The synthesis of a polypeptide product has been the ultimate goal in the genetic engineering process. Most of the polypeptide products have therapeutic or industrial uses. This process has been referred to as gene expression.
Like cloning hosts and vectors, suitable expression hosts and vectors are assent the expression of cloned genes. Unlike a cloning vector, an expression vector has some exp signaling elements preceding the cloned gene. These elements are: the promoter, the tee and the ribosome binding site.
The simplest expression vector is an E. coli plasmid that carries just an E. coli pro The choice of the promoter is of utmost importance in the construction of expression vet The choice of the expression host is of equal importance.
Prokaryotic Expression Hosts: E. coli is the simplest of all prokaryotic expression hoes to its relative structural simplicity. Despite having many advantages, prokaryotic host cells many drawbacks.
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1. Some polypeptides fail to fold properly and precipitate as inclusion bodies.
2. Foreign polypeptides are toxic to the host cells and therefore, cell cultures sec them cannot grow in a high density. This problem is circumvented by the use inducible promoter that is turned on only after the culture has been grown.
3. Most eukaryotic polypeptides undergo some posttranslational modifications us addition of phosphates and sugars to achieve functional specificity. They cannot when they are synthesized in a prokaryotic host, since this host lacks the enzyme problem is addressed by isolating and adding the necessary eukaryotic enzymes growth medium.
Eukaryotic Expression Host: Yeast is a simple eukaryotic cell that has the nice posttranslational modification enzymes like those of higher eukaryotic cells however, ye a disadvantage in having active proteases, which degrade the secreted polypeptides.
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Thispr is overcome by designing yeast hosts, in which the protease genes have been deleted. Hereto proteins are synthesized in insect cells by baculovirus expression vector. The advantage high level of expression, correct folding and posttranslational modifications like those in mama; cells. A vaccine for AIDS virus has been prepared by this method.
Despite the significant advantages of producing human proteins in heterologou cells, the best place to produce mammalian proteins is the mammalian cell itself. STEP11. Screening of the Expressed Product:
The screening for the expressed polypeptide product is earned out in a similar ma that of the immunological screening method of the right clone of transformed cells.
The re is simple. The clone of transformed cells will express the polypeptide product and not the containing untransformed cells. Radio labeled / fluorescent antibodies are exposed against the clones. The clones expressing the product will react with the antibodies in a species specific manner and are detected by autoradiography. This method of screening is also known as plaque e lift method of screening.