Molecular Biotechnology. Bernard R. Glick
Figure 3.9 Expression of a soluble fusion protein. To avoid formation of insoluble inclusion bodies, the gene encoding the target protein is cloned adjacent to, and in the same reading frame as, the thioredoxin gene and expressed as a single polypeptide. ATG encodes the start codon; TGA encodes a stop codon; the arrow indicates the transcription start site.
Decreasing Protein Degradation
Specific amino acids at the N terminus can increase the stability of a protein, probably by making it less susceptible to degradation by cellular proteases. For example, alteration of the N terminal amino acids increased the in vitro survival time of β-galactosidase from approximately 2 minutes to more than 20 hours (Table 3.7). Amino acid additions that extend the intrinsic survival of a protein can be readily incorporated into cloned genes. Often the presence of a single extra amino acid at the N-terminal end is sufficient to stabilize a target protein. Long-lived proteins can accumulate in cells and thereby increase the yield of the product. This phenomenon occurs in both prokaryotes and eukaryotes.
Table 3.7 Stability of β-galactosidase with certain amino acids added to its N terminus
Specific internal amino acid sequences can also make a protein more susceptible to proteolytic degradation. For example, proteins that contain PEST sequences, which are rich in proline (P), glutamic acid (E), serine (S), and threonine (T), and are often flanked by clusters of positively charged amino acids such as lysine and arginine, are marked for degradation within the cell. In some instances, it is possible to enhance the stability of a protein by altering its PEST regions by genetic manipulation. Such changes, of course, must not alter the function of the target protein.
In a number of studies, proteins synthesized from cloned genes have been found to be more resistant to degradation by host cell proteases when they are part of a fusion protein, in which the fusion partner is not especially susceptible to proteolysis. Alternatively, stable foreign proteins can be produced in bacterial host strains that are deficient in the production of proteases. However, this is not as simple as it might appear as E. coli has at least 25 different proteases. Moreover, these proteases are important for the degradation of abnormal or defective proteins, which is a housekeeping function that is necessary for the continued viability of the cells. In one study, strains with mutations in one or more protease genes were constructed. Generally, the strains that were most deficient in overall protease activity grew most slowly. Thus, decreasing protease activity caused cells to be debilitated. However, an E. coli strain with mutations in both the gene for the RNA polymerase sigma factor that is responsible for heat shock protein synthesis (rpoH) and the gene for a protease that is required for cell growth at high temperatures (degP) secreted target proteins that had a 36-fold-greater specific activity than when they were produced in wild-type host cells. This increase in activity reflects a decrease in the proteolytic degradation of these secreted proteins.
Increasing Protein Secretion
E. coli and other Gram-negative bacteria have multiple pathways for the secretion of various proteins. One of these systems (general secretion pathway) uses a membrane-bound protein complex for transmitting a secretory protein through the inner membrane into the periplasm (Fig. 3.10A). Some secretory proteins are then secreted across the outer membrane to the external environment. Directing a foreign protein to the periplasm or the growth medium makes its purification easier and less costly, as many fewer proteins are present outside the cytoplasmic membrane than in the cytoplasm. Moreover, the stability of a cloned protein depends on its cellular location in E. coli. For example, recombinant proinsulin is approximately 10 times more stable if it is exported into the periplasm than if it is localized in the cytoplasm. Secretion of proteins to the periplasm facilitates the correct formation of disulfide bonds because the periplasm provides an oxidative environment, in contrast to the more reducing environment of the cytoplasm. Table 3.8 indicates the amounts of secreted recombinant pharmaceutical proteins attainable in various bacterial systems.
Figure 3.10 Protein secretion in bacteria. (A) Type II secretion pathway in Gram-negative bacteria. The SecB protein binds to a secretory protein in the cytoplasm (1). SecB attaches to the SecA protein that is part of the Sec complex of the inner membrane (2), and the secretory protein is translocated through the inner membrane (3). A signal peptidase removes the signal peptide and the secretory protein is properly folded in the periplasm (4). The secretory protein combines with the protein complex of the general secretory pathway (Gsp) (5) and is translocated across the outer membrane to the external environment (6). (B) Protein secretion in gram-positive bacteria. A signal recognition particle (SRP) binds to the signal peptide of a secretory protein, and this complex binds to a membrane protein that directs the secretory protein (1) to the Sec complex. There is also an SRP-independent pathway (2), where a signal peptide alone makes contact with the Sec complex. The secretory protein is translocated through a channel within the Sec complex (3), and the signal peptide is removed by a signal peptidase. Proper folding of the secretory protein occurs as it passes through the cell wall (4).
Table 3.8 Yields of several secreted recombinant proteins produced in different bacteria
Secretion into the Periplasm
An amino acid sequence called the signal peptide (also called the signal sequence, or leader peptide), located at the N-terminal end of a secretory protein, facilitates its export by enabling the protein to pass through the secretion (Sec) complex in the cell membrane (Fig. 3.10). It is sometimes possible to engineer a protein for secretion to the periplasm by adding the DNA sequence encoding a signal peptide to the cloned gene. When the recombinant protein is secreted into the periplasm, the signal peptide is precisely removed by a signal peptidase that is a component of the secretion apparatus so that the N-terminal end of the target protein is identical to that of the natural protein.
The presence of a signal peptide sequence does not necessarily guarantee a high rate of secretion. When the fusion of a target gene to a DNA fragment encoding a signal peptide is ineffective in producing a secreted protein product, alternative strategies need to be employed. One approach that was found to be successful for the secretion of the protein interleukin-2 (a cytokine that stimulates both T-cell growth and B-cell antibody synthesis) was the fusion of the interleukin-2 gene downstream from the gene for the entire maltose-binding protein, rather than just the maltose-binding protein signal sequence (Fig. 3.11). When this genetic fusion was introduced into E. coli cells on a plasmid vector, a large fraction of the fusion protein was localized in the periplasm. DNA encoding the recognition site for a protease (such as factor Xa) was included between the two genes to release functional interleukin-2 from the fusion protein by digestion with the protease (see below).
Figure 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