Molecular Biotechnology: Principles and Applications - Glick, B. R., Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Chimeric Proteins

Very often, foreign Proteins—especially small ones—are detected in heterologous host Cells only in minimal amounts. This seemingly low level of expression of their encoding genes is in many cases explained by the degradation of foreign proteins within the host cells. One way to solve this problem involves the covalent attachment of the cloned Gene product to some stable host Cell protein. In such a construct, known as a “fusion protein,” the cloned gene product is protected from Cleavage by host cell proteases, as demonstrated in experimental studies.

Protein fusion is programmed at the DNA level by ligating the coding Regions of the respective genes. In its simplest form, a fusion vector system involves the insertion of a target gene or its segment into the coding region of a cloned host gene. It is critical that the RNA transcribed from the cloned target gene has the correct nucleotide sequence to ensure the proper production of the cloned gene product. If a shift in the reading frame occurs during DNA segment joining—that is, if the codon sequence determines a truncated or incorrect Translation product—a functionally active form of the protein cannot be formed either. The correctness of the reading frame can be verified in various ways. Typically, this requires knowing the exact nucleotide sequence of the ligated DNA fragments.

Cleavage of Fusion Proteins

Depending on the intended use of the cloned gene protein product, it may be used as is or as part of a fusion protein, although the latter option is less common. For instance, due to the presence of the host protein fragment, most fusion proteins prove unsuitable for clinical Applications, and the target gene product itself may turn out to be inactive. Furthermore, fusion proteins are subject to more complex testing Procedures that they must pass to obtain regulatory approval from relevant agencies. All this necessitates finding ways to remove extraneous Amino acid sequences from the resulting product molecule. One such approach is based on attaching the protein encoded by the target gene to a host cell protein containing a short peptide recognized by a specific non-bacterial protease. This attachment is also programmed at the DNA level. Oligonucleotide linkers bearing protease cleavage sites can be appended to the cloned gene before this construct is introduced into an expressing fusion vector system. A linker can be, for example, an oligonucleotide encoding the peptide Ile-Glu-Gly-Arg. Following the synthesis and purification of the fusion protein, Blood Coagulation factor Xa can be used to separate the protein product encoded by the cloned gene; factor Xa is a specific proteinase that cleaves peptide bonds exclusively at the C-terminus of the Ile-Glu-Gly-Arg sequence (Fig. 6.6). Moreover, since this peptide is relatively rare in natural proteins, this approach can be applied to the Separation of many other products encoded by cloned genes.

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Fig. 6.6. Proteolytic cleavage of a fusion protein by blood coagulation factor X_a. Factor X_a recognizes the Amino Acid Sequence separating the two Components of the fusion protein. Cleavage releases the functional protein encoded by the cloned gene.

Applications of Fusion Proteins In some cases, the final product intended for use is the fusion protein itself. For example, there is frequently a need to produce Antibodies that recognize a specific region of a protein molecule. To accomplish this, a DNA segment encoding the protein domain against which the desired antibodies will be raised can be inserted into a suitable vector. The resulting fusion protein then serves as the antigen. Antibodies directed against the stabilizing host-derived protein component can be removed by absorption on the pure stabilizing protein, leaving only the antibodies that bind to the desired amino acid sequence.

One cloning fusion vector designed to produce specific antibodies contains the 5'-terminal segment of the E. coli ompF gene, which encodes an outer membrane protein, and the adjacent portion of the E. coli lacZ (ß-galactosidase) gene (Fig. 6.7). This segment contains the information required to initiate METABOLISM/31.html">Transcription and Introduction/27.html">Translation of the fusion gene, as well as for the secretion of the fusion protein. Although the truncated lacZ gene lacks the codons for the first Eight Amino Acids, the protein it encodes retains enzymatic activity. In this form, (β-galactosidase) is able to function regardless of which Peptides are attached to its N-terminus. The lacZ gene is inserted into the vector in such a way that it is out of frame with the leader sequence of the ompF gene; consequently, active ß-galactosidase is not produced. However, if the reading frame of a cloned DNA fragment matches that of the ompF and lacZ genes, a three-component fusion protein is formed, consisting of the OmpF fragment, the protein encoded by the cloned gene, and the functionally active C-terminal portion of ß-galactosidase. It can be used as an antigen to raise antibodies cross-reacting with the cloned gene protein, or as a tool to obtain small fragments of specific proteins.

