High copy number self-replicating plasmids in pseudomonas

US8318481B2 · kind B2 · utility

Assignee

• Pfenex Inc. · US

Inventors

• Lawrence Chew · San Diego, US
• Stacey L. Lee · San Diego, US

Key dates

• Filing date: Dec 5, 2008
• Grant date: Nov 27, 2012
• Expiry date: Mar 22, 2031

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/78
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Provided herein are improved copy number plasmids, particularly those plasmids capable of replication in a bacterial cell. The improved copy number plasmid contain a deletion, insertion, or substitution in the replication control region, particularly a Pseudomonas-specific replication control region, that results in an increase in plasmid copy number in comparison to a control plasmid. Also provided are host cells containing the improved copy number plasmids, as well as methods of using the improved copy number plasmids for the recombinant production of a protein of interest. Further provided are methods for generating plasmids with improved copy number. The methods disclosed herein involve the reiterative selection of improved copy number plasmids by the growth and selection of plasmids capable of growth under increasing selective pressure, wherein the selective pressure is applied utilizing a selection agent to which the control plasmid confers resistance.