The formation of pSoin, plasmid vehical

The formation of pSoin  plasmid vehical
PLasmid pSH2O was digested with a mixture of Eco RI and Barn HI to produce a large and small DNA fragment. The sinai fragment was ks  cardedand the large fragment ercatcd with alkaline phosphatasc to present subsequent sell-ligation (see p. 18). T4 ligate was then used to join the synthetic somalosialin gene to the large fragment of pBH2O and Transformer were selected by inue of their ampicillin resistance, The DNA sequence of pSom I indicated that the clone carrying this plasmid should produce a peptide containing somatostatin. but no somatostatin was found. I1owcver in rcconstructton experiments it was obsened
that exogenous somatostatin was degraded rapidly in C. cub extracts. Thusthe laduec to find somatoseasin actisity could be accounted for by intracellular degradation by endogenous proteolytic enzymes, Such proteolyticdegradation might be prcs-entcd by attachment of the somatostalin to alarge protcin e.g. a-gabctosidasc. The a-gaiaciosidasc structural gene hasan Eco RI site near the COOH-ierminus and the asailablc data on the amino-acid sequence of this protein suggested that It would be poisibk to
Insert the synthetic gene utso this site and still mainiarn the proper reading frame. In order (0 do this. Iwo new plasmids psom II and pSom 11-3 were created.