Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes
- 25 February 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Biomacromolecules
- Vol. 11 (4), 944-952
- https://doi.org/10.1021/bm901387t
Abstract
This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.Keywords
This publication has 37 references indexed in Scilit:
- Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteinsProtein Science, 2009
- Temperature Triggered Self-Assembly of Polypeptides into Multivalent Spherical MicellesJournal of the American Chemical Society, 2007
- Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cyclingAnalytical Biochemistry, 2007
- Synthesis and Characterization of Multiblock Copolymers Based on Spider Dragline Silk ProteinsBiomacromolecules, 2006
- Molecular Cloning of Protein-Based PolymersBiomacromolecules, 2006
- Ultra-High Expression of a Thermally Responsive Recombinant Fusion Protein in E. coliBiotechnology Progress, 2006
- Recombinant extracellular matrix-like proteins with repetitive elastin or collagen-like functional motifsBiotechnology Letters, 2005
- Potential Use of Chitosan as a Cell Scaffold Material for Cartilage Tissue EngineeringTissue Engineering, 2002
- Sequential amplification of cloned DNA as tandem multimers using class-IIS restriction enzymesGenetic Analysis: Biomolecular Engineering, 1996
- Phase‐structure transitions of the elastin polypentapeptide–water system within the framework of composition–temperature studiesPeptide Science, 1985