and its stability in bacteria, that is, the N-end rule. They reported protein half-lives of only 2 minutes when the following amino acids were present at the amino terminus: Arg, Lys, Phe Leu, Trp, and Tyr. In contrast, all other amino acids conferred half-lives of >10 hours when present at the amino terminus of the protein examined. This suggests that one should examine the sequence to be expressed for the residue in the +2 position. If the residue is among those that destabilize the protein, it may be worth the effort to change this residue to one that confers stability WHICH PROTEIN EXPRESSION SYSTEM SUITS YOUR NEEDS? Track record What systems are currently used in the laboratory or by others in the field? If the protein coding sequence of interest is well characterized, and the protein or its close relatives have been expressed successfully by others in the field, it is wise to try the same expression system. Go with what has worked in the past If nothing else, results obtained using the familiar system will serve as a starting point. As an example, most of the recombinant expression of mammalian src homology SH2 protein interaction domains has been done using the pGeX vector series, and sim ilar examples of preferred systems are found in other fields of research. If little is known about the protein to be expressed, it is best to take stock of what information there is before entering the lab Before beginning any experimentation, it is wise to answer the following question What Do You Know about the gene to Be Expressed Source In general, simple globular proteins from prokaryotic and eukaryotic sources are good candidates for expression in e coli Monomeric proteins with few cysteines or prosthetic gr heme and metals)and of average size(<60kDa) will likely give good production. Secreted eukaryotic proteins and membrane bound proteins, especially those with several transmembrane domains, are likely to be problematic in E. coli Solubility of recombinant proteins in E coli can also be estimated by a math- ematical analysis of the amino acid sequences (wilkinson and Harrison. 1991 E coli Expression System 465and its stability in bacteria, that is, the “N-end rule.”They reported protein half-lives of only 2 minutes when the following amino acids were present at the amino terminus: Arg, Lys, Phe, Leu, Trp, and Tyr. In contrast, all other amino acids conferred half-lives of >10 hours when present at the amino terminus of the protein examined. This suggests that one should examine the sequence to be expressed for the residue in the +2 position. If the residue is among those that destabilize the protein, it may be worth the effort to change this residue to one that confers stability. WHICH PROTEIN EXPRESSION SYSTEM SUITS YOUR NEEDS? Track Record What systems are currently used in the laboratory or by others in the field? If the protein coding sequence of interest is well characterized, and the protein or its close relatives have been expressed successfully by others in the field, it is wise to try the same expression system. Go with what has worked in the past. If nothing else, results obtained using the familiar system will serve as a starting point. As an example, most of the recombinant expression of mammalian src homology SH2 protein interaction domains has been done using the pGEX vector series, and sim￾ilar examples of preferred systems are found in other fields of research. If little is known about the protein to be expressed, it is best to take stock of what information there is before entering the lab. Before beginning any experimentation, it is wise to answer the following question: What Do You Know about the Gene to Be Expressed? Source In general, simple globular proteins from prokaryotic and eukaryotic sources are good candidates for expression in E. coli. Monomeric proteins with few cysteines or prosthetic groups (e.g., heme and metals) and of average size (<60kDa) will likely give good production. Secreted eukaryotic proteins and membrane￾bound proteins, especially those with several transmembrane domains, are likely to be problematic in E. coli. Solubility of recombinant proteins in E. coli can also be estimated by a math￾ematical analysis of the amino acid sequences (Wilkinson and Harrison, 1991). E. coli Expression Systems 465