Fig. 6.7. A fusion cloning vector. It contains an ampicillin resistance gene (Amp^r) as a selectable marker, the 5'-terminal segment of the ompF gene encoding the N-terminus of an outer membrane protein, an AbcI restriction endonuclease site, and a truncated ß-galactosidase (lacZ) gene. The gene to be cloned is inserted into the AbcI site. Following transcription and translation of this genetic construct, a three-component fusion protein is produced.

Table 6.3. Purification of fusion proteins produced by E. coli¹⁾

¹⁾ Based on Nygren et al., 1994, Trends Biotechnol. 12: 184—188.

²⁾ ZZ is a protein A fragment from Staphylococcus aureus; Strep-tag is a peptide with an affinity for streptavidin; PinPoint is a protein fragment biotinylated in E. coli in vivo; MBP is maltose-binding protein; GST is glutathione S-transferase; Flag is a peptide recognized by enterokinase.

Fusion proteins are utilized not only to stabilize Polypeptides but also to simplify the purification Procedure of recombinant proteins (Table 6.3). For example, a Saccharomyces cerevisiae plasmid construct harboring the human interleukin-2 gene with an attached DNA segment encoding the marker peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (sold under the trade name Flag) serves a dual function: it ensures the stabilization of the interleukin-2 gene product and facilitates its purification. Interleukin-2 is a biological factor that stimulates T-cell growth and B-cell antibody synthesis. The fusion protein resulting from the expression of this genetic construct in Yeast cells can be purified in a single step via immunoaffinity Chromatography. To achieve this, Monoclonal Antibodies against the marker peptide are immobilized on a polypropylene support, and the fusion protein is passed through a Column, where it binds to these antibodies (Fig. 6.8). The marker peptide is a small molecule, consuming only a minimal fraction of cellular resources. The fusion protein exhibits the same biological activity as native interleukin-2. However, if it is intended for clinical use, the marker peptide must be removed, as mandated by regulatory agencies overseeing pharmaceutical applications. Bovine enterokinase can be employed for this purpose.

Many proteins produced by E. coli accumulate within cells in the form of insoluble, biologically inactive inclusion bodies. Although biologically active protein can often be recovered from such structures in small quantities, this requires prolonged solubilization. The poor Solubility of proteins in vivo is frequently caused by improper folding, and various strategies have been attempted to resolve this issue. For instance, fusion proteins in which one of the components is thioredoxin—an 11.7 kDa protein—remain in solution even when they account for 40% of the total cellular protein. With this in mind, the target gene was inserted into the polylinker immediately downstream of the thioredoxin gene, placing both genes under the control of the p^L promoter in an E. coli plasmid vector (Fig. 6.9). The chromosome of host E. coli cells used in this system contains a genetic construct specifying The production of the cI repressor—a copy of the cI gene under the transcriptional control of the trp promoter. In the absence of Tryptophan (Fig. 6.9, A), the repressor is produced in sufficient quantities to block transcription from the p^L promoter, and the fusion protein is not synthesized. When tryptophan is added to the medium (Fig. 6.9, B), the trp promoter is switched off, the repressor protein is no longer synthesized, and the fusion protein Genes are transcribed from the plasmid p^L promoter. The synthesized fusion protein, consisting of thioredoxin and the target protein, accumulates primarily in specialized regions on the inner side of the E. coli Plasma Membrane known as adhesion zones, and is released from the cells upon osmotic Shock. Subsequently, the target protein can be cleaved from the fusion protein using enterokinase. Thioredoxin-containing fusion Proteins can also be purified by another method. If the target protein remains stable at elevated temperatures—since thioredoxin is not denatured by heating up to 80 °C—the fusion protein can be incubated at high temperatures to eliminate the majority of other cellular proteins that denature under these conditions.

Fig. 6.8. Purification of a fusion protein via immunoaffinity chromatography. Antibodies against the marker peptide of the fusion protein are immobilized on a solid support, and the fusion protein is passed through the column. The marker peptide, as part of the fusion protein, binds to the antibodies, while all other proteins pass freely through the column. The purified fusion protein is then eluted from the column.

Incorporation of Proteins into Surface Structures

To screen large complementary DNA (cDNA) libraries (up to 5–10¹⁰ clones) encoding rare proteins, specialized fusion systems have been developed. Typically, cDNA is inserted into The genes of surface proteins (filament or pilus proteins) of filamentous Bacteriophages (such as M13) or Bacteria; following transcription and translation, fusion proteins are produced that become part of the Surface structures of these microorganisms. Here, they are identified using immunological Methods. Often, the surface protein gene pIII of phage M13, which binds to E. coli F-pili and initiates infection, is used for fusions. For the cloning of cDNA and other coding sequences, a plasmid (phagemid) was constructed containing a small fragment of M13 DNA ensuring its in vitro packaging into phage particles, the pIII protein gene under the control of a regulated bacterial promoter (such as the E. coli lac promoter), and a cloning site near the 5'-end of the pIII gene. After Replication of the recombinant M13 phage in E. coli, the target protein becomes fused to the N-terminus of the phage protein, and plaques containing it can be identified immunologically. Recombinant phagemids isolated from such plaques can serve as a source of the corresponding cDNA. This highly efficient Selection system enables the detection of cDNAs for rare yet crucial proteins.

Fig. 6.9. Expression of a plasmid vector carrying the thioredoxin gene–target protein gene genetic construct in the absence (A) and presence (B) of tryptophan. Arrows labeled p^Trp and p^L indicate the direction of transcription. Abbreviations and designations: o^Trp, operator bound by the trp repressor; o^L, operator bound by the cI repressor; p^Trp, trp promoter; p^L, bacteriophage λ left promoter; TT, transcription termination signal. A nucleotide sequence encoding an enterokinase-cleavable peptide is located between the thioredoxin and target protein genes. Horseshoe-shaped curves depict the binding of repressors to their respective operators.

Libraries containing bacterial surface protein genes can also be used to identify clones carrying specific nucleotide sequences. To incorporate a gene of interest into the surface structures of a Gram-negative bacterium such as E. coli, its genes are fused with the genes encoding the proteins of that Structure. Bacterial proteins commonly employed for this purpose include outer membrane protein A (OmpA) and peptidoglycan-associated lipoprotein (PAL) of E. coli, as well as outer membrane protein F (OprF) of Pseudomonas aeruginosa. In such constructs, the target protein is usually located at either the C- or N-terminus of the fusion protein, although short polypeptides are occasionally inserted within the middle of the bacterial protein molecule (Fig. 6.10).

Fig. 6.10. Fusion proteins consisting of a bacterial surface protein and a foreign target protein attached to its N- or C-terminus (A) or inserted into exposed regions of the molecule (B). In both cases, the foreign peptides or protein end up displayed On the surface of the bacterial cell.

Fusion systems with target protein localization on the bacterial cell surface can also be employed for the overproduction of certain PROTEINS AND PEPTIDES. For instance, in one study, the gene encoding the major outer membrane protein of Pseudomonas aeruginosa (OprF) was engineered to incorporate the gene for an antigenic determinant of the malaria pathogen Plasmodium falciparum. Bacterial cells synthesizing the corresponding chimeric protein tested positive with monoclonal antibodies against P. falciparum. Consequently, surface chimeric Proteins can be utilized as Vaccines (Ch. 11).