Novel Plasmid Vectors for the Soluble Expression of Recombinant Proteins in Escherichia coli
Posted Feb 20 2013 7:00pm
Description of Invention: A series of novel plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli (E. coli) are provided. Because of its ease of growth and generally low cost to cultivate, E. coli is often employed as the host for vectors expressing recombinant proteins. In an ideal situation, the recombinant protein is expressed from a strong promoter, highly soluble, and recovered in high yield and activity. Unfortunately, it is quite common that the overproduced recombinant protein is either detrimental to the cell or simply compartmentalized into insoluble inclusion bodies. Recently, NIH investigators have developed plasmid vectors that enable the recovery and purification of recombinant proteins that have previously proven to be difficult to express in soluble form. These vectors have a pSC101 origin of replication and, therefore, are maintained in E. coli at approximately five (5) copies per cell (plasmid details and maps will be provided upon request). These vectors express the recombinant proteins at low basal levels and this feature facilitates higher solubility and correct folding of the expressed protein. The utility of these vectors is verified by expressing and purifying full-length human DNA polymerases from E. coli and showing that the purified DNA polymerases are catalytically active in vitro.
Obtain recombinant proteins that were previously hard to purify
Produce recombinant proteins from a number of sources and with different catalytic activities
Express multimeric protein complexes
Dramatically increase the proportion of soluble protein that can be obtained in E. coli
Fully compatible with the replicons of conventional high-expression systems (e.g., pET vectors, EMD Biosciences
Facilitate the correct folding of the recombinant protein and increases its solubility
In vitro data available
Inventors: Roger Woodgate (NICHD) John P Mcdonald (NICHD) Kiyonobu Karata (NICHD)
Patent Status: HHS, Reference No. E-028-2010/0
Research Tools – Patent protection is not being pursued for this technology.
For Licensing Information Please Contact: Suryanarayana Vepa Ph.D. NIH Office of Technology Transfer 6011 Executive Blvd. Suite 325, Rockville, MD 20852 United States Email: email@example.com Phone: 301-435-5020 Fax: 301-402-0220