[Purification and characteristics of an RNA-ligase preparation from bacteriophage T4]

A purification techniques was developed to obtain a preparation of the bacteriophage T4 RNA-ligase (EC 6.5.1.3) free of contaminating nuclease activities. It includes fractioning on phosphocellulose and DEAE-cellulose, chromatography on DEAE-cellulose, isoelectric sedimentation, gel filtration through Sephadex G-100 and chromatography on hydroxylapatite and aminohexyl-sepharose. The enzyme yield amounts to 30-37%. By means of biochemical tests, the preparation of RNA-ligase was found to be good for molecular biology and gene engineering, in particular for such areas where oligodeoxyribonucleotides and high molecular-weight RNA are used as substrates. The ligating ability of the enzyme was demonstrated by means of the preparative synthesis of nonaribonucleotide ApUpG(pU)6 from trinucleoside diphosphate ApUpG and hexoribouridylic acid, as well as by insertion of [5'-32P]-labelled cytidine-3',5'-diphosphate in the 3'-end of the bacteriophage MS 2 RNA.