Restriction digests of the clone give the following sizes (kb): BamHI/HindIII--4.4, 0.5, 0.4; KpnI/SalI--4.5, 1.0. pPFZ-R2 (ATCC 39544) and pPFZ-R4 (ATCC 39543) are the same except that the ATG initiation codon follows the ribosomal binding site of the trp leader peptide by 5 bp in pPFZ-R2 and by 11 bp in pPFZ-R4. The insert contains BamHI sites. A KpnI/SalI digest should give a probe of about 970 bp which contains the sequences encoding aa 83 through the TGA stop codon. pPFZ-R2 (ATCC 39544) shows significantly higher expression of the protein product than pPFZ-R4 (ATCC 39543). Constructed from two fragments of pCR101, a 235 bp BamHI/KpnI fragment (encoding aa 6-83) and a 4772 bp HindIII/KpnI fragment (encoding aa 83 through TGA stop codon, pMB1, ampR) ligated with a 370 bp HindIII/BamHI fragment of ptrpLI-R2-B48. The 370 bp HindIII/BamHI fragment of ptrpLI-R2-B48 contains trp promoter-operator, ribosome binding site, artificial ATG initiation codon, and chemically derived sequence encoding the first 5 amino acids of the protein. |
