TECHNIQUES

An expression vector is a plasmid that contains the properly positioned transcriptional and translational control sequences for protein expression. In order to produce bacterial proteins, a cloned structural gene must be inserted into the expression vector. With the use of a relaxed control plasmid and an efficient promoter (a type of transcriptional control element), the production of a protein of interest may reach a high level of the host's total cellular protein. Expression vectors may also be used to isolate specific cDNAs. We can identify specific eukaryotic cDNAs by looking for their gene products in bacteria after cloning the cDNA into appropriately constructed plasmids or phages (expression vectors). cDNAs are inserted into these vectors within regions that promote their expression in the cell (often E. coli). Often regulated bacterial promoters are used. Proteins may be expressed as fusion proteins in which amino acids from a prokaryotic protein are incorporated at one end of the eukaryotic protein. Fusion proteins are often more stable than the corresponding eukaryotic protein in bacteria and are therefore produced at higher levels.