Rare codons

These experiments make it clear that removing competition with release factors leads to more efficient incorporation of the desired amino acid. Unfortunately, the technology to incorporate nonstandard nucleotides into mRNAs coding for full-length proteins is not yet available. Alternatives that have been tested include using (i) a 4-nucleotide codon-anticodon pair, dubbed frame-shift suppression (Sect. 6.1), (ii) a rare codon, and (iii) cell-free extracts from organisms that are either deficient in a release factor (Sect. 5.1) or unable to translate one or more codons (Sect. 6.2). 

In an effort to reduce the competition encountered with naturally occurring tRNAs, even when rare codons are used, Hardesty and co-workers have devised a strategy based on 4-nucleotide codon-anticodon pairs [48]. An extra thymidine was inserted either 5 or 3 to the rare arginine codon AGG to yield TAGG... 

Kane, J. E. (1995). Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6,494-500. 

LysS, LysE and Lad are all available in conjunction with additional rare codon tRNAs on the same chloramphenicol resistant plasmid (see Rosettas strain). 

The computer program presented here can generate very different relative frequencies of occurrence of all amino acids (and stop codons) within a target set. One-pot, two-pot, and three-pot syntheses can be simulated. Optionally, correction factors can be included to compensate for the (possible) differences in chemical coupling efficiencies of the nucleotide synthons. Finally, the codon usage of three major expression microorganisms can be considered, treating rare codons as pseudo stop codons. 

A wide variety of E coli strains designed for protein expression are commercially available, derived from the E. coli strain BL21, that are tailored to facilitate disulfide bond formation, fine-tune protein expression levels, enhance the expression of proteins that contain rare codons, and other specific requirements. These strains are available as lambda DE3 lysogens, which carry a chromosomal copy of the T7 RNA polymerase gene under control of the lac promoter (inducible by IPTG). Once induced, the T7 RNA polymerase drives expression of genes that are under control of the T7 promoter, such as those cloned into the pET family of expres-... 

Some authors have been concerned that the use of codons for which there is a low level oftRNA in E. coli might limit expression. This rare codon effect has only been observed in extreme cases where a protein is massively overproduced and contains several rare Arg-codons (e.g. Ref. 99). This completely depletes the concentration of charged tRNA in the cell. Since an effect will only be seen if translation undergoes chain termination at the slowing point, it would not be expected to play any role where only a few hundred molecules are being synthesized per cell, as in phage display. 

Organism-biased codon usage can be remedied by manipulating the interplay between a codon and its encoded amino acid. It is evident that several codons encode a particular amino acid, but only one amino acid is encoded by a particular codon. This redundancy is, thus, unidirectional in this context. Consequently, one could either increase the availability of the amino acid corresponding to the rare codon or change the transcript sequence to reflect the codon preferences of the host (analogous to market forces, one can either manipulate the supply or the demand sides). 

There are many commercially available E. coli cell strains for the heterologous expression of recombinant proteins. By far the most common promoter system is the T7 expression system and the most commonly used expression host is BL21(DE3). If one is using a promoter that is recognized by the E. coli RNA polymerase, any cell strain can be used. Frequently useful are cell strains that supplement rare codons and strains that alter the ability of E. coli to produce disulfide bonds in the cytoplasm. However, there are cell strains that have been developed for their ability to express toxic proteins149,150 and a wide range of other strains that may be useful for any particular protein. 

Fig. 2. Main window of the software package ANACONDA for two-codon context analysis in total genomes at the gene view level. Individual ORFs that were used to calculate codon context bias are shown in the hierarchical left panel. Clicking on one of them changes the main panel into the gene view layout. This is composed of a header with the name of the ORF as stated in the original file and the sequence itself. This sequence is colored according to the residual color scale obtained for that ORFeome, i.e., each codon pair is colored in the ORF sequence with the same color scale that it had in the ORFeome map for two-codon contexts. Rare codons are highlighted using circles.

Filters. Searching for specific ORFeome features can be performed using subsets of ORFs. The sequences that comply with the imposed rules are presented in a special tab in the left panel (Filtered). The available filters include (1) searching for special color patterns or codon/amino acid sequences (2) searching for runs of up to six rare codons (3) looking for ORFs rich in bad contexts or rare codons and (4) finding ORFs whose G + C% is included in a chosen interval. This filter... 

Rare codons are highlighted by a blue circle in the sequence view layout, and will be considered in future versions of ANACONDA as codons to be preferentially optimized. 

The header of the gene view layout includes (1) the ORF name, (2) the total number of codons of that ORF, (3) the number of codons whose frequency is below the chosen threshold for rare codons, (4) the percentage of rare codons in the ORF,... 

D. discoideum, but are used at about 50% frequency in humans. Particularly rare codons in D. discoideum are CUG, UCG, AGG, GGG, GGG, AGG, GGG, GGG, GGA, CGG, GGG (see Table 5.2). By analyzing these data, it becomes clear that adaptation of codons in human cDNAs prepared for expression in D. discoideum is demanding. It is an open debate whether or not codon bias serves a gene regulatory function, but it has been observed in E. coli that most... 

Zahn K. Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth. J Bacteriol 1996 178 2926-2933. 

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