be necessary but not sufficient to direct a target protein to the periplasm. Translocation across the outer membrane and into the growth medium is inefficient. In most cases target proteins found in the growth medium are the result of damage to the cell enve- pe and do not represent true secretion (Stader and Silhavy, 1990). Translocation across the inner cell membrane of E coli is incompletely understood (reviewed by Wickner, Driessen, and Hartl, 1991), and the efficiency of export will depend on the indi idual target protein. Currently the export cannot be predicted based on protein sequence, although some generalizations have been made about the sequence immediately following the sigr peptide(Boyd and vith. 1990: Yamane and mizushima 1988). Therefore it is possible to find target proteins in the cyto- plasm(with uncleaved signal sequence) or in the periplasm in partially processed form, in place of or in addition to the expected periplasmic processed species. In some cases the proportion of protein that is exported can be increased by lowering the tem- perature 15 to 30oC during induction Post-translational Modification E. coli does not glycosylate or phosphorylate proteins or cognize proteolytic processing signals from eukaryotes, so take this into account when designing the cloning strategy. If proteolytic processing is needed, it is best to express only the coding sequences for the fully processed protein. If the protein of interest requires glycosylation for activity, and full activity is important in the final der a eukaryotic host, such Pichia, insect cells, or mammalian cells. Is the protein potentially Toxic? Consider whether the protein of interest is likely to have a tox effect on the host cell. Where the function of the protein is known this can be guessed at with some accuracy. For example, non specific proteases, nucleases, or pore-forming membrane proteins might all be expected to have some toxic effect on E coli. Expres sion of toxic proteins may be very low, and there will be strong selective pressure on cells to eliminate the gene of interest by point mutation to change the translation frame, insertion of a stop codon, or change in an amino acid residue critical to the proteins function. Larger deletion of parts of the plasmid may also be seen. If there is a suggestion that the gene product will be toxic, use an expression vector with a tightly regulated promoter (e.g, T7, PET E coli Expression Systems 469be necessary but not sufficient to direct a target protein to the periplasm. Translocation across the outer membrane and into the growth medium is inefficient. In most cases target proteins found in the growth medium are the result of damage to the cell enve￾lope and do not represent true secretion (Stader and Silhavy, 1990). Translocation across the inner cell membrane of E. coli is incompletely understood (reviewed by Wickner, Driessen, and Hartl, 1991), and the efficiency of export will depend on the indi￾vidual target protein. Currently the export cannot be predicted based on protein sequence, although some generalizations have been made about the sequence immediately following the signal peptide (Boyd and Beckwith, 1990; Yamane and Mizushima, 1988). Therefore it is possible to find target proteins in the cyto￾plasm (with uncleaved signal sequence) or in the periplasm in partially processed form, in place of or in addition to the expected periplasmic processed species. In some cases the proportion of protein that is exported can be increased by lowering the tem￾perature 15 to 30°C during induction. Post-translational Modification E. coli does not glycosylate or phosphorylate proteins or recognize proteolytic processing signals from eukaryotes, so take this into account when designing the cloning strategy. If proteolytic processing is needed, it is best to express only the coding sequences for the fully processed protein. If the protein of interest requires glycosylation for activity, and full activity is important in the final use, consider a eukaryotic host, such as Pichia, insect cells, or mammalian cells. Is the Protein Potentially Toxic? Consider whether the protein of interest is likely to have a toxic effect on the host cell.Where the function of the protein is known, this can be guessed at with some accuracy. For example, non￾specific proteases, nucleases, or pore-forming membrane proteins might all be expected to have some toxic effect on E. coli. Expres￾sion of toxic proteins may be very low, and there will be strong selective pressure on cells to eliminate the gene of interest by point mutation to change the translation frame, insertion of a stop codon, or change in an amino acid residue critical to the protein’s function. Larger deletion of parts of the plasmid may also be seen. If there is a suggestion that the gene product will be toxic, use an expression vector with a tightly regulated promoter (e.g., T7, pET E. coli Expression Systems 469