The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells

Standard recombinant DNA procedures (21) were followed to generate the hybrid molecules. HI fragment encoding the B-lactamase signal peptide-c~-globin (SIG-GLO) was generated from pSP125E by the addition of Barn HI linkers at a blunt-ended Bgl Ill site; this fragment was ligated directly into the Barn HI site of pLJ to generate pLJSIG-GLO. pLJPRO-GLO Construction. pLaS1 was digested with Eco RI to generate a 462-bp fragment encoding preproSRIF (41, 46) . This fragment was ligated into the Eco RI site of a pBR322 derivative in which all four Nae I sites were deleted by digestion with Nae I and Bal I generating plasmid pDWS18 ( Fig. 1 A) . Plasmid pSP125E was digested with Nco I, filled-in using the Klenow fragment of DNA polymerase I, 12-mer Bam HI linkers were added, and following digestion with Bam HI and blunt-ending with Klenow, the DNA fragment encoding tx-globin was ligated into pDWSI8 which was partially digested with Nae I. This generated a hybrid gene encoding the preproSRIF signal peptide and proregion in frame with c~-globin at the 3' end; four amino acids (AlaAspProArg) were introduced (double lines) by this construction. Plasmid pPRO-GLO was digested with Barn HI and the appropriate DNA fragment ligated into the unique Bam HI site of pLJ to generate pI_JPRO-GLO.