Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Use of DNA Clones
Use of E. coli Expression Systems

Most hormonal Proteins and other signaling or regulatory proteins are expressed in the body in such small quantities that it is virtually impossible to isolate them in pure form in significant amounts using standard biochemical techniques. The widespread THERAPEUTIC USE OF such proteins, as well as fundamental research into their Structure and function, depends on The ability to synthesize them in large quantities at reasonable production costs.

Today, recombinant DNA techniques are widely employed to transform E. coli Cells into "factories" for the synthesis of such proteins. For example, commercial production has been established for factor VIII (a Blood-clotting factor), granulocyte colony-stimulating factor (G-CSF), Insulin, Growth Hormone, and other human proteins for therapeutic Applications.

For instance, G-CSF stimulates The production of granulocytes, which are phagocytic white Blood Cells responsible for defending the body against bacterial infections. Administering G-CSF to Cancer patients helps counteract the Chemotherapy-induced decline in granulocyte production, thereby protecting patients from severe infections during their chemotherapy courses.

To produce "exotic" proteins in large quantities, it is first necessary to synthesize a cDNA clone encoding the protein of interest. Second, one must construct a plasmid vector capable of expressing high levels of this protein when introduced into an E. coli Cell. A key element in creating such expression vectors is the inclusion of a promoter—a nucleotide sequence (DNA fragment) where cDNA METABOLISM/31.html">Transcription initiates.

As an example, let us consider a relatively simple expression vector designed for the expression of the G-CSF protein (Figure 107).

The original plasmid vector contains an E. coli chromosomal fragment that includes the lac promoter and the lacZ Gene, which encodes the protein ß-galactosidase. When lactose (or a lactose analog such as isopropyl β-D-1-thiogalactopyranoside, IPTG (Figure 23)) is present in The Cell, RNA polymerase efficiently transcribes the lacZ gene, synthesizing lacZ mRNA, which is subsequently translated into the ß-galactosidase protein (Figure 107, top).

The Modification of the plasmid vector involves the following steps: the lacZ gene is excised from the original vector using restriction Enzymes, and the cloned cDNA—in our case, the gene encoding the G-CSF protein—is inserted in its place.

Following E. coli transformation (the uptake of the plasmid vector into the cell), The addition of lactose or IPTG to the culture medium induces transcription from the lac promoter to produce G-CSF mRNA, which is then efficiently translated into the G-CSF protein.

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Figure 107 - Expression of G-CSF cDNA in E. coli cells

Similarly, more sophisticated expression vectors can be engineered to establish advanced expression systems in E. coli for the synthesis of large quantities of desired proteins.

To simplify the Isolation of the synthesized recombinant protein from the culture medium, a cDNA modification method is commonly employed. Typically, a short nucleotide sequence is added to the end of the cDNA so that the expressed protein carries a stretch of six Histidine residues (a hexahistidine tag) at its C-terminus.

Proteins modified in this manner bind tightly to an affinity matrix containing chelated nickel atoms, whereas the majority of endogenous E. coli proteins fail to immobilize on this matrix. The nickel-bound proteins can subsequently be eluted by lowering the pH of the medium.

In most cases, this approach yields a pure, functionally active recombinant protein, since the addition of a short nucleotide sequence to either the C- or N-terminus of the polypeptide chain generally does not affect the protein's biochemical activity.