Presence of a Start Codon Some expression vectors provide the start codon for translation tiation, while others rely on the start codon of the gene you're rying to express. Note that in E coli, 5 to 12 base pairs or less sep arate the ribosome binding site and the start codon. So you would incorporate this requirement into your cloning strategy when the start codon is provided by the gene you plan to express GC Content Coding sequences with high GC (70%) content may reduce level of expression of a protein in E coli. Check the sequence ing a DNa analysis program Codon usage may also affect the level of protein expression. If the gene of interest contains codons not commonly used in E coli, low expression may result due to the depletion of tRNAs for the rarer codons. When one or more rare codons is encountered ranslational pausing may result, slowing the rate of protein synthesis and exposing the mRNA to degradation. This potential problem is of particular concern when the sequence encodes a protein >60kDa, when rare codons are found at high frequency or when multiple rare codons are found over a short distance of the coding sequence. For example, rare codons for arginine found in tandem can create a recognition sequence for ribosome binding(e.g,_AGGAGG) that closely approximates a Shine Dalgarno sequence UAAGGAGG. This may bind ribosomes non productively and block translation from the bona fide ribosome binding site(RBS)at the initiator codon further upstream Nonetheless, the appearance of a rare codon does not necessarily lead to poor expression. It is best to try expression of the native gene, and then make changes if these seem warranted later Strate gies include mutating the gene of interest to use optimal codons for the host organism, and co-transforming the host with rare tRNA genes. In one example, introduction into the E coli host of a rare arginine(AGG) tRNA resulted in a several-fold increase in the expression of a protein that uses the AGG codon(Hua et al., 1994). In another case, substitution of the rare arginine codon AGG with the E. coli-preferred CGU improved expression Robinson et aL., 1984). Other work has shown that rare codons account for decreased expression of the gene of interest in E. coli (Zhang, Zubay, and Goldman, 1991; Sorensen, Kurland, and Pederson, 1989). Rare codons may have an even more dramatic 466 BellPresence of a Start Codon Some expression vectors provide the start codon for translation initiation, while others rely on the start codon of the gene you’re trying to express. Note that in E. coli, 5 to 12 base pairs or less sep￾arate the ribosome binding site and the start codon. So you would incorporate this requirement into your cloning strategy when the start codon is provided by the gene you plan to express. GC Content Coding sequences with high GC (>70%) content may reduce the level of expression of a protein in E. coli. Check the sequence using a DNA analysis program. Codon Usage Codon usage may also affect the level of protein expression. If the gene of interest contains codons not commonly used in E. coli, low expression may result due to the depletion of tRNAs for the rarer codons. When one or more rare codons is encountered, translational pausing may result, slowing the rate of protein synthesis and exposing the mRNA to degradation. This potential problem is of particular concern when the sequence encodes a protein >60kDa, when rare codons are found at high frequency, or when multiple rare codons are found over a short distance of the coding sequence. For example, rare codons for arginine found in tandem can create a recognition sequence for ribosome binding (e.g., _AGGAGG) that closely approximates a Shine￾Dalgarno sequence UAAGGAGG.This may bind ribosomes non￾productively and block translation from the bona fide ribosome binding site (RBS) at the initiator codon further upstream. Nonetheless, the appearance of a rare codon does not necessarily lead to poor expression. It is best to try expression of the native gene, and then make changes if these seem warranted later. Strate￾gies include mutating the gene of interest to use optimal codons for the host organism, and co-transforming the host with rare tRNA genes. In one example, introduction into the E. coli host of a rare arginine (AGG) tRNA resulted in a several-fold increase in the expression of a protein that uses the AGG codon (Hua et al., 1994). In another case, substitution of the rare arginine codon AGG with the E. coli-preferred CGU improved expression (Robinson et al., 1984). Other work has shown that rare codons account for decreased expression of the gene of interest in E. coli (Zhang, Zubay, and Goldman, 1991; Sorensen, Kurland, and Pederson, 1989). Rare codons may have an even more dramatic 466 Bell