3.11 Engineering the secretion of interleukin-2. (A) Interleukin-2 fused to the E. coli maltose-binding protein (MBP) signal peptide is not secreted. (B) When interleukin-2 is fused to the E. coli maltose-binding protein and its signal peptide, with the two proteins joined by a linker peptide, secretion occurs. Subsequently, the maltose-binding protein and the linker peptide are removed by digestion with factor Xa.
In many instances, when foreign proteins engineered for secretion are overproduced in E. coli, the precursor form is only partially processed, with about half of the secreted proteins retaining the leader peptide and the other half being fully processed to the mature form. This is probably the result of overloading some of the components involved in the secretion process. If this is the case, then it might be possible to increase the ratio of processed to unprocessed proteins by increasing the level of expression of some of the limiting components of the protein secretion pathway. This hypothesis was tested in a series of experiments in which a plasmid containing both the prlA4 and secE genes, which encode major components of the molecular apparatus that physically moves proteins across the membrane, was introduced into E. coli host cells. Following this augmentation of the host cell secretory machinery, the fraction of the cloned protein (in this case, the cytokine interleukin-6) that was secreted to the periplasm as the processed form with the signal peptide removed increased from about 50% to more than 90%.
Secretion into the Medium
The outer membrane of Gram-negative bacteria restricts the secretion of proteins into the surrounding medium. One solution is to produce the secretory protein in a Gram-positive bacterium such as L. lactis which lacks an outer membrane and therefore can secrete proteins directly into the medium (Fig. 3.10B). Alternatively, the Gram-negative bacterium can be modified to secrete proteins directly into the growth medium.
In general, relatively few proteins pass through the outer membrane of E. coli. However, some Gram-negative bacteria can secrete a bacteriocidal protein called a bacteriocin into the medium. A bacteriocin release protein activates phospholipase A, which is present in the bacterial inner membrane and cleaves membrane phosopholipids so that both the inner and outer membranes are permeabilized. Some cytoplasmic and periplasmic proteins are also released into the culture medium. Thus, by expressing the bacteriocin release protein gene from a strong regulatable promoter, E. coli cells may be permeabilized at will. The cells that carry the bacteriocin release protein gene are transformed with another plasmid carrying a cloned gene that has been fused to a secretion signal peptide sequence for transport across the inner membrane. The cloned gene is placed under the same transcriptional-regulatory control as the bacteriocin release protein gene so that the two genes can be induced simultaneously. Once in the periplasm, the target protein is released to the external medium via the permeablized outer membrane (Fig. 3.12).
Figure 3.12 E. coli cells engineered to secrete a foreign protein to the periplasm by fusing the gene of interest (green) to a secretion signal (A) and to the growth medium by permeabilizing cell membranes with a bacteriocin release protein encoded on another plasmid (red) (B).
Although secretion of E. coli proteins to the growth medium is quite rare, the small protein YebF is naturally secreted to the medium without lysing the cells or permeabilizing the membranes. When various proteins are fused to the C-terminal end of YebF, the entire fusion protein is secreted to the medium by an unidentified outer membrane receptor (Fig. 3.13). To date, researchers have reported the secretion to the medium of human interleukin-2 (a 15-kDa hydrophobic protein), bacterial α-amylase (a 48-kDa hydrophilic protein), and alkaline phosphatase (94 kDa), demonstrating that a wide range of proteins may be secreted to the medium using this system. It may be possible to employ other naturally secreted proteins in a similar manner.
Figure 3.13 Secretion, following expression in E. coli, of YebF–interleukin-2 fusion protein into the growth medium. The protein synthesized in the cytoplasm includes a signal peptide (yellow) that is excised when the fusion protein is secreted to the periplasm. The YebF–interleukin-2 fusion protein is then secreted from the periplasm, across the outer membrane, to the growth medium. YebF is shown in blue and interleukin-2 in green.
Facilitating Protein Purification
A number of purification tags have been developed to simplify the purification of recombinant proteins (Table 3.9). The basis for this approach is the expression of a target protein as a fusion protein with a short peptide sequence that has a high affinity for a protein, antibody, carbohydrate, or other ligand; for this reason, the peptide tags are often referred to as affinity tags. For example, the coding sequence for a target protein such as human interleukin-2 is joined to DNA encoding the affinity tag sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (where Asp, Tyr, and Lys indicate aspartic acid, tyrosine, and lysine, respectively) that has the dual function of reducing the degradation of the expressed interleukin-2 and then enabling the product to be purified. Following expression and secretion (during which the signal peptide is removed), the target protein can be purified from the medium in a single step by immunoaffinity chromatography. Monoclonal antibodies that bind to the affinity tag are immobilized on a polypropylene (or other solid) support and specifically capture the fusion protein (Fig. 3.14). In this case, the fusion protein was found to have the same biological activity as native interleukin-2.
Table 3.9 Some protein fusion systems used to facilitate the purification of foreign proteins in E. coli and other host organisms
Figure 3.14 Purification of a fusion protein by immunoaffinity chromatography. (A) An antibody (anti-tag antibody) that binds to a short peptide sequence (affinity tag) of the fusion protein (interleukin-2 in this example) is attached to a solid polypropylene support. The mixture of secreted proteins are passed through the column containing the bound antibody. (B) The affinity tag of the fusion protein binds to the antibody, and the other proteins pass through. The immunopurified fusion protein can then be selectively eluted from the column by the addition of pure affinity tag peptide.
In many instances antigen−antibody complexes that form during the immunoaffinity process are difficult to separate without the use of denaturing chemicals. As an alternative, it has become very popular to express a protein with an affinity tag that contains six or eight histidines attached to either the N- or C-terminal end of the target protein. Following expression and host cell lysis, the histidine-tagged protein, along with other cellular proteins, is then passed over an affinity column of nickel–nitrilotriacetic acid. The histidine-tagged protein, but not the other cellular proteins, binds tightly to the column. The bound protein is eventually eluted from the column by the addition of imidazole (the side chain of histidine). With this protocol, some cloned and overexpressed proteins have been purified up to 100-fold with greater than 90% recovery in a single step. In addition, this system can be utilized to purify denatured proteins, for example, following solubilization of inclusion bodies and before the solubilized proteins are renatured.
While purification tags and other fusion partners may not disrupt the function of a protein, to satisfy the government agencies that regulate the use of pharmaceuticals, it is still necessary to remove these sequences if the product is to be used for human immunotherapy or other
