AMIDATING ENZYME GENE

PURPOSE:To obtain the subject gene, capable of coding an enzyme amidating the C-terminal of a peptide derived from a porcine atrium and useful for producing the enzyme amidating the C-terminal alpha-carboxyl groups of the peptide, etc., important to physiological activity and providing a physiologically active substance. CONSTITUTION:A guanidine thiocyanate solution is added to a fresh porcine atrium and cells are then broken while being cooled with ice. Centrifugation is carried out and the supernatant is further layered to carry out centrifugation at 20 deg.C and 35Krpm. for 18hr. Thereby, the supernatant is removed and precipitated RNA is dissolved in a buffer solution and precipitated with ethanol to separate the whole RNA, which is then passed through an oligo(dT)cellulose column to collect mRNA. The resultant mRNA is subsequently used as a template to synthesize a cDNA with a reverse transcriptase. A cDNA library is then prepared by using a cDNA cloning system and further cultured to carry plaque hybridization with a DNA probe. A DNA is then extracted from a positive clone to afford the objective gene capable of coding an enzyme amidating the porcine atrium.