effect on translation when they occur close to the initiator codon Chen and Inouye, 1990). While codon usage is not the only or most important factor, be aware that it may influence translation Secondary Structure Secondary structures that occur near the start codon may block translation initiation(Gold et al., 1981; Buell et al., 1985) or serve as translation pause sites resulting in premature termi nation and truncated protein. These can be found using DNA or RNA analysis software Structures with clear stem structures greater than eight bases long may be disrupted by site-specific mutation or by making all or a portion of the coding sequence synthetically Depending on the size of the gene, and the importance of obtaining high-expression levels, it may be worth synthesizing the gene. This has been generally done by synthesizing overlapping oligonucleotides that when annealed can be extended using PCr and ligated to form the full-length coding sequence. There are several examples where this approach has been used to optimize codon usage for E coli (Koshiba et al., 1999; Beck von Bodman et aL., 1986). In addition, if one takes on the work and expense of synthesizing a gene, secondary structures in the predicted RnA that might stall translation can be removed and sites for restric tion endonucleases can be introduced Size of a Gene or protein As a rule, very large(>100k Da)and very small(<5kDa) pro teins are more difficult to express in E coli. Small polypeptides with little secondary structure tend to be rapidly degraded in E. coli Degradation can be minimized by expressing such short oligopeptides as concatemers with proteolytic or chemical cleav age sites in between the monomeric units(Hostomsky, Smrt, and Paces, 1985). Short peptides are also successfully expressed as fusion proteins. Fusion with GST, MalB or other larger, well folded partners will tend to stabilize a short peptide, making expression possible and purification relatively simple. One publi- cation has shown MBP to be superior to other large fusion pro- teins at stabilizing short polypeptides(Kapust and Waugh, 1999) At the other extreme, proteins that are above 60kDa are best made using smaller affinity tags, such as FLAG, his, or on their own, without any fusion. While there is no clear upper limit, the larger the protein, the lower the yield is likely to be E coli Expression System 467effect on translation when they occur close to the initiator codon (Chen and Inouye, 1990). While codon usage is not the only or most important factor, be aware that it may influence translation efficiency. Secondary Structure Secondary structures that occur near the start codon may block translation initiation (Gold et al., 1981; Buell et al., 1985), or serve as translation pause sites resulting in premature termi￾nation and truncated protein. These can be found using DNA or RNA analysis software. Structures with clear stem structures greater than eight bases long may be disrupted by site-specific mutation or by making all or a portion of the coding sequence synthetically. Depending on the size of the gene, and the importance of obtaining high-expression levels, it may be worth synthesizing the gene. This has been generally done by synthesizing overlapping oligonucleotides that when annealed can be extended using PCR and ligated to form the full-length coding sequence. There are several examples where this approach has been used to optimize codon usage for E. coli (Koshiba et al., 1999; Beck von Bodman et al., 1986). In addition, if one takes on the work and expense of synthesizing a gene, secondary structures in the predicted RNA that might stall translation can be removed, and sites for restric￾tion endonucleases can be introduced. Size of a Gene or Protein As a rule, very large (>100kDa) and very small (<5kDa) pro￾teins are more difficult to express in E. coli. Small polypeptides with little secondary structure tend to be rapidly degraded in E. coli. Degradation can be minimized by expressing such short oligopeptides as concatemers with proteolytic or chemical cleav￾age sites in between the monomeric units (Hostomsky, Smrt, and Paces, 1985). Short peptides are also successfully expressed as fusion proteins. Fusion with GST, MalB or other larger, well￾folded partners will tend to stabilize a short peptide, making expression possible and purification relatively simple. One publi￾cation has shown MBP to be superior to other large fusion pro￾teins at stabilizing short polypeptides (Kapust and Waugh, 1999). At the other extreme, proteins that are above 60kDa are best made using smaller affinity tags, such as FLAG, his6, or on their own, without any fusion. While there is no clear upper limit, the larger the protein, the lower the yield is likely to be. E. coli Expression Systems 467