Is Screening Necessary Prior to Expression 476 What Aspects of Growth and Induction Are Critical to Success? What Are the Options for Lysing Cells? Troubleshooting No Expression of the Protein The Protein Is Expressed, but It Is Not the Expected Size Based on Electrophoretic Analysis The protein is Insoluble. now what? 48 Solubility Is Essential. What Are Your Options? The Protein Is Made, but Very Little Is Full-Length; Most of It Is Cleaved to Smaller fragments 483 Your Fusion Protein Wont Bind to Its Affinity Resin 484 Your Fusion Protein Won't Digest Cleavage of the Fusion Protein with a Protease Produced tra bands 485 Extra Protein Bands Are Observed after Affinity Must the Protease Be Removed after Digestion of the..485 Purification Fusion protein? Bibliography.... Over the past decade the variety of hosts and vector systems for recombinant protein expression has increased dramatically Researchers now select from among mammalian, insect, yeast, and prokaryotic hosts, and the number of vectors available for use in these organisms continues to grow. With the increased availabil ding sequencing information, it is certain that these and other, yet to be developed systems will be important in the future. Despite the development of eukaryotic systems, E coli remains the most widely used host for recombi nant protein expression. E. coli is easy to transform, grows quickly in simple media, and requires inexpensive equipment for growth and storage. And in most cases, E coli can be made to produce adequate amounts of protein suitable for the intended application The purpose of this chapter is to guide the user in selecting the appropriate host and troubleshooting the process of recombinant protein expression. EXPRESSION VECTOR STRUCTURE What Makes a Plasmid an Expression Vector? Vectors for expression in E. coli contain at a minimum the following elements BellIs Screening Necessary Prior to Expression? . . . . . . . . . . . . 476 What Aspects of Growth and Induction Are Critical to Success? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478 What Are the Options for Lysing Cells? . . . . . . . . . . . . . . . . 479 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480 No Expression of the Protein . . . . . . . . . . . . . . . . . . . . . . . . . 480 The Protein Is Expressed, but It Is Not the Expected Size Based on Electrophoretic Analysis . . . . . . . . . . . . . . . . . . . 480 The Protein Is Insoluble, Now What? . . . . . . . . . . . . . . . . . . 481 Solubility Is Essential. What Are Your Options? . . . . . . . . . . . 482 The Protein Is Made, but Very Little Is Full-Length; Most of It Is Cleaved to Smaller Fragments . . . . . . . . . . . 483 Your Fusion Protein Won’t Bind to Its Affinity Resin . . . . . . 484 Your Fusion Protein Won’t Digest . . . . . . . . . . . . . . . . . . . . . 485 Cleavage of the Fusion Protein with a Protease Produced Several Extra Bands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 Extra Protein Bands Are Observed after Affinity Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 Must the Protease Be Removed after Digestion of the Fusion Protein? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486 Over the past decade the variety of hosts and vector systems for recombinant protein expression has increased dramatically. Researchers now select from among mammalian, insect, yeast, and prokaryotic hosts, and the number of vectors available for use in these organisms continues to grow. With the increased availabil￾ity of cDNAs and protein coding sequencing information, it is certain that these and other, yet to be developed systems will be important in the future. Despite the development of eukaryotic systems, E. coli remains the most widely used host for recombi￾nant protein expression. E. coli is easy to transform, grows quickly in simple media, and requires inexpensive equipment for growth and storage. And in most cases, E. coli can be made to produce adequate amounts of protein suitable for the intended application. The purpose of this chapter is to guide the user in selecting the appropriate host and troubleshooting the process of recombinant protein expression. EXPRESSION VECTOR STRUCTURE What Makes a Plasmid an Expression Vector? Vectors for expression in E. coli contain at a minimum, the following elements: 462 Bell