Restriction digests of the clone give the following sizes (kb): EcoRI/SalI--3.3, 1.75; EcoRI--5.1; SalI--5.1; BamHI--4.9, 0.2. Runaway vector contains a temperature-sensitive mutation in the copy-control gene. Maintained at a low copy number below 34C; replication increases 50-fold at temperatures greater than 34C. Growth at 37C is lethal. The 1.75 kb EcoRI/SalI fragment contains a trp promoter/regulator and the EGF/urogastrone coding region. The complete coding sequence of the mature peptide, plus a "pro" sequence of 8 aa, is contained in a 0.2 kb XbaI/SalI fragment. Directs the synthesis of a peptide of 61 aa, including an 8-residue "leader" sequence. Expresses the urogastrone analog product on the order of 50 mg per O. D. liter as determined by SDS-PAGE. The microbially expressed product is biologically active. Constructed from oligonucleotides selected to reflect the codon usage of Escherichia coli. Contains a HinfI site interrupting Asp3, an SphI site interrupting Cys31, and an NruI site after Tyr44. |
