Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis

Area of Science:

• Molecular biology
• Microbial genetics

Background:

• pUB110-based plasmids are widely used for protein expression and secretion in Bacillus subtilis.
• Existing vectors like pUB18P43 and pWB705 have limitations in flexibility and compatibility.
• A new pE194-based vector (pE18) was developed for enhanced expression studies.

Purpose of the Study:

• To compare the efficacy of pUB110-based and pE194-based expression vectors in Bacillus subtilis.
• To identify the reasons for differences in protein production yields between the two vector systems.
• To develop improved expression vectors for greater gene expression.

Main Methods:

• Construction of a novel pE194-based plasmid vector (pE18).
• Expression analysis of three model genes (cytoplasmic BirA, extracytoplasmic PrsA, extracellular staphylokinase) using both pUB110 and pE194-based vectors.
• Investigation of plasmid stability and copy number.
• Determination of protein production yields through multiple analytical approaches.

Main Results:

• pE194-based vectors consistently yielded 2-3 fold higher protein production compared to pUB110-based vectors.
• Differences in protein yield were not attributed to plasmid rearrangement or copy number variations.
• Lower yields from pUB110 vectors were identified as resulting from transcription interference from plasmid-encoded genes.
• Gene orientation within pUB110 vectors significantly impacted expression levels.

Conclusions:

• The pE194-based expression system offers superior protein production in Bacillus subtilis over pUB110-based systems.
• Transcription interference is a key factor limiting gene expression in pUB110 vectors.
• Optimized vector design, including new constructs like pUB19P43 and pWB980, can enhance gene expression capabilities.