Distance Learning: Techniques in Structural Molecular Biology (TSMB)

This section provides an introduction to recombinant protein expression in the bacterial host E.coli. This includes a description of tools available and factors influencing the successful production of pure, monodisperse recombinant protein. Protocols for production and handling of selenomethionine-labelled recombinant proteins are also covered. Case studies are described illustrating typical problems that can be encountered during protein expression together with current approaches to overcome them.

• Recombinant Gene Expression

Tools of the Trade
Construct Design
Expression Vectors
Control Elements, Promoters and Ribosome-Binding Sites
Affinity Tags
Site-specific Proteases for Affinity Tag Removal
E.coli Host Strains

• Factors Affecting Protein Yields

Culture conditions
Purification
Troubleshooting
In vitro Refolding of Recombinant Proteins from Inclusion Bodies

• Labelling Expressed Proteins

Expression of Selenomethionine-labelled Proteins
Radioisotope Labelling for NMR
Cell Free Protein Synthesis

Objectives

To give a practical introduction to the expression of recombinant proteins, including selenomethionine labelled recombinants, in E. coli. To describe the materials and methods employed and to explain the factors that affect the yield obtained. To illustrate the theoretical material with case studies. At the end of this section, students should

• Understand the theoretical background behind the practical technique of recombinant protein expression
• Know some of the tools and techniques used in practice
• Be able to "trouble-shoot" protein expression and to design simple experiments (on paper)
• Understand the importance of Selenomethionine labelled recombinants in structural biology, and know how they are produced
• Have studied case studies of protein expression in practice