In the last section, we saw how protein-coding genes can be identified in genome sequences. Using the genetic code (and provided the intron and exon boundaries are known), the amino acid sequence of any protein coded in a genome can be deduced. As was discussed earlier, this sequence can often provide an important clue to the protein’s function if found to be similar to the amino acid sequence of a protein that has already been studied (see Figure 8–23). Although this strategy is often successful, it typically provides only the likely biochemical function of the protein; for example, whether the protein resembles a kinase or a protease. It usually remains for the experimenter to verify (or refute) this assignment and, most importantly, to discover the protein’s biological function in the whole organism; that is, to what attributes of the organism does the kinase or the protease contribute and in what molecular pathways does it function? Nowadays, most new proteins are “discovered” through genome sequencing, and it often remains a great challenge to ascertain their functions.
An important approach in determining gene function is to alter the gene (or in some cases, its expression pattern), to put the altered copy back into the germ line of the organism, and to deduce the function of the normal gene by the changes caused by its alteration. Various techniques to implement this strategy are discussed in the next section of this chapter. But it is equally important to study the biochemical and structural properties of a gene product, as outlined in the first part of this chapter. One of the most important contributions of DNA cloning to cell and molecular biology is the ability to produce any protein, even the rare ones, in nearly unlimited amounts—as long as the gene coding for it is known. Such high-level production is usually carried out in living cells using expression vectors (Figure 8–41). These are generally plasmids that have been designed to produce a large amount of stable mRNA that can be efficiently translated into protein when the plasmid is introduced into bacterial, yeast, insect, or mammalian cells. To prevent the high level of the foreign protein from interfering with the cell’s growth, the expression vector is often designed to delay the synthesis of the foreign mRNA and protein until shortly before the cells are harvested and lysed (Figure 8–42).
Because the desired protein made from an expression vector is produced inside a cell, it must be purified away from the host-cell proteins by chromatography following cell lysis; but because it is such a plentiful species in the cell (often 1–10% of the total cell protein), the purification is usually easy to accomplish in only a few steps. As we saw in the first part of this chapter, many expression
Figure 8–41 Production of large amounts of a protein from a protein-coding DNA sequence cloned into an expression vector and introduced into cells. A plasmid vector has been engineered to contain a highly active promoter, which causes unusually large amounts of mRNA to be produced from an adjacent protein-coding gene inserted into the plasmid vector. Depending on the characteristics of the cloning vector, the plasmid is introduced into bacterial, yeast, insect, or mammalian cells, where the inserted gene is efficiently transcribed and translated into protein. If the gene to be overexpressed has no introns (typical for genes from bacteria, archaea, and simple eukaryotes), it can simply be cloned from genomic DNA by PCR. For cloned animal and plant genes, it is often more convenient to obtain the gene as cDNA, either from a cDNA library (see Figure 8–32) or cloned directly by PCR from RNA isolated from the organism (see Figure 8–37). Alternatively, the DNA coding for the protein can be made by chemical synthesis (see p. 472).
vectors have been designed to add a molecular tag—a cluster of histidine residues or a small marker protein—to the expressed protein to facilitate easy purification by affinity chromatography (see Figure 8–11). A variety of expression vectors is available, each engineered to function in the type of cell in which the protein is to be made.