These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Optimizing scaleup yield for protein production: Computationally Optimized DNA Assembly (CODA) and Translation Engineering.
    Author: Hatfield GW, Roth DA.
    Journal: Biotechnol Annu Rev; 2007; 13():27-42. PubMed ID: 17875472.
    Abstract:
    Translation Engineering 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. Here, we describe 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. We have patented Translation Engineering technologies to manage the complexity of gene design to account for codon pair usage, translational pausing signals, RNA secondary structure and user-defined sequences such as restriction sites. Failure to optimize for codon pair-encoded translation pauses often results in the relatively common occurrence of a slowly translated codon pair that slows the rate of protein elongation and decreases total protein production. Translation Engineering technology improves heterologous expression by tuning the gene sequence for translation in any well-characterized host, including cell-free expression techniques characterized by "broken"Escherichia coli systems used in kits for today's molecular tools market. In addition, we have patented a novel gene assembly method (Computationally Optimized DNA Assembly; CODA) that uses the degeneracy of the genetic code to design oligonucleotides with thermodynamic properties for self-assembly into a single, linear DNA product. Fast translational kinetics and robust protein expression are optimized in synthetic "Hot Rod" genes that are guaranteed to express in E. coli at high levels. These genes are optimized for codon usage and other properties known to aid protein expression, and importantly, they are engineered to be devoid of mRNA secondary structures that might impede transcription, and over-represented codon pairs that might impede translation. Hot Rod genes allow translating ribosomes and E. coli RNA polymerases to maintain coupled translation and transcription at maximal rates. As a result, the nascent mRNA is produced at a high level and is sequestered in polysomes where it is protected from degradation, even further enhancing protein production. In this review we demonstrate that codon context can profoundly influence translation kinetics, and that over-represented codon pairs are often present at protein domain boundaries and appear to control independent protein folding in several popular expression systems. Finally, we consider that over-represented codon pairs (pause sites) may be essential to solving problems of protein expression, solubility, folding and activity encountered when genes are introduced into heterologous expression systems, where the specific set of codon pairs controlling ribosome pausing are different. Thus, Translation Engineering combined with synthetic biology (gene synthesis) techniques may allow us to manipulate the translation kinetics of genes to restore or enhance function in a variety of traditional and novel expression systems.
    [Abstract] [Full Text] [Related] [New Search]