Abstract
Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223–3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor ( lacI Q) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by ‘forced cloning’ into the appropriately selected vector.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.