observe any expression of this protein.
[full text PDF]