bp sphi bamhi fragment specifying the c terminal 5 amino acids of tgf alpha and the n terminal 252 amino acids of pe 40 was isolated m13 mp19 vector dna was cut with sphi and bamhi and the vector dna was isolated the 750 bp sphi bamhi tgf alpha pe 40 fragment was ligated into the m13 vector dna overnight at 15 u00b0 c bacterial host cells were transformed with this ligation mixture candidate clones were isolated and their plasmid dna was sequenced to insure that these clones contained the proper recombinant dnas single stranded dna was prepared for mutagenesis an oligonucleotide oligo 133 was synthesized and used in site directed mutagenesis to introduce a bsteii site into the tgf alpha pe 40 dna at amino acid position 369 of pe 40 one consequence of this mutagenesis was the conversion of the serine residue at position 369 of pe 40 to a threonine a dna clone containing the newly created bsteii site was identified isolated and sequenced to ensure the presence of the proper recombinant dna this clone was next digested with apai and sali restriction enzymes a 120 bp insert dna fragment containing the newly created bsteii site was isolated and ligated into ptac tgf57 pe40 that had also been digested with apai and sali bacterial host cells were transformed and a candidate clone was isolated and sequenced to insure that the proper recombinant dna was present this newly created plasmid dna was called ptac tgf57.