vectors). Minimize propagation of the cells to avoid opportunities for mutation and recombination Each requirement placed on a recombinant protein will affect the choice of expression system. If a protein is to be used only to prepare antibody, it need not be soluble or active, and the pro- duction of inclusion bodies(aggregates of improperly folded protein)in E. coli may be all that is needed. Alternatively, if a proteins biological activity will be assayed, or if it is to be used in structural studies(NMR, crystallography, etc. ) a properly folded and soluble form will be required Will Structural Changes(Additional or Fewer Amino Acids) Affect Your Application? Depending on the way that a gene is inserted in an expression vector, additional sequences may be added to the clone, and these may lead to extra amino acid residues at the N-or C-termini of the final expressed protein. In many cases these will have no dele terious effect, but if structural studies or precise comparisons to a native protein are to be done, it is wise to eliminate amino acids added by cloning steps. PCR amplification is the most commonly used method to generate inserts for expression, and proper desigN of PCR primers can eliminate most or all additional residues in he protein. Is the Sequence of Your protein Recognized by Specific Proteases? If you plan to express your gene in a fusion vector that prov an internal protease cleavage site for removal of the affinity tag (discussed below ), check that your native protein is not recognized by the protease. Most proteases are highly specific, but thrombin has a variety of secondary cleavage sites( Chang, 1985). Advertisements for Commercial Expression vectors Are Very Promising. What Levels of Expression Should You Expect? There are several systems available for protein expressio mammalian, insect, yeast, and E. coli. While it is impossible to predict the yields of protein from these systems for any given protein, some rough guidelines can be given. For any vector it is possible that no expression will be seen! Reported yields in stably transfected mammalian cells are in the range of 1 to 100 ug/10 470 Bellvectors). Minimize propagation of the cells to avoid opportunities for mutation and recombination. Must Your Protein Be Functional? Each requirement placed on a recombinant protein will affect the choice of expression system. If a protein is to be used only to prepare antibody, it need not be soluble or active, and the pro￾duction of inclusion bodies (aggregates of improperly folded protein) in E. coli may be all that is needed. Alternatively, if a protein’s biological activity will be assayed, or if it is to be used in structural studies (NMR, crystallography, etc.), a properly folded and soluble form will be required. Will Structural Changes (Additional or Fewer Amino Acids) Affect Your Application? Depending on the way that a gene is inserted in an expression vector, additional sequences may be added to the clone, and these may lead to extra amino acid residues at the N- or C-termini of the final expressed protein. In many cases these will have no dele￾terious effect, but if structural studies or precise comparisons to a native protein are to be done, it is wise to eliminate amino acids added by cloning steps. PCR amplification is the most commonly used method to generate inserts for expression, and proper design of PCR primers can eliminate most or all additional residues in the protein. Is the Sequence of Your Protein Recognized by Specific Proteases? If you plan to express your gene in a fusion vector that provides an internal protease cleavage site for removal of the affinity tag (discussed below), check that your native protein is not recognized by the protease. Most proteases are highly specific, but thrombin has a variety of secondary cleavage sites (Chang, 1985). Advertisements for Commercial Expression Vectors Are Very Promising.What Levels of Expression Should You Expect? There are several systems available for protein expression in mammalian, insect, yeast, and E. coli. While it is impossible to predict the yields of protein from these systems for any given protein, some rough guidelines can be given. For any vector it is possible that no expression will be seen! Reported yields in stably transfected mammalian cells are in the range of 1 to 100mg/106 470 Bell