Walter Fiers

Expression, purification and crystallization of fully active, glycosylated human interleukin-5

Recombinant human interleukin‐5 (hIL‐5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno‐affinity chromatography and gel filtration. Purified hIL‐5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 Å.

[26] Expression of heterologous unfused protein in Escherichia coli

Publisher Summary This chapter describes the use of plasmid expression vectors based on the inducible leftward promoter of coliphage λ. These vectors have been engineered to incorporate bacterial ribosome binding sites designed for the easy insertion of adventitious coding sequences. The chapter discusses examples of tailoring cloned cDNA to obtain a precise fusion of coding sequences for the prokaryotic translation signals. Expression vectors have been developed that incorporate the essential control elements to ensure an efficient transcription and translation of essentially any coding region in E. coli . The salient features of such an expression vector are (1) the presence of a strong and preferably regulatable promoter and (2) the presence of an efficient ribosome-binding site and initiation codon, which are easily accessible for the insertion of adventitious coding sequences. The most widely used strategy to clone eukaryotic genes is the reverse transcription of purified mRNA. The cDNA clones obtained in this way contain sequences upstream from the coding region that are not suitable for expression in bacterial systems. In cases where the mRNA codes for a secreted protein, the coding region of the mature protein is preceded by the signal peptide, which is removed during the process of secretion. To obtain efficient expression of the mature protein in E. coli , the cDNA needs to be tailored in such a way that the codon for the first amino acid of the mature protein can be precisely fused to the initiation codon of the bacterial expression vector. The chapter discusses the the two aspects to be considered in the overall design of an expression system for unfused proteins: (1) the construction of an expression vector suitable for easy insertion of adventitious coding sequences and (2) methods to tailor coding sequences such that these can be precisely joined to the expression signals of the vector. The chapter discusses human immune interferon, human tumor necrosis factor, and human interleukin 2.