High-level Expression and Secretion of Serratia marcescens Metalloprotease Inhibitor in $Bacillus$ $subtilis$ by Aid of Subtilisin Promoter and signal Sequence

Abstract

Metalloprotease inhibitor (SmaPI) of a gram-negative bacterium, Serratia marcescens, was expressed and secreted In a heterologous host, Bacillus subtilis DB431, by aid of a subtilisin promotor and signal sequence. The DNA fragment containing the coding sequence for SmaPI was amplified by polymerase chain reaction, and the amplified-DNA product was inserted into the downstream region of a subtilisin signal sequence. The recombinant SmaPI expressed in Bacillus subtilis DB431 was secreted into the culture medium in a large amount. After cultivation for 32 h, the amount of SmaPI secreted into the culture medium reached about 100 mg/liter when estimated by measuring inhibitory activities toward Serratia marcescens metalloprotease (SMP). The NH2-terminal amino acid sequencing analysis confirmed that authentic SmaPI and the recombinant SmaPI have the same NH2-terminal amino acid sequences. The inhibitory activity of the purified recombinant SmaPI was found to be nearly equivalent to that of authentic SmaPI.