High-level expression vectors for Caulobacter crescentus incorporating the transcription/translation initiation regions of the paracrystalline surface-layer-protein gene.
A number of plasmid vectors were constructed for high-level gene expression in the dimorphic gram-negative bacterium Caulobacter crescentus. These vectors incorporate the transcription and translation initiation regions of the C. crescentus CB15A rsaA gene, which codes for the abundantly synthesized protein comprising the bacterium's paracrystalline surface layer. The expression vectors are based on the broad-host-range IncQ plasmid RSF1010 (R300B) and incorporate the rsaA promoter and transcription start site. Some vectors also contain translation initiation information; these can result in the addition of as little as a single glycine residue to the protein encoded by the cloned segment. The vectors can be introduced into C. crescentus by electroporation at high frequency (ranging up to 10(6)-10(7) electroporants/micrograms DNA with surface-layer-deficient mutant C. crescentus CB2A) or conjugal transfer. They range in size from 10 to 12 kb, specify either chloramphenicol or kanamycin resistance, and possess the restriction sites EcoRI, BamHI, KpnI, and SstI for cloning genes downstream of the rsaA gene sequences. For a number of the vectors, the complete nucleotide sequence is known. A comparison was made between the expression of an endoglucanase gene from these plasmids in C. crescentus CB2A and CB15A and similar constructions under the control of lacZ alpha transcription and translation initiation signals carried on a pUC9 vector in an Escherichia coli host. The two expression systems compared favorably; cell lysates prepared from C. crescentus CB2A exhibited 40% of the endoglucanase activity of similarly prepared lysates from E. coli JM101. Lysates prepared from C. crescentus CB15A exhibited only 8% of the endoglucanase activity of E. coli lysates.
Bingle
W H
WH
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Smit
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002480:Cellulase