New chromatographic and biochemical strategies for quick preparative isolation of tRNA.
A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.
Cayama
E
E
Centro de Investigaciones Biomédicas (BIOMED), Facultad de Ciencias de la Salud, Universidad de Carabobo, Núcleo Aragua, Final Avenida Leonardo Ruiz Pineda, La Morita II, Maracay 2103, Estado Aragua, Venezuela.
Yépez
A
A
Rotondo
F
F
Bandeira
E
E
Ferreras
A C
AC
Triana-Alonso
F J
FJ
D000215:Acylation; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography / Q000379:methods*; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D012329:RNA, Bacterial / Q000302:isolation & purification; D012331:RNA, Fungal / Q000302:isolation & purification; D012343:RNA, Transfer / Q000737:chemistry / Q000302:isolation & purification*; D012355:RNA, Transfer, Ile / Q000302:isolation & purification; D012358:RNA, Transfer, Met / Q000302:isolation & purification; D012360:RNA, Transfer, Phe / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
D012329:RNA, Bacterial; D012331:RNA, Fungal; D012355:RNA, Transfer, Ile; D012358:RNA, Transfer, Met; D012360:RNA, Transfer, Phe; C089238:fMet-tRNA(fMet); D012343:RNA, Transfer; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase