Screening and optimizing protein production in E. coli.
Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects. These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost. The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years. For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster. The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups. This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format.
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Lorraine
L
Hewitt
James M
JM
McDonnell
D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D005782:Gels; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D005782:Gels; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA