PEPTIDASE

Purification Methods

The dipeptidase is purified 700-fold by homogenising the tissue, extracting with Triton X-100, elimination of insoluble material, and ion-exchange, size exclusion and affinity chromatography. [Hitchcock et al. Anal Biochem 163 219 1987.] Restriction enzymes (endonucleases). These are enzymes which cleave double-stranded DNA (linear or circular) at specific nucleotide sequences within the DNA strands which are then used for cloning (by ligating bits of DNA sequences together) or for identifying particular DNA materials, e.g. plasmids, genes etc. A very large number of restriction enzymes are now available commercially and are extensively used in molecular biology. They are highly specific for particular nucleotide arrangements and are sensitive to the reaction conditions, e.g. composition of the medium, pH, salt concentration, temperature etc, which have to be strictly adhered to. Endonucleases are essentially of two types. One type cleaves the DNA producing blunt ends, i.e. the nucleotides are paired at each end. The second type cleaves the DNA leaving hanging ends, i.e. the nucleotides at the ends of each strand (usually four nucleotides) of the double strands protrude ahead of the paired nucleotides. The latter enzymes particularly allow ligation of the cleaved sequence to occur in a desired direction. The enzymes do not require further purification, and the reaction conditions are also provided by the suppliers from which the necessary reaction media can also be purchased (see commercial catalogues).