title: A strategy for the expression of recombinant proteins traditionally hard to purify.
abstract: We have developed a series of 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. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.
authors: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Ekaterina G
authors: EG
authors: Frank
authors: John P
authors: JP
authors: McDonald
authors: Kiyonobu
authors: K
authors: Karata
authors: Donald
authors: D
authors: Huston
authors: Roger
authors: R
authors: Woodgate
mesh_terms: D002846:Chromatography, Affinity; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C546205:POLN protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases