Separation of human from mouse and monkey adenosine deaminase by ion-exchange chromatography following retroviral-mediated gene transfer.
A method for the chromatographic separation of human adenosine deaminase (ADA) from murine and monkey ADA is described. This procedure was developed in order to detect the expression of low or moderate levels of human ADA following retroviral-mediated gene transfer of cloned human ADA gene sequences into both mouse and monkey cells. Protein separation was achieved on a Mono Q (HR 5/5) anion-exchange column using the Pharmacia fast protein liquid chromatography system and was found to be a highly reproducible method yielding enzymatically active protein. An increasing linear gradient extending from 0.05 to 0.5 M potassium chloride (pH 7.5) was used to elute the enzyme. Under these conditions, most human ADA does not bind to the column and elutes in the low-salt buffer (0.05 M KCl), while murine ADA elutes at 0.12 M KCl and monkey ADA at 0.15 M KCl. The column fractions were assayed for ADA activity, and the characteristic isozyme banding patterns for human, mouse, and monkey ADA were confirmed by starch gel electrophoresis. This procedure allows the rapid and reproducible separation of human ADA from that of other species and yields partially purified enzymatically active protein.
D000243:Adenosine Deaminase / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D003001:Cloning, Molecular; D004592:Electrophoresis, Starch Gel; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification*; D008251:Macaca; D051379:Mice; D009700:Nucleoside Deaminases / Q000302:isolation & purification*; D012190:Retroviridae
D007527:Isoenzymes; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase