pBE-S枯草芽孢桿菌分泌型表達載體質粒

Recombinant protein production using Bacillus subtilis as host facilitates soluble expression
and secretory expression. The B. subtilis expression host is particularly effective for proteins that
have complex structures, such as S-S bonds. Unlike the E. coli expression systems, recombinant
proteins produced by B. subtilis are free of endotoxin.
T. Eggert et al. have reported that the level of secretion of recombinant proteins is significantly
influenced by the type of signal peptide used.1) Takara Bio has developed a system to screen for
efficient expression of secreted proteins using B. subtilis. This system makes it possible to identify
the signal peptides suitable for secretory expression of a target protein from among a library of
173 types of B. subtilis-derived secretory signal peptides. However, because the plasmids of B.
subtilis have extremely low copy number, plasmid construction within B. subtilis is difficult. This
system uses pBE-S DNA as a shuttle vector of E. coli and B. subtilis, which makes it possible to
construct and propagate expression plasmids in E. coli.
This system includes:
? SP DNA mixture - the DNA library of the secretory signal peptides of B. subtilis
? The B. subtilis / E. coli shuttle vector pBE-S DNA
? B. subtilis strain RIK1285
The SP DNA mixture contains DNA fragments (for In-Fusion? cloning) that encode 173 types
of B. subtilis secretory signal peptides. The pBE-S DNA vector includes the pUB110-derived
replication ori (pUB ori) and a kanamycin-resistant gene (Kanr
) which functions in B. subtilis, as
well as the pUC-derived replication ori (ColE1 ori) along with an ampicillin-resistant gene (Ampr
)
which functions in E. coli. Additionally, it includes a B. subtilis-derived subtilisin promoter (aprE
promoter) and secretory signal peptide (aprE SP), which are located upstream from the multicloning site (MCS) and the His tag sequence. Once linearization of pBE-S DNA containing a
target gene in the MCS is performed through the use of the restriction enzymes Mlu I and Eco52 I,
the Clontech In-Fusion? cloning method is used to introduce the DNA library of 173 types of
secretory signal peptides in the SP DNA mixture in place of the aprE SP (see vector map below).
This makes it possible to screen for an efficient secretory signal peptide suitable for the target protein.
Because the MCS of pBE-S DNA is compatible with the E. coli cold-shock expression system
pCold vector (Cat. #3360 - 3365 and 3371), one can easily transfer the target gene between these
vectors. The host strain B. subtilis RIK1285, provided with this kit, is deficient in two kinds of
proteases,2) and therefore is suitable for the secretory expression of target proteins.

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