Expression of α-Lytic Protease in Bacillus Subtilis

Abstract

We describe the expression of α-lytic protease, the serine gram negative protease of Lysobacter enzymogenes in Bacillus subtilis. When direct cloning of the α-lytic protease gene into the B. subtilis vector pUB110 failed to show detectable expression, alternative strategies were adopted. These included using the transcription, translation and post translational modification elements of a native Bacillus protein (Subtilisin BPN') in hybrid α-lytic protease gene constructs. The constructs were ligated into the B. subtilis multi copy plasmid pUB110 and the ligation product used to transform competent cells of the protease deficient B. subtilis strain DB104. Making these constructs involved the swapping of large sequence domains between the two genes. This was accomplished by using PCR generated cassettes of different leader sequence segments of the subtilisin BPN' gene that could be attached to the α-lytic protease structural gene precisely and reproducibly, without the use of restriction sites. The gene combination which resulted in detectable expression and secretion of α-lytic protease had the promoter, ribosome binding site and signal sequence of subtilisin BPN' followed in frame by the pro and mature protein sequences of α-lytic protease. It was important to insert the gene into the plasmid in a counterclockwise direction to preserve plasmid stability and hence transcription and expression. Transcription, translation and post translational modification and secretion from this construct was established using mRNA and Western analysis as well as kinetic substrate assays.

Key Words:

• α-lytic protease
• B. subtilis
• E. coli
• Mutagenesis
• Polymerase chain reaction
• Pre sequence
• Pro sequence
• Protein expression
• Protein secretion
• Subtilisin