Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis.
pUb110-based vectors are commonly used for expression and secretion studies in Bacillus subtilis. Two of these plasmids, pUB18P43 and pWB705, have been applies to produce several proteins of interest. To offer greater flexibility and compatibility in this system, the authors have also constructed a pE194-based plasmid vector (pE18). To determine whether the pUB110-based or the pE194-based vector serves as a better expression system, three structural genes encoding cytoplasmic BirA, extracytoplasmic PrsA and extracellular staphylokinase, respectively, were used as models. Production of these products using pUB110-based vectors was consistently 2--3-fold lower than that using the pE194-based vectors. The observed difference in the protein yield did not result from either the rearrangement of the plasmid or the difference in the plasmid copy number. By using three different approaches, the lower production yield from the pUB110-based vectors was found to be due to the transcription interference from the plasmid encoded genes. These findings illustrate that the orientation of the inserted gene in pUB110-based vector can greatly affect gene expression. Two new expression vectors, pUB19P43 and pWB980, were constructed to allow better gene expression.
Wu
S C
SC
Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, Alberta T2n 1N4, Canada.
Wong
S L
SL
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008666:Metalloendopeptidases / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C082160:prsA protein, bacteria; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli