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The DNA was digested with restriction enzymes BamHI and XhoI to check the LLT1 insert. Plasmids containing cDNA from human 17[beta]-HSD1 or 17[beta]-HSD2, kindly provided by Stefan Andersson, were recloned into pcDNA3 vector by PCR with primers at the 5' end containing a HindIII restriction site (17[beta]-HSD1) or a BamHI restriction site (17[beta]-HSD2), a Kozak consensus sequence (Kozak 1989) and the initiation codon, and primers at the 3' end containing a stop codon followed by an XbaI restriction site. The PCR amplification product was digested with BamHI and XhoI and cloned into the pET15b vector (Novagen, Madison, WI, USA) as previously described (16). |
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