Is an Insect Cell System Suitable for the Expression of Your protein Should You express Your Protein in an insect Cell Line or Recombinant baculovirus? Procedures for Preparing Recombinant Baculovirus 524 Criteria for Selecting a Transfer Vector Which Insect Cell Host Is Most Appropriate for Your 525 Implementing the Baculovirus Experiment 527 What Special Considerations Are There for Expressin..527 What's the Best Approach to Scale-Up? What Special Considerations Are There for Expressing..527 Secreted Proteins? Glycosylated Proteins 528 What Are the Options for Expressing More Than One Protein? How Can You obtain maximal Protein yields? 529 x What ls the Best Way to Process Cells for Purification..530 Troubleshooting 530 Suboptimal Growth Conditions 530 Viral Production problems 53 Mutation Solubility Problems 532 532 Bibliography∴ 533 SECTION A: A PRACTICAL GUIDE TO EUKARYOTIC EXPRESSION Recombinant gene expression in eukaryotic systems is often the nly viable route to the large-scale production of authentic, po translationally modified proteins. It is becoming increasingly easy to find a suitable system to overexpress virtually any gene pre provided that it is properly engineered into an appropriate expres- sion vector. Commercially available systems provide a wide range of possibilities for expression in mammalian, insect, and lower eukaryotic hosts, each claiming the highest possible expression levels with the least amount of effort. Indeed, many of these systems do offer vast improvements in their ease of use and rapid end points over technologies available as re ago. In addition methods of transferring dna into cells have advanced in parallel enabling transfection efficiencies approach ing 100%. However, one still needs to carefully consider the most 492 Trill et alIs an Insect Cell System Suitable for the Expression of Your Protein? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 Should You Express Your Protein in an Insect Cell Line or Recombinant Baculovirus? . . . . . . . . . . . . . . . . . . . . . . . . . . 522 Procedures for Preparing Recombinant Baculovirus . . . . . . 524 Criteria for Selecting a Transfer Vector . . . . . . . . . . . . . . . . . 524 Which Insect Cell Host Is Most Appropriate for Your Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 Implementing the Baculovirus Experiment . . . . . . . . . . . . . . . . 527 What’s the Best Approach to Scale-Up? . . . . . . . . . . . . . . . . 527 What Special Considerations Are There for Expressing Secreted Proteins? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527 What Special Considerations Are There for Expressing Glycosylated Proteins? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528 What Are the Options for Expressing More Than One Protein? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 How Can You Obtain Maximal Protein Yields? . . . . . . . . . . . 529 What Is the Best Way to Process Cells for Purification?. . . 530 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530 Suboptimal Growth Conditions . . . . . . . . . . . . . . . . . . . . . . . . 530 Viral Production Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531 Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531 Solubility Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 SECTION A: A PRACTICAL GUIDE TO EUKARYOTIC EXPRESSION Recombinant gene expression in eukaryotic systems is often the only viable route to the large-scale production of authentic, post￾translationally modified proteins. It is becoming increasingly easy to find a suitable system to overexpress virtually any gene product, provided that it is properly engineered into an appropriate expres￾sion vector. Commercially available systems provide a wide range of possibilities for expression in mammalian, insect, and lower eukaryotic hosts, each claiming the highest possible expression levels with the least amount of effort. Indeed, many of these systems do offer vast improvements in their ease of use and rapid end points over technologies available as recently as 5 to 10 years ago. In addition methods of transferring DNA into cells have advanced in parallel enabling transfection efficiencies approach￾ing 100%. However, one still needs to carefully consider the most 492 Trill et al