Restriction digests of the clone give the following sizes (kb): HindIII--3.2, 2.7; SmaI--5.9; BamHI--5.7, 0.2; XhoI--5.9. Construct permitting expression and secretion of alkaline phosphatase using the chitinase promoter and signal sequences. The insert contains the complete coding sequence for phoA, as well as the induction signals, ribosome-binding site and the signal sequence for S. plicatus chitinase-56 (also called chitinase-63). The preferred cleavage site is after Ala30. A HindIII digest yields a 3.2 kb fragment, containing approximately 250 bp of Streptomyces DNA (the chitinase regulatory sequences), and coding for a chitinase-phoA fusion protein. The phoA sequences can be removed vy a SmaI/XhoI digest and replaced with other sequences to be expressed and secreted. |
