Restriction digests of the clone give the following sizes (kb): HindIII--6.2, 1.9, 1.1; EcoRI--8.0, 1.2; PstI--9.2. The pDS472 5' oligo changed the ATG of GST to TTG (M1L) in order to prevent translation of only the GST portion of a fusion protein. The vector was designed for C-terminal tagging with GST (glutathione S transferase). The vector contains a thrombin cleavage site (LVPR/GS) immediately following the GST moiety, to allow removal of the tag following purification. The vector was constructed by 1) amplification of the Schistosoma japonicum glutathione S-transferase gene with primers designed to flank the gene and modify the polylinker, 2) gel purification and digest of the PCR product with XhoI, 3) ligation into pREP4X cleaved with XhoI and SmaI; the SmaI site was lost during cloning, 4) digestion with NotI & SalI, purified GST-containing fragment was cloned into pSLF172, digested with NotI and SalI. |
