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We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability....
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... are known to be relatively resistant to auto-digestion, but introducing amino acid changes might make them more susceptible to auto-digestion. Since some commercial serine proteases are supplied in propylene glycol we tested if this additive would suppress the activity of the V24 variants of SubC. The data presented in Fig. 5 shows that concentrations of 10% or higher of propylene glycol inhibit the activity of V24. We have not systematically tested the length of time that the V24 resists auto-digestion but we have stored this protease variant for over 2 years at −80 • C and protease activity results show that the stored enzyme has approximately the same ...
Citations
... In one experiment, we assembled three fragments (see Supplementary Fig. S3) of the gusA gene into plasmid p110S:: subC-V25 using Esp3I-based GGA. The subC-V25 gene encodes a temper atur e-sensitiv e serine pr otease (Thompson et al. 2020 ) derived from subtilisin Carlsberg. Expression of SubC leads to a zone of clearing on agar plates supplemented with milk and this phenotype allo w ed us to detect transformation of template DN A follo wing a GGA reaction; gusA activity was detected by the blue colour generated by the cleavage of X-gluc. ...
In order to facilitate Golden Gate DNA assembly, we have constructed a collection of Bacillus subtilis replicative plasmids representing five origins of replication derived from plasmids pUB110, pE194, pWV01, pBS72, and pTH1030. The first three of these plasmids use rolling circle replication and the latter two use theta replication. All of the plasmids carry the same multiple cloning site surrounded by transcriptional terminators. The plasmids are about three kilobases in size, allowing them to be easily amplified by inverse PCR using a common set of primers to generate cloning-ready amplicons. This plasmid PCR amplification approach also facilitates a workflow that eliminates Escherichia coli as a shuttle intermediate. All of the plasmids lack a site for at least three of the type IIS restriction enzyme BbsI, BsaI, Esp3I, PaqCI or SapI, making them compatible with Golden Gate DNA assembly. We have demonstrated the utility of the plasmids by performing Golden Gate assembly of gusA and bgaB-reporter gene fragments and in expressing plasmid-borne red fluorescent protein under the control of RNA polymerase from bacteriophage K1E.
Bacillus subtilis is a robust industrial workhorse for the production of heterologous proteins. Chromosomal integration-based protein production has advantages over plasmid-based methods. Considering that the expression level of a gene is affected by its location in the chromosome, it is important to find an optimal integration site for the gene to be expressed. This work establishes a method for random insertion of a gene expression cassette into chromosomes, enabling the screening of optimal integration sites for high-level protein production. Specifically, a gene expression cassette and a chloromycetin-resistance marker are assembled into a transposon. This transposon is inserted between the promoter and the ribosomal binding site of the zeocin-resistance marker in the chromosome, which blocks the transcription of the zeocin-resistance gene. Transposase Himar1-mediated transposition of this transposon activates the zeocin-resistance marker, which can be selected on plates containing both chloromycetin and zeocin. The transposition frequency was over 10⁻⁵. This method was used to select proper insertion sites for the expression cassette of methyl parathion hydrolase (MPH). Compared with the common integration site amyE, the expression level of MPH was increased up to 50% at the yjbH site.
