This technology is also used to make large amounts of many medically useful proteins, including hormones (such as insulin and growth factors) used as human pharmaceuticals, and viral coat proteins for use in vaccines. Expression vectors also allow scientists to produce many proteins of biological interest in large enough amounts for detailed structural studies. Nearly all three-dimensional protein structures depicted in this book are of proteins produced in this way. Recombinant DNA techniques thus allow scientists to move with ease from protein to gene, and vice versa, so that the functions of both can be explored on multiple fronts (Figure 8–43).
DNA cloning allows a copy of any specific part of a DNA or RNA sequence to be selected from the millions of other sequences in a cell and produced in unlimited amounts in pure form. DNA sequences can be amplified after breaking up chromosomal DNA and inserting the resulting DNA fragments into the chromosome of a self-replicating genetic element such as a plasmid. The resulting “genomic DNA library” is housed in millions of bacterial cells, each carrying a different cloned DNA fragment. Individual cells from this library that are allowed to proliferate produce large amounts of a single cloned DNA fragment. Bypassing cloning vectors and bacterial cells altogether, the polymerase chain reaction (PCR) allows DNA cloning to be performed directly with a DNA polymerase and DNA primers—provided that the DNA sequence of interest is already known.
The procedures used to obtain DNA clones that correspond in sequence to mRNA molecules are the same, except that a DNA copy of the mRNA sequence, called cDNA, is first made. Unlike genomic DNA clones, cDNA clones lack intron sequences, making them the clones of choice for analyzing the protein product of a gene.
Nucleic acid hybridization reactions provide a sensitive means of detecting any nucleotide sequence of interest. The enormous specificity of this hybridization reaction allows any single-stranded sequence of nucleotides to be labeled with a radioisotope or chemical and used as a probe to find a complementary partner strand, even in a cell or cell extract that contains millions of different DNA and RNA sequences. DNA hybridization also makes it possible to use PCR to amplify any section of any genome once its sequence is known.
direction of electrophoresis
Figure 8–42 Production of large amounts of a protein by using a plasmid expression vector. In this example, an expression vector that overproduces a DNA helicase has been introduced into bacteria. In this expression vector, transcription from this coding sequence is under the control of a viral promoter that becomes active only at a temperature of 37°C or higher. The total cell protein, either from bacteria grown at 25°C (no helicase protein made) or after a shift of the same bacteria to 42°C for up to 2 hours (helicase protein has become the most abundant protein species in the lysate), has been analyzed by SDS polyacrylamide-gel electrophoresis. (Courtesy of Jack Barry.) sequence of a peptide fragment
E. coli or otherPROTEIN host cell to produce protein DNA primers for PCR insert protein-coding region of gene into expression vector (from cDNA clone)
Figure 8–43 Recombinant DNA techniques make it possible to move experimentally from gene to protein and from protein to gene. If a gene has been identified (right), its protein-coding sequence can be inserted into an expression vector to produce large quantities of the protein (see Figure 8–41), which can then be studied biochemically or structurally. If a protein has been purified based on its biochemical properties, mass spectrometry (see Figure 8–18) can be used to obtain a partial amino acid sequence, which is used to search a genome sequence for the corresponding nucleotide sequence. The complete gene can then be cloned by PCR from a sequenced genome (see Figure 8–37). The gene can also be manipulated and introduced into cells or organisms to study its function, a topic covered in the next section of this chapter.