E. coli Expression Systems Can Produce Large Quantities of Proteins from Cloned Genes
Many protein hormones and other signaling or regulatory proteins are normally expressed at very low concentrations, which precludes their isolation and purification in large quantities by standard biochemical techniques. Widespread therapeutic use of such proteins, as well as basic research on their structure and functions, depends on efficient procedures for producing them in large amounts at reasonable cost. Recombinant DNA techniques that turn E. coli cells into factories for synthesizing low-abundance proteins are now used to produce granulocyte colony-stimulating factor (G-CSF), insulin, growth hormone, erythropoietin, and other human proteins with therapeutic uses commercially. For example, G-CSF stimulates the production of granulocytes, the phagocytic white blood cells that are critical to defense against bacterial infections. Administration of G-CSF to cancer patients helps offset the reduction in granulocyte production caused by chemotherapeutic agents, thereby protecting patients against serious infection while they are receiving chemotherapy.
The first step in producing large amounts of a low-abundance protein is to obtain a cDNA clone encoding the full-length protein by the methods discussed previously. The second step is to engineer plasmid vectors that will express large amounts of the encoded protein when they are inserted into E. coli cells. The key to designing such expression vectors is inclusion of a promoter, a DNA sequence from which transcription of the cDNA can begin. Consider, for example, the relatively simple system for expressing G-CSF shown in Figure 6-28. In this case, G-CSF is expressed in E. coli transformed with plasmid vectors that contain the lac promoter adjacent to the cloned cDNA encoding G-CSF. Transcription from the lac promoter occurs at high rates only when lactose, or a lactose analog such as isopropylthiogalactoside (IPTG), is added to the culture medium. Even larger quantities of a desired protein can be produced in more complicated E. coli expression systems.
EXPERIMENTAL FIGURE 6-28 Some eukaryotic proteins can be produced in E. coli cells from plasmid vectors containing the lac promoter. (a) This plasmid expression vector contains a fragment of the E. coli chromosome containing the lac promoter and the neighboring lacZ gene. In the presence of the lactose analog IPTG, RNA polymerase normally transcribes the lacZ gene, producing lacZ mRNA, which is translated into the encoded protein, β-galactosidase. (b) The lacZ gene can be cut out of the expression vector with restriction enzymes and replaced by a cloned cDNA, in this case, one encoding granulocyte colony-stimulating factor (G-CSF). When the resulting plasmid is inserted into E. coli cells, addition of IPTG and subsequent transcription from the lac promoter produce G-CSF mRNA, which is translated into G-CSF protein.
To aid in purification of a eukaryotic protein produced in an E. coli expression system, researchers often modify the cDNA encoding the recombinant protein to facilitate its separation from endogenous E. coli proteins. A commonly used modification of this type is the addition of a short nucleotide sequence to the end of the cDNA, so that the expressed protein will have six histidine residues at the C-terminus. Proteins modified in this way bind tightly to an affinity matrix that contains chelated nickel atoms, whereas E. coli proteins do not bind to such a matrix. The bound proteins can be released from the nickel atoms by decreasing the pH of the surrounding medium. In most cases, this procedure yields a pure recombinant protein that is functional, since addition of short amino acid sequences to either the C-terminus or the N-terminus of a protein usually does not interfere with the protein’s biochemical activity.