A strategy for efficientin vitrotranslation of cDNAs using the rabbit β-globin leader sequence

Analysis of in vitro synthesized proteins is frequently hampered by inefficient translation especially of large proteins. It had been shown that replacement of the GC-rich leader of human SRF by an in frame fusion to the human /3-globin leader sequence including the AUG initiation codon strongly stimulated translation in a reticulocyte lysate (1). We expanded on this observation and show that the /3-globin leader alone can enhance translation efficiencies both in rabbit reticulocyte lysates and in wheat germ extracts. The region homologous to the 5' end of rabbit /3-globin RNA but lacking the AUG initiation codon was inserted into the pBluescript KS vector (Stratatene) to generate plasmid pBAT (Figure 1A). The cRNAs tested lack most of their natural leader but retain the authentic initiation codon. Upon translation in a reticulocyte lysate the correct translation product of a cRNA encoding human TF-IID (A.A. & T.W., unpublished) is only detectable in the presence of the /3-globin leader (Figure IB, lanes 3 and 4) and translation of mouse Oct2.2 (2) is stimulated 25 fold (Figure IB, lanes 5 and 6). For a variety of applications in vitro translation in wheat germ extracts is preferred. Again translation of TF-IID is strongly enhanced by the leader insertion (Figure 1C, lanes 3 and 4) but even the level of Oct2.2 translation can be increased (Figure 1C, lanes 7 and 8). Full length human retinoblastoma Rbl05 (3) translation product is only detectable when the natural leader is replaced by the j3-globin leader (Figure 1C, lanes 5 and 6). The identical result is obtained upon translation in reticulocyte lysates (data not shown). Our results demonstrate that the /3-globin leader sequence alone increases in vitro translation efficiencies with various cDNAs/RNAs in both reticulocyte lysates and wheat germ extracts. This strategy is thus extremely useful for a variety of applications, e.g. when high levels of full length protein synthesis are required or when functional analyses of cDNAs lacking a leader sequence are performed.