One of the most useful applications of recombinant DNA technology is the ability to artificially synthesise large quantities of natural or modified proteins in a host cell such as bacteria or yeast. The benefits of these techniques have been enjoyed for many years since the first insulin molecules were cloned and expressed in 1982 (Table 1). Contamination of proteins purified from native sources, such as in the case of the blood product factor VIII which was often contaminated with infectious agents, has also increased the need to develop effective vectors for production of foreign genes. In general, the expression of foreign genes is carried out in specialised cloning vectors in a host such as E.coli (Figure 1). It is possible to use cell-free transcription and translation systems that direct the synthesis of proteins without the need to grow and maintain cells. Cell-free in vitro protein expression is carried out with the appropriate amino acids, ribosomes, tRNA molecules, cofactors and isolated template mRNA or DNA. Wheat germ extracts or rabbit reticulocyte lysates can provide the necessary components and are usually the systems of choice for eukaryotic protein production. The resulting proteins may be detected by polyacrylamide gel electrophoresis or by immunological detection using Western blotting. These systems are ideal for the rapid production of proteins on a smaller scale than in vivo systems and are particularly useful in the study of protein functional analysis, such as post-translational modification, or folding and stability studies. Furthermore, it is possible to produce proteins that would be toxic to expression hosts in vivo.

Table1. A number of recombinant DNA-derived human therapeutic reagents

Fig1. Components of a typical prokaryotic expression vector. To produce a transcript (coding sequence) and translate it, a number of sequences in the vector are required. These include the promoter and ribosome binding site (RBS). The activity of the promoter may be modulated by a regulatory gene (R), which acts in a way similar to that of the regulatory gene in the lac operon. T indicates a transcription terminator.

Prokaryotic Expression Vectors

For a foreign gene to be expressed in a bacterial cell, it must have particular promoter sequences upstream of the coding region, to which the RNA polymerase will bind prior to transcription of the gene. The choice of promoter is vital for correct and efficient transcription, since the sequence and position of promoters are specific to a particular host such as E. coli. It must also contain a ribosome-binding site , placed just before the coding region. Unless a cloned gene contains both of these sequences, it will not be expressed in a bacterial host cell. If the gene has been produced via cDNA from a eukaryotic cell, then it will certainly not have any such sequences. Consequently, expression vectors have been developed that contain promoter and ribosome-binding sites positioned just before one or more restriction sites for the insertion of foreign DNA ( Figure 1 ). These regulatory sequences , such as that from the lac operon of E. coli , are usually derived from genes that, when induced, are strongly expressed in bacteria. Since the mRNA produced from the gene is read as triplet codons, the inserted sequence must be placed so that its reading frame is in phase with the regulatory sequence. This can be ensured by the use of three vectors that differ only in the number of bases between promoter and insertion site, the second and third vectors being respectively one and two bases longer than the first. If an insert is cloned in all three vectors then in general it will subsequently be in the correct reading frame in one of them. The resulting clones can be screened for the production of a functional foreign protein. The final clone should then be checked by Sanger sequencing.

Expression of Eukaryotic Genes

 It is not only possible, but usually essential, to use cDNA instead of a eukaryotic genomic DNA to direct the production of a functional protein by bacteria. This is because bacteria are not capable of processing RNA to remove introns, and so any foreign genes must be pre-processed as cDNA if they contain introns. A further problem arises if the protein must be glycosylated, by the conjugation with oligosaccharides at specific sites, in order to become functional. Although the use of bacterial expression systems is somewhat limited for eukaryotic systems, there are a number of eukaryotic expression systems based on plant, mammalian, insect and yeast cells. These types of cell can perform such post-translational modifications, producing a correct glycosylation pattern or phosphorylation. It is also possible to include a signal or address sequence at the 5′ end of the mRNA that directs the protein to a particular cellular compartment or even out of the cell altogether into the supernatant. This makes the recovery of expressed recombinant proteins much easier, since the super natant may be drawn off while the cells are still producing protein.

One useful eukaryotic expression system is based on the monkey COS cell line. These cells each contain a region derived from a mammalian monkey virus termed simian virus 40 ( SV40). A defective region of the SV40 genome has been stably integrated into the COS cell genome. This allows the expression of a protein termed the large T antigen, which is required for viral replication. When a recombinant vector having the SV40 origin of replication and carrying foreign DNA is inserted into the COS cells, viral replication takes place. This results in high-level expression of foreign proteins. The disadvantage of this system is the ultimate lysis of the COS cells and limited insert capacity of the vector. Much interest has thus been focussed on other modified viruses, vaccinia virus and baculovirus. These have been developed for high-level expression in mammalian cells and insect cells, respectively. The vaccinia virus in particular has been used to correct defective ion transport by introducing a wild-type cystic fibrosis gene into cells bearing a mutated cystic fibrosis (CFTR) gene. There is no doubt that the further development of these vector systems will enhance eukaryotic protein expression in the future.

Phage Display Techniques

As a result of the production of phagemid vectors and as a means of overcoming the problems of screening large numbers of clones generated from genomic libraries of antibody genes, a method for linking the phenotype or expressed protein with the genotype has been devised. This is termed phage display, since a functional protein is linked to a major coat protein of a coliphage, whilst the single-stranded gene encoding the protein is packaged within the virion. The initial steps of the method rely on the PCR to amplify gene fragments that represent functional domains or subunits of a protein such as an antibody. These are then cloned into a phage display vector, which is an adapted phagemid vector and used to transform E. coli . A helper phage is then added to provide accessory proteins for new phage molecules to be constructed. The DNA fragments representing the protein or polypeptide of interest are also transcribed and translated, but linked to the gene for major coat protein III (gIII). Thus when the phage is assembled, the protein or polypeptide of interest is incorporated into the coat of the phage and displayed, whilst the corresponding DNA is encapsulated ( Figure 2).

Fig2. Flow diagram indicating the main steps in the phage display technique.

There are numerous applications for the display of proteins on the surface of bacteriophage viruses, bacteria and other organisms, and commercial organisations have been quick to exploit this technology. One major application is the analysis and pro duction of engineered antibodies from which the technology was mainly developed. In general, phage-based systems have a number of novel applications in terms of ease of selection rather than screening of antibody fragments, allowing analysis by methods such as affinity chromatography. In this way, it is possible to generate large numbers of antibody heavy and light chain genes by PCR amplification and mix them in a random fashion. This recombinatorial library approach may allow new or novel partners to be formed, as well as naturally existing ones. This strategy is not restricted to antibodies and vast libraries of peptides may be used in this combinatorial chemistry approach to identify novel compounds of use in biotechnology and medicine.

Phage-based cloning methods also offer the advantage of allowing mutagenesis to be performed with relative ease. This may allow the production of antibodies with affinities approaching that derived from the human or mouse immune system. This may be brought about by using an error-prone DNA polymerase in the initial steps of constructing a phage display library . It is possible that these types of libraries may provide a route to high-affinity recombinant antibody fragments that are difficult to produce by more conventional hybridoma fusion techniques. Surface display libraries have also been prepared for the selection of ligands, hormones and other polypeptides in addition to allowing studies on protein–protein or protein–DNA interactions or determining the precise binding domains in these receptor–ligand interactions.

Alternative Display Systems

 A number of display systems have been developed based on the original phage display technique. One interesting method is ribosome display, where a sequence or even a library of sequences are transcribed and translated in vitro; however, in the DNA library, the sequences are fused to spacer sequences lacking a stop codon. During translation at the ribosome, the protein protrudes from the ribosome and is locked in with the mRNA. The complex can be stabilised by adding salt. In this way, it is possible to select the appropriate protein through binding to its ligand. Thus a high- affinity protein–ligand can be isolated that has the mRNA that originally encoded it. The mRNA may then be reverse transcribed into cDNA and amplified by PCR to allow further methods, such as mutagenesis, to be undertaken. In the similar technique of mRNA display, the association between the protein and mRNA is through a more stable covalent puromycin link rather than the salt-induced link as in ribosome display. Further display systems, based on yeast or bacteria, have also been developed and provide powerful in vitro selection methods.

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مواضيع ذات صلة

Applications of Gene Cloning

cDNA Libraries

Genomic DNA Libraries

Ligating DNA Molecules

Knockout of Leptin Gene

Engineered CRISPR–Cas9 Systems Allow Precise Genome Editing

Plasmid Expression Vectors Can Be Designed for Use in Animal Cells

Engineering Antibody Synthesis in Bacteria

Heritable Genome Editing

Challenges to in Vivo Editing of Hematopoietic Stem Cells

EX Vivo Manufacturing of Therapeutic Cells

Using Engineered Nucleases to Initiate the Genome Editing Process