High throughput cloning and expression strategies for protein production.
Traditionally, the production of a recombinant protein requires a preliminary cloning step of the target gene into an expression vector before evaluating its cellular expression. Among current methods, site-specific recombination cloning techniques, which eliminate the use of restriction endonucleases and ligase, offer several advantages in the context of high throughput (HT) procedures. Rapid and highly efficient, the recombinational cloning technology is largely used for structural genomics and functional proteomics. However, the correct expression of some genes requires further optimization steps that are time-consuming and carried out at relatively late stages in the cloning-expression process. An alternative strategy is described where expression is tested in vitro before cloning the target gene. This technology, amenable to automation for HT studies, makes the expression of several hundreds of genes possible from PCR products in cell-free transcription-translation systems. Once this preliminary step is achieved, the PCR product, which gives satisfying expression levels, is selected, and then cloned in a plasmid for its cellular expression and perpetuation.
Betton
Jean-Michel
JM
Unité de Repliement et Modélisation des Protéines, Institut Pasteur, CNRS-URA2185, 28 rue du Docteur Roux, 75724 Paris cedex 15, France. jmbetton@pasteur.fr
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins