Following table contains several popular restriction endonucleases and their sequence specificities.
Selection of the recombinant plasmid
How can one select only those host organisms that contain a properly constructed vector? In the case of plasmid transformation, this is usually done through the use of antibiotics and/or chromogenic substrates. For example, E. coli transformed with a plasmid containing a foreign DNA insert in its polylinker region that lacks b-galactosidase activity because the insert interrupts the protein-encoding sequence of the lacZ' gene. Thus, when grown in the presence of blue dye X-gal bacterial colonies that have an insert in their polylinker region form colorless colonies, whereas bacteria containing only plasmids that lack an insert form blue colonies. Addition of an antibiotic (such as ampicillin) will eliminate any colonies that did not take up the plasmid. The plasmids intact gene contains a gene that confers antibiotic resistance.
Production of proteins coded by target DNA
One of the greatest advantages of recombinant DNA technology is in the production of large quantities of scarce and/or novel proteins. This is a relatively straightforward procedure for bacterial proteins: A cloned structural gene must be inserted into an expression vector, a plasmid that contains the properly positioned transcriptional and translational control sequences for the protein's expression. With the use of a relaxed control plasmid and an efficient promoter, the production of protein of interest may reach 30% of the host's total cellular protein. Such genetically engineered organisms are called overproducers. Bacterial cells often sequester such large amounts of useless and possibly toxic protein as insoluble and denatured inclusions result. Protein extracted from these inclusions must therefore be renatured, usually dissolving it in a guanidinium chloride or urea solution and then dialyzing away the denaturant. A strategy for circumventing this difficulty is to precede the protein of interest with the signal sequence of a bacterial protein. Such a protein is secreted into the bacterial periplasm with the concomitant removal of its signal sequence by a bacterial protease. Secreted proteins, which are relatively few in number, can be released into the medium by hypotonic disruption of the bacterial outer membrane, so their purification is greatly simplified relative to that of intracellular proteins. The ability to synthesize a given protein in large quantities is already having enormous medical, agricultural, and industrial impact. Those that are in routine clinical use include human insulin, human growth hormone, and many other beneficial stimulators of human cells and organs.
REFERENCES
1. Voet, D. and Voet, J. G. Biochemistry John Wiley and Sons, Inc. New York, 1995.
GLOSSARY Clone: A large number of cells or molecules that are identical with a single parental cell or molecule. Endonuclease: An enzyme that cleaves internal phosphodiester bonds in DNA or RNA. Ligation: The enzyme-catalyzed joining in phosphodiester linkage of 2 stretches of DNA or RNA into one; the respective enzymes are DNA and RNA ligases. Palindrome: A sequence of duplex DNA that is the same when the 2 strands are read in the opposite directions. Plasmid: A small, extrachromosomal, circular molecule of DNA that replicates independently of the host DNA. Recombinant DNA: The altered DNA that results from the insertion of a sequence of deoxynucleotides not previously present into an existing molecule of DNA by enzymatic or chemical means. Restriction enzyme: An endonuclease that causes cleavage of both strands of DNA at highly specific sites dictated by the base sequence. Sticky-ended DNA: DNA with complementary single strands that protrude from opposite ends of a DNA duplex. Vector: A plasmid, bacteriophage or virus into which foreign DNA can be introduced for the purposes of cloning.
