Abstract
We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61-nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids. Tandem plasmids are formed in a ligation-independent manner, propagate efficiently, and produce protein pairs in high quantities. This greatly facilitates co-expression for structural genomics projects that produce thousands of clones bearing identical origins and antibiotic markers.
Highlights
We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids
To generate tandem plasmids from expression plasmids containing the LINK sequence, each ORF-expressing plasmid is cleaved at the LINK site with a restriction enzyme, and the plasmids are joined in a ligation-independent manner
The LINK method is ideal for structural genomics applications for two major reasons
Summary
We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids. Tandem plasmids are formed in a ligation-independent manner, propagate efficiently, and produce protein pairs in high quantities This greatly facilitates co-expression for structural genomics projects that produce thousands of clones bearing identical origins and antibiotic markers. Because the presence of an identical replication origin and antibiotic resistance marker precludes stable propagation of plasmid pairs in the same cell, one or both of the ORFs must be moved to a new plasmid to achieve co-expression To this end, a number of methods have been developed, including the use of two plasmids with different selectable markers and compatible origins of replication [3, 4], a single plasmid containing two ORFs under control of separate promoters [5], or a single plasmid containing ORFs arranged in a polycistronic message [6]. From the Structural Genomics of Pathogenic Protozoa (SGPP) Consortium and ‡Center for Human Genetics and Molecular Pediatric Disease, University of Rochester Medical Center, Rochester, NY 14642; §Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642; ¶Departments of Genome Sciences and Medicine, University of Washington, Seattle, WA 98195; ʈHoward Hughes Medical Institute, University of Washington, Seattle, WA 98195; and **Department of Biochemistry, University of Washington, Seattle, WA 98195
